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Methods in
Molecular Biology 2566

Carlo Pellicciari · Marco Biggiogera

Manuela Malatesta Editors

Histochemistry
of Single
Molecules
Methods and Protocols
Second Edition
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
 Histochemistry of Single
Molecules

Methods and Protocols

Second Edition

Edited by

Carlo Pellicciari
Department of Biology and Biotechnology, University of Pavia, PAVIA, Italy

Marco Biggiogera
Department of Biology and Biotechnology, University of Pavia, PAVIA, Italy

Manuela Malatesta
Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, VERONA, Italy
Editors
Carlo Pellicciari Marco Biggiogera
Department of Biology and Department of Biology and Biotechnology
Biotechnology University of Pavia
University of Pavia PAVIA, Italy
PAVIA, Italy

Manuela Malatesta
Department of Neurosciences,
Biomedicine and Movement Sciences
University of Verona
VERONA, Italy

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-2674-0 ISBN 978-1-0716-2675-7 (eBook)
https://doi.org/10.1007/978-1-0716-2675-7
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part
of Springer Nature 2023
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and
retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter
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even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
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The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to
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made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Cover Illustration: Combined lectin- and immune-histochemistry on a semithin cryosection of normal human
urothelium. For labelling the proteins, the primary polyclonal rabbit antibody against transmembrane proteins
uroplakins and Alexa Flour 588 conjugated secondary goat anti-rabbit antibody were used (red fluorescence), while
for labelling the sugar residues, the FITC-conjugated Amaranthus caudatus agglutinin (ACA) was used (green
fluorescence). Colocalization of uroplakins and ACA binding is seen as orange to yellow fluorescence. Nuclear DNA
was counterstained with DAPI (blue fluorescence). (Courtesy of Daša Zupančič, Mateja Erdani Kreft and Rok Romih.)

This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface

As a distinctive feature, histochemical techniques allow localizing different chemical species

in the very place (in a tissue, or a cell or an organelle) they exist, are synthesized, or function
in vivo; this makes histochemistry a unique tool for basic and applied bio-medical research,
where it provides topological evidence of the biochemical and molecular data.
The story of histochemistry goes a long way back and, in the last 150 years, has evolved
in parallel with the detection instruments, from light and electron microscopy to cytometry
and super-resolution microscopy. Since the year 2000, about 325,000 articles, in which
histochemistry was used, have been published in qualified international journals (according
to the Web of Science database). This demonstrates that histochemistry is central for inves-
tigating very different research subjects (from cell and tissue biology to anatomy and
pathology, from zoology and botany to ecology, from developmental biology to nanotech-
nology), where it is principally applied to locate and quantify single molecules or molecular
complexes in situ, in the attempt to relate structural organization and function.
This second edition of Histochemistry of Single Molecules aims at updating and improving
the first edition’s overview of histochemical techniques, through a series of discursive
chapters and lab-tested protocols for the detection of specific molecules or metabolic
processes, at light and electron microscopy.
The book opens with a review chapter on the evolution of histochemical markers in
cytometry, and then it is divided into seven parts dealing with an assortment of chemical
targets.
Part I is on vital histochemistry, and it includes four chapters on autofluorescence
imaging, lysosome imaging by carbon dots, the detection of oxidative and nitrosative stress,
and the identification of adipogenic and osteogenic differentiation of living stem cells. Part
II, Carbohydrate Histochemistry, is comprises an updated overview on lectin histochemis-
try, followed by two chapters on the histochemical and immunohistochemical labelling of
proteoglycans, and on combined lectin- and immuno-histochemistry for fluorescence
microscopy. Five chapters form Part III, Protein Histochemistry: in the first four chapters,
immunohistochemistry is used to detect proteins marking myogenic differentiation or
autophagy at light microscopy, or milk proteins at electron microscopy, while in the last
one potassium permanganate is rediscovered as a stain for basic proteins on ultrathin
sections at transmission electron microscopy. Part IV, Lipid Histochemistry, offers an update
of basics in fixation and tissue processing, and gives protocols for the staining of myelin in
the nervous system or of lipid droplets in mouse oocytes and embryos. Part V, Nuclear
Histochemistry, contains a protocol for assessing DNA damage in cervical epithelial cells and
two contrasting methods for transmission electron microscopy: a uranyl-free technique for
nuclear structures and a staining procedure for the specific visualization of RNA by terbium
citrate vapors. Part VI is on plant histochemistry: three chapters describe protocols for the
detection of different molecules in plant cell walls, one is on starch staining with iodine
solution, and the last one on plant secretory structures. The book ends with Part VII,
Histochemistry for Nanoscience: this part, which was not present in the first edition,
includes protocols for the visualization of chemically different nanoconstructs in animal
and plant cells at bright-field, fluorescence, and transmission electron microscopy.

v
vi Preface

These 28 chapters demonstrate that histochemical techniques may be effectively used to

visualize, with high specificity, a plethora of molecules in differently processed tissues and
cells, thus confirming that histochemistry still ranks high among the methodological
approaches in life science research.

Pavia, Italy Carlo Pellicciari

Pavia, Italy Marco Biggiogera
Verona, Italy Manuela Malatesta
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1 Histochemistry in Advanced Cytometry: From Fluorochromes
to Mass Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Giuliano Mazzini and Marco Danova

PART I VITAL HISTOCHEMISTRY

2 Autofluorescence Label-Free Imaging of the Liver Reticular Structure. . . . . . . . . 29
Anna C. Croce, Giuseppina Palladini, Andrea Ferrigno,
and Mariapia Vairetti
3 Lysosome Imaging Based on Fluorescent Carbon Dots . . . . . . . . . . . . . . . . . . . . . . 37
Shuo Guo, Yuanqiang Sun, and Zhaohui Li
4 Oxidative and Nitrosative Stress Detection in Human Sperm
Using Fluorescent Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Sara Escada-Rebelo and João Ramalho-Santos
5 Simultaneous Labeling of Adipogenic and Osteogenic Differentiating
Stem Cells for Live Confocal Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Patrizia Vaghi, Amanda Oldani, Paola Fulghieri, Lidia Pollara,
Enza Maria Valente, and Virginie Sottile

PART II CARBOHYDRATE HISTOCHEMISTRY

6 Lectin Histochemistry: Historical Perspectives, State of the Art,

and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Susan Ann Brooks
7 Histochemical and Immunohistochemical Methods for the
Identification of Proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
David Sánchez-Porras, Juan Varas, Carlos Godoy-Guzmán,
Fabiola Bermejo-Casares, Sebastián San Martı́n, and Vı́ctor Carriel
8 Combined Lectin- and Immuno-histochemistry (CLIH) for
Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Daša Zupančič, Mateja Erdani Kreft, and Rok Romih

PART III PROTEIN HISTOCHEMISTRY

9 Immunofluorescence Labeling of Skeletal Muscle in Development,
Regeneration, and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Marie E. Esper, Kasun Kodippili, and Michael A. Rudnicki

vii
viii Contents

10 Immunohistochemical Detection of the Autophagy Markers LC3

and p62/SQSTM1 in Formalin-Fixed and Paraffin-Embedded Tissue . . . . . . . . . 133
Sabina Berezowska and José A. Galván
11 Immunohistochemical Detection of the Chaperone-Mediated
Autophagy Markers LAMP2A and HSPA8 in Formalin-Fixed and
Paraffin-Embedded Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Tereza Losmanová, Mario P. Tschan, José A. Galván,
and Sabina Berezowska
12 Immunogold Labeling of Milk Proteins at Transmission
Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Paolo D’Incecco
13 Rediscover Potassium Permanganate as a Stain for Basic Proteins
on Ultrathin Sections at Transmission Electron Microscopy . . . . . . . . . . . . . . . . . . 159
Lorena Zannino, Claudio Casali, and Marco Biggiogera

PART IV LIPID HISTOCHEMISTRY

14 Tissue Fixation and Processing for the Histological Identification

of Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
David Sánchez-Porras, Fabiola Bermejo-Casares, Ramon Carmona,
Tamara Weiss, Fernando Campos, and Vı́ctor Carriel
15 Staining Methods for Normal and Regenerative Myelin in the
Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Óscar D. Garcı́a-Garcı́a, Tamara Weiss, Jesús Chato-Astrain,
Stefania Raimondo, and Vı́ctor Carriel
16 Nile Red and BODIPY Staining of Lipid Droplets in Mouse
Oocytes and Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Simona Bisogno, Łukasz Ga˛sior, and Grażyna E. Ptak

PART V NUCLEAR HISTOCHEMISTRY

17 Chromatin Dispersion Test to Asses DNA Damage in Cervical

Epithelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Elva I. Cortés-Gutiérrez, José L. Fernández, Martha I. Dávila-Rodrı́guez,
Carlos Garcı́a de la Vega, and Jaime Gosálvez
18 Uranyl-Free Staining as a Suitable Contrasting Technique for
Nuclear Structures at Transmission Electron Microscopy . . . . . . . . . . . . . . . . . . . . 225
Maria Assunta Lacavalla and Barbara Cisterna
19 Specific RNA Visualization at Electron Microscopy via Terbium
Citrate Vapors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Claudio Casali, Lorena Zannino, and Marco Biggiogera

PART VI PLANT HISTOCHEMISTRY

20 Localizing Molecules in Plant Cell Walls Using Fluorescence Microscopy . . . . . . 243

Lloyd A. Donaldson
 Contents ix

21 Ratiometric Fluorescent Safranin-O Staining Allows the

Quantification of Lignin Contents In Muro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Oriane Morel, Corentin Spriet, Cédric Lion, Fabien Baldacci-Cresp,
Garance Pontier, Marie Baucher, Christophe Biot, Simon Hawkins,
and Godfrey Neutelings
22 Live Fluorescence Visualization of Cellulose and Pectin in
Plant Cell Walls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Youssef Chebli and Anja Geitmann
23 Staining Starch with Iodine Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Shengnan Zhao, Yinhui Ren, and Cunxu Wei
24 Histochemical Analysis of Plant Secretory Structures . . . . . . . . . . . . . . . . . . . . . . . . 291
Diego Demarco

PART VII HISTOCHEMISTRY FOR NANOSCIENCE

25 Alcian Blue Staining to Visualize Intracellular Hyaluronic

Acid-Based Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Mathieu Repellin, Flavia Carton, Giovanna Lollo,
and Manuela Malatesta
26 Prussian Blue Staining to Visualize Iron Oxide Nanoparticles . . . . . . . . . . . . . . . . 321
Valeria Bitonto, Francesca Garello, Arnaud Scherberich,
and Miriam Filippi
27 Diaminobenzidine Photooxidation to Visualize Fluorescent
Nanoparticles in Adhering Cultured Cells at Transmission
Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
Manuela Costanzo and Manuela Malatesta
28 Fluorescent Labeling of Lignin Nanocapsules with Fluorol
Yellow 088 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
Franco Cheli, Sara Falsini, Maria Cristina Salvatici,
Sandra Ristori, Silvia Schiff, Emilio Corti, Irene Costantini,
Cristina Gonnelli, Francesco Saverio Pavone, and Alessio Papini

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Contributors

FABIEN BALDACCI-CRESP • Université de Lille, CNRS, UMR 8576, UGSF – Unité de

Glycobiologie Structurale et Fonctionnelle, Lille, France
MARIE BAUCHER • Laboratoire de Biotechnologie Végétale (LBV), Université Libre de
Bruxelles, Gosselies, Belgium
SABINA BEREZOWSKA • Department of Laboratory Medicine and Pathology, Institute of
Pathology, Lausanne University Hospital and University of Lausanne, Lausanne,
Switzerland; Institute of Pathology, University of Bern, Bern, Switzerland
FABIOLA BERMEJO-CASARES • Department of Histology (Tissue Engineering Group), Faculty
of Medicine, University of Granada, Granada, Spain; Instituto de Investigacion
Biosanitaria, Ibs.GRANADA, Granada, Spain
MARCO BIGGIOGERA • Department of Biology and Biotechnology “Lazzaro Spallanzani”,
Laboratory of Cell Biology and Neurobiology, University of Pavia, Pavia, Italy
CHRISTOPHE BIOT • Université de Lille, CNRS, UMR 8576, UGSF – Unité de Glycobiologie
Structurale et Fonctionnelle, Lille, France
SIMONA BISOGNO • Malopolska Centre of Biotechnology, Jagiellonian University, Krakow,
Poland
VALERIA BITONTO • Department of Molecular Biotechnology and Health Sciences, University
of Turin, Torino, Italy
SUSAN ANN BROOKS • Department of Biological & Medical Sciences, Oxford Brookes
University, Oxford, UK
FERNANDO CAMPOS • Department of Histology (Tissue Engineering Group), Faculty of
Medicine, University of Granada, Granada, Spain; Instituto de Investigacion
Biosanitaria, Ibs.GRANADA, Granada, Spain
RAMÓN CARMONA • Department of Cell Biology, Faculty of Science, University of Granada,
Granada, Spain
VÍCTOR CARRIEL • Department of Histology (Tissue Engineering Group), Faculty of
Medicine, University of Granada, Granada, Spain; Instituto de Investigacion
Biosanitaria, Ibs.GRANADA, Granada, Spain
FLAVIA CARTON • Department of Neurosciences, Biomedicine and Movement Sciences,
Anatomy and Histology Section, University of Verona, Verona, Italy; University of Eastern
Piedmont, Department of Health Sciences, Novara, Italy
CLAUDIO CASALI • Department of Biology and Biotechnology “Lazzaro Spallanzani”,
Laboratory of Cell Biology and Neurobiology, University of Pavia, Pavia, Italy
JESÚS CHATO-ASTRAIN • Department of Histology (Tissue Engineering Group), Faculty of
Medicine, University of Granada, Granada, Spain; Instituto de Investigacion
Biosanitaria, Ibs.GRANADA, Granada, Spain
YOUSSEF CHEBLI • Department of Plant Science and ECP3-Multi-Scale Imaging Facility,
McGill University, Sainte-Anne-de-Bellevue, QC, Canada
FRANCO CHELI • LENS – European Laboratory for Non-linear Spectroscopy, University of
Florence, Florence, Italy
BARBARA CISTERNA • Department of Neurosciences, Biomedicine and Movement Sciences,
University of Verona, Verona, Italy

xi
xii Contributors

ELVA I. CORTÉS-GUTIÉRREZ • Universidad Autonoma de Nuevo Leon, México Faculty of

Biological Sciences, Monterrey, Mexico
EMILIO CORTI • Department of Biology, University of Florence, Florence, Italy
IRENE COSTANTINI • LENS – European Laboratory for Non-linear Spectroscopy, University of
Florence, Florence, Italy
MANUELA COSTANZO • Department of Neurosciences, Biomedicine and Movement Sciences,
Anatomy and Histology Section, University of Verona, Verona, Italy
ANNA C. CROCE • Institute of Molecular Genetics “Luigi Luca Cavalli Sforza” (IGM) –
CNR, Pavia, Italy; Department of Biology and Biotechnology “Lazzaro Spallanzani”,
University of Pavia, Pavia, Italy
PAOLO D’INCECCO • Department of Food, Environmental and Nutritional Sciences,
University of Milan, Milan, Italy
MARCO DANOVA • Department of Internal Medicine and Oncology, ASST Pavia and
LIUCC University, Castellanza, Varese, Italy
MARTHA I. DÁVILA-RODRÍGUEZ • Universidad Autonoma de Nuevo Leon, Faculty of Public
Health and Nutrition, Monterrey, Mexico
DIEGO DEMARCO • Departamento de Botânica, Instituto de Biociências, Universidade de São
Paulo, São Paulo, Brazil
LLOYD A. DONALDSON • Scion Crown Research Institute, Rotorua, New Zealand
SARA ESCADA-REBELO • PhD Programme in Experimental Biology and Biomedicine (BEB),
IIIUC- Institute for Interdisciplinary Research, University of Coimbra, Coimbra,
Portugal; Biology of Reproduction and Stem Cell Group, Center for Neuroscience and Cell
Biology, University of Coimbra, Coimbra, Portugal
MARIE E. ESPER • The Sprott Centre for Stem Cell Research, Regenerative Medicine Program,
Ottawa Hospital Research Institute, Ottawa, ON, Canada; Department of Cellular and
Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada
SARA FALSINI • Department of Biology, University of Florence, Florence, Italy
JOSÉ L. FERNÁNDEZ • Genetics Unit, INIBIC, Complejo Hospitalario Universitario A
Coruña, As Xubias, La Coruña, Spain; Laboratorio de Genética Molecular y
Radiobiologı́a Centro Oncologico de Galicia, La Coruña, Spain
ANDREA FERRIGNO • Department of Internal Medicine and Therapeutics, University of
Pavia, Pavia, Italy
MIRIAM FILIPPI • Soft Robotics Laboratory, ETH Zurich, Zurich, Switzerland
PAOLA FULGHIERI • Department of Molecular Medicine, University of Pavia, Pavia, Italy
JOSÉ A. GALVÁN • Institute of Pathology, University of Bern, Bern, Switzerland
CARLOS GARCÍA DE LA VEGA • Unit of Genetics, Department of Biology, Universidad
Autonoma de Madrid, Madrid, Spain
ÓSCAR D. GARCÍA-GARCÍA • Department of Histology (Tissue Engineering Group), Faculty of
Medicine, University of Granada, Granada, Spain; Instituto de Investigacion
Biosanitaria, Ibs.GRANADA, Granada, Spain
FRANCESCA GARELLO • Department of Molecular Biotechnology and Health Sciences,
University of Turin, Torino, Italy
ŁUKASZ GA˛SIOR • Malopolska Centre of Biotechnology, Jagiellonian University, Krakow,
Poland
ANJA GEITMANN • Department of Plant Science and ECP3-Multi-Scale Imaging Facility,
McGill University, Sainte-Anne-de-Bellevue, QC, Canada
 Contributors xiii

CARLOS GODOY-GUZMÁN • Centro de Investigacion Biomédica y Aplicada (CIBAP), Escuela

de Medicina, Universidad de Santiago de Chile, (USACH), Santiago, Chile
CRISTINA GONNELLI • Department of Biology, University of Florence, Florence, Italy
JAIME GOSÁLVEZ • Unit of Genetics, Department of Biology, Universidad Autonoma de
Madrid, Madrid, Spain
SHUO GUO • College of Chemistry, Institute of Analytical Chemistry for Life Science,
Zhengzhou University, Zhengzhou, China
SIMON HAWKINS • Université de Lille, CNRS, UMR 8576, UGSF – Unité de Glycobiologie
Structurale et Fonctionnelle, Lille, France
KASUN KODIPPILI • The Sprott Centre for Stem Cell Research, Regenerative Medicine
Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada; Department of
Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa,
ON, Canada
MATEJA ERDANI KREFT • Institute of Cell Biology, Faculty of Medicine, University of
Ljubljana, Ljubljana, Slovenia
MARIA ASSUNTA LACAVALLA • Department of Neurosciences, Biomedicine and Movement
Sciences, University of Verona, Verona, Italy
ZHAOHUI LI • College of Chemistry, Institute of Analytical Chemistry for Life Science,
Zhengzhou University, Zhengzhou, China
CÉDRIC LION • Université de Lille, CNRS, UMR 8576, UGSF – Unité de Glycobiologie
Structurale et Fonctionnelle, Lille, France
GIOVANNA LOLLO • Laboratoire d’Automatique, de Génie des Procédés et de Génie
Pharmaceutique, Université Claude Bernard Lyon 1, Villeurbanne, France
TEREZA LOSMANOVÁ • Institute of Pathology, University of Bern, Bern, Switzerland
MANUELA MALATESTA • Department of Neurosciences, Biomedicine and Movement Sciences,
Anatomy and Histology Section, University of Verona, Verona, Italy
GIULIANO MAZZINI • Institute of Molecular Genetics – CNR (National Research Council),
Pavia, Italy; Department of Biology and Biotechnology “Lazzaro Spallanzani”, University
of Pavia, Pavia, Italy
ORIANE MOREL • Université de Lille, CNRS, UMR 8576, UGSF – Unité de Glycobiologie
Structurale et Fonctionnelle, Lille, France; Institute of Biophysics, University of Natural
Resources and Life Sciences Vienna, Vienna, Austria
GODFREY NEUTELINGS • Université de Lille, CNRS, UMR 8576, UGSF – Unité de
Glycobiologie Structurale et Fonctionnelle, Lille, France
AMANDA OLDANI • PASS-Bio Med, Centro Grandi Strumenti, University of Pavia, Pavia,
Italy
GIUSEPPINA PALLADINI • Fondazione IRCCS Policlinico San Matteo, Pavia, Italy;
Department of Internal Medicine and Therapeutics, University of Pavia, Pavia, Italy
ALESSIO PAPINI • Department of Biology, University of Florence, Florence, Italy
FRANCESCO SAVERIO PAVONE • LENS – European Laboratory for Non-linear Spectroscopy,
University of Florence, Florence, Italy
LIDIA POLLARA • Department of Molecular Medicine, University of Pavia, Pavia, Italy
GARANCE PONTIER • Université de Lille, CNRS, UMR 8576, UGSF – Unité de Glycobiologie
Structurale et Fonctionnelle, Lille, France
GRAŻYNA E. PTAK • Malopolska Centre of Biotechnology, Jagiellonian University, Krakow,
Poland
xiv Contributors

STEFANIA RAIMONDO • Dipartimento di Scienze Cliniche e Biologiche, Università di Torino,

Torino, Italy; Neuroscience Institute Cavalieri Ottolenghi (NICO), Torino, Italy
JOÃO RAMALHO-SANTOS • Biology of Reproduction and Stem Cell Group, Center for
Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; Department of
Life Sciences, University of Coimbra, Coimbra, Portugal
YINHUI REN • Key Laboratory of Crop Genetics and Physiology of Jiangsu Province/Key
Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou
University, Yangzhou, China; Co-Innovation Center for Modern Production Technology of
Grain Crops of Jiangsu Province/Joint International Research Laboratory of Agriculture
and Agri-Product Safety of the Ministry of Education, Yangzhou University, Yangzhou,
China
MATHIEU REPELLIN • Department of Neurosciences, Biomedicine and Movement Sciences,
Anatomy and Histology Section, University of Verona, Verona, Italy; Laboratoire
d’Automatique, de Génie des Procédés et de Génie Pharmaceutique, Université Claude
Bernard Lyon 1, Villeurbanne, France
SANDRA RISTORI • Department of Chemistry, University of Florence, Florence, Italy
ROK ROMIH • Institute of Cell Biology, Faculty of Medicine, University of Ljubljana,
Ljubljana, Slovenia
MICHAEL A. RUDNICKI • The Sprott Centre for Stem Cell Research, Regenerative Medicine
Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada; Department of
Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa,
ON, Canada
MARIA CRISTINA SALVATICI • Institute of Chemistry of Organometallic Compounds
(ICCOM)-Electron Microscopy Centre (Ce.M. E.), National Research Council (CNR),
Florence, Italy
SEBASTIÁN SAN MARTÍN • Centro de Investigaciones Biomédicas, Escuela de Medicina,
Facultad de Medicina, Universidad de Valparaı́so, Valparaı́so, Chile
DAVID SÁNCHEZ-PORRAS • Department of Histology (Tissue Engineering Group), Faculty of
Medicine, University of Granada, Granada, Spain; Instituto de Investigacion
Biosanitaria, Ibs.GRANADA, Granada, Spain
ARNAUD SCHERBERICH • Department of Biomedicine, University and University Hospital of
Basel, Basel, Switzerland; Department of Biomedical Engineering, University of Basel,
Allschwil, Switzerland
SILVIA SCHIFF • Department of Biology, University of Florence, Florence, Italy
VIRGINIE SOTTILE • Department of Molecular Medicine, University of Pavia, Pavia, Italy
CORENTIN SPRIET • Université de Lille, CNRS, UMR 8576, UGSF – Unité de Glycobiologie
Structurale et Fonctionnelle, Lille, France; Université de Lille, CNRS, Inserm, CHU Lille,
Institut Pasteur de Lille, US 41-UMS 2014-PLBS, Lille, France
YUANQIANG SUN • College of Chemistry, Institute of Analytical Chemistry for Life Science,
Zhengzhou University, Zhengzhou, China
MARIO P. TSCHAN • Institute of Pathology, University of Bern, Bern, Switzerland
PATRIZIA VAGHI • PASS-Bio Med, Centro Grandi Strumenti, University of Pavia, Pavia,
Italy
MARIAPIA VAIRETTI • Department of Internal Medicine and Therapeutics, University of
Pavia, Pavia, Italy
 Contributors xv

ENZA MARIA VALENTE • Department of Molecular Medicine, University of Pavia, Pavia,

Italy; Neurogenetics Research Centre, IRCCS Mondino Foundation, Pavia, Italy
JUAN VARAS • Centro de Investigaciones Biomédicas, Escuela de Medicina, Facultad de
Medicina, Universidad de Valparaı́so, Valparaı́so, Chile
CUNXU WEI • Key Laboratory of Crop Genetics and Physiology of Jiangsu Province/Key
Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou
University, Yangzhou, China; Co-Innovation Center for Modern Production Technology of
Grain Crops of Jiangsu Province/Joint International Research Laboratory of Agriculture
and Agri-Product Safety of the Ministry of Education, Yangzhou University, Yangzhou,
China
TAMARA WEISS • Department of Plastic, Reconstructive and Aesthetic Surgery, Medical
University of Vienna, Vienna, Austria
LORENA ZANNINO • Department of Biology and Biotechnology “Lazzaro Spallanzani”,
Laboratory of Cell Biology and Neurobiology, University of Pavia, Pavia, Italy
SHENGNAN ZHAO • Key Laboratory of Crop Genetics and Physiology of Jiangsu Province/Key
Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou
University, Yangzhou, China; Co-Innovation Center for Modern Production Technology of
Grain Crops of Jiangsu Province/Joint International Research Laboratory of Agriculture
and Agri-Product Safety of the Ministry of Education, Yangzhou University, Yangzhou,
China
DAŠA ZUPANČIČ • Institute of Cell Biology, Faculty of Medicine, University of Ljubljana,
Ljubljana, Slovenia
 Chapter 1

Histochemistry in Advanced Cytometry: From

Fluorochromes to Mass Probes
Giuliano Mazzini and Marco Danova

Abstract
For over half a century, fluorescence has been the milestone of most of the quantitative approaches in
various fields from chemistry and biochemistry to microscopy. This latter also evolved into cytometry,
thanks to the development of fluorescence techniques. The dyes of classical cytochemistry were replaced by
fluorochromes, and the pioneer microphotometry was replaced by microfluorometry. The latter has great
advantages in terms of simplicity, sensitivity, and accuracy. The extensive research and availability of new
fluorochromes as well as the technological evolution contributed to the success of microfluorometry. The
development of flow cytometry in the 1960s gave a giant boost to cell analysis and in particular to the
clinical diagnostics. The synergy between flow cytometry and the subsequent development of monoclonal
antibodies allowed the setup of multiparametric analytical panels that are today popular and irreplaceable in
many clinical and research laboratories. Multiparametric analysis has required the application of an increas-
ing number of fluorochromes, but their simultaneous use creates problems of mutual contamination, hence
the need to develop new fluorescent probes. Semiconductor and nanotechnology research enabled the
development of new probes called nanocrystals or quantum dots, which offered great advantages to the
multiparametric analysis: in fact, thanks to their spectrofluorometric peculiarities, dozens of quantum dots
may be simultaneously used without appreciable crosstalk between them. New analytical horizons in
cytometry seem to be associated with a new concept of analysis that replaces fluorescence toward new
markers with (non-radiative) isotopes of heavy metals. Thus, the mass flow cytometry was born, which
seems to guarantee the simultaneous compensation-free analysis of up to 100 markers on a single sample
aliquot.

Key words Quantitative microscopy, Cytometry, Fluorescence, Flow cytometry, Mass flow cytometry

1 Introduction

The great advances of microscopic techniques, in the second half of

the last century, led to the birth of quantitative microscopy. The
microscope became not only an instrument for the observation of
microstructures and morphologies but also an instrument capable
of quantitate some of the main cellular components. Thus, cyto-
metry was born, an analytical approach that, initially based on
microscopy, was aimed to the quantitative determination of cellular

Carlo Pellicciari et al. (eds.), Histochemistry of Single Molecules: Methods and Protocols,
Methods in Molecular Biology, vol. 2566, https://doi.org/10.1007/978-1-0716-2675-7_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

1
2 Giuliano Mazzini and Marco Danova

constituents. In addition to cytometry, cytochemistry also evolved:

from the empirical science that used salts and natural pigments
(from both the animal and plant worlds), cytochemistry became a
refined technique based on defined and controlled chemical reac-
tions. The central core of this first methodological and later instru-
mental evolution is DNA on which an immense research work was
carried out in the years from 1950 to 1970 [1–7]. DNA, as a
fundamental cell component, immediately became the subject of
quantitative study. From the first observations of cell proliferation
and neoplastic transformation, the need derived for a procedure to
exactly measure the amount of DNA per cell. The first approaches
of DNA cytometry were based on the evaluation of the Feulgen
reaction products by absorption measurement [8–12]. The com-
plexity and limitations of this procedure were soon overcome by the
development of fluorescence microscopy [13] and related fluoro-
chromes. Quantitative cytochemistry thus underwent a great devel-
opment with the availability of new fluorescent molecules that have
made cytofluorometry the technique of choice for increasingly
sensitive, precise, and reliable quantitative determinations [14–16].
After decades of research on DNA, other cellular components
have also been the subject of quantitative interest, e.g., RNA and
proteins.
In addition to the quantitative fluorescence probes, several
markers have been developed to study cellular functions. In this
case, measurements are not strictly quantitative but rather aimed at
acquiring information on the cell functional states (viability, death/
apoptosis occurrence, oxidative state, membrane potential, pH,
etc.) under both physiological and pathological conditions.
A milestone in the development of cytometry, however, is
linked to the technological evolution that led to the birth of flow
cytometry (FC). The ability to directly analyze cells in suspension
led to enormous advantages. In fact, classical cytometry on a slide
(even after various attempts for automation) allowed the analysis of
a few hundred cells in very long times, whereas FC made it possible
to analyze thousands of cells in a few seconds [17–22].
A further boost (this is very great too) was linked to the
development of immunofluorescence and, immediately after, to
the advent of monoclonal antibodies. Thanks to the conjugation
with a variety of fluorochromes, monoclonal antibodies became a
very powerful investigative tool in various fields of immunology,
both in biological research and in the clinical diagnostics. The
combination of fluorescent monoclonal antibodies and FC is
today an unbeatable tool in many clinical laboratories [23–25].
Modern diagnostic approaches in the immunohematological
areas often require the use of a large panel of antibodies. These
multiparametric investigations, therefore, require the simultaneous
use of multiple fluorescent markers. To avoid errors due to the
mutual interference between probes, it is necessary that the labeling
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 3

fluorochromes are spectrally separated or, in other words, with

fluorescent emissions in fairly narrow and sufficiently distinct
bands. Today, using traditional fluorochromes (in addition to the
new ones called “tandem”), the FC techniques allow to discrimi-
nate up to 12 different colors; to do this, however, the sample must
be divided into aliquots analyzed with instruments equipped with
several sequential excitation sources (from the UV to the far-red
wavelengths).
To further expand the potential of multiparametric analysis, a
family of innovative fluorescent probes called nanocrystals
(or QDs) have been developed in recent years. Their chemical/
physical characteristics make them unique in the broad scenario of
the commercially available fluorescent markers, today.
Finally, after almost a century of fluorescence, cytometry is
evolving toward new analytical horizons offered by the use of
markers based on heavy metal isotope. FC is going to leave the
concept of optical (spectrofluorometric) analysis and uses the time-
of-flight mass spectrometry (so-called CyTOF) to recognize new
markers. This technology utilizes rare metal isotopes instead of
fluorophores to label antibodies, thus breaking the limit of multi-
plexing capability of conventional cytometry, and allows the multi-
ple simultaneous labelling of dozens of “targets” (actually near a
hundred) without mutual interference. Obviously, in parallel with
the instruments’ technological evolution, a commercial fight has
also begun for the development of new diagnostic kits based on
these markers. Terminology is also changing and the historical
“fluorescence flow cytometry” becomes today “mass flow
cytometry.”

2 Fluorochromes and Cytometry

Although the first prototypes of flow cytometers were based on the

measurement of scattered light parameters, the instrumentation
that later became commercially available was instead based on the
use of fluorochromes labeling the cellular components to be
detected and measured. It is worth noting that although the vast
majority of (clinical) applications of FC are presently directed to
measure immunofluorescence markers, DNA was instead the start-
ing cellular target.
The first experimental contributions to the automated analysis
of cells in suspension were aimed at measuring the volume (through
the evaluation of scattered light) and immediately afterward the
DNA content (through the measurement of fluorescence). The
goal was in fact to automate the cytometric analyses of DNA
(traditionally carried out on a slide), and, more or less in the same
years, FC was replacing the more laborious and imprecise cytopho-
tometric determination [8–12]. In other words, fluorochromes
4 Giuliano Mazzini and Marco Danova

were replacing the classic dyes of cytochemistry, at least as far as

quantitative aspects were concerned.
In the years around the 1970s, FC was an advanced analytical
technique for cell proliferation studies, mainly based on the analysis
of fluorescence emitted by a stoichiometrically bound fluoro-
chrome to DNA. It is important to remember the first attempts
to adapt to FC some important classical reactions of cytochemistry
in fluorescence [26–29]. Subsequently, FC was significantly
improved by extensive methodological researches that made it
available, in the timespan of a few years, a wide range of methodol-
ogies with often specially developed procedures and fluoro-
chromes. The impact of the phenanthridine fluorochromes,
ethidium bromide (EB) and propidium iodide (PI), on DNA FC
was huge [30–32], and these fluorochromes are still nowadays
among the most widely used probes for the quantitative analysis
of DNA [33–37]. From the methodological point of view, also the
so-called fluorescent antibiotics (mithramycin, olivomycin, cromo-
mycin) have provided important contributions to DNA
quantitation.
After DNA, the interest of FC was addressed to cellular pro-
teins, and the methodological evolution exactly follows what has
just been described for DNA. At first, traditional techniques were
adapted to samples of cells in suspension, and then FC-tailored
methods and fluorochromes were, on purpose, developed. Starting
from eosin, fluorescamine, and ortho-phthalaldehyde, then fluores-
cein isothiocyanate (FITC) became the leader probe in this field
(still today, it is the most widely used in immunofluorescence). The
transfer to FC of the first immunofluorescence methods (initially
used in fluorescence microscopy) then gave a major boost toward
spreading the FC techniques in most of the biomedical labora-
tories. The methodological research subsequently made a series of
fluorochromes available, either synthetic (e.g., the Hoechst series)
or derived from algae (e.g., phycobiliprotein).
Other fluorochromes were later proposed with the aim not to
strictly quantitate the amount of molecules but to monitor cellular
functions or markers of cell viability or death, oxidation state, pH,
etc. A new family of probes were recently developed, called nano-
crystals or QDs. They are characterized by very peculiar spectroflu-
orometric properties allowing a dedicated application in
multiparametric FC. Lastly, cytometry evolved toward an innova-
tive analytical strategy that would even replace fluorescence with
the labeling by heavy metal mass isotopes; hence, mass cytometry
was born.
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 5

3 Fluorescence and Autofluorescence

The evolution of cytometry in fluorescence also conditioned a series

of researches on the importance of bioluminescence with mainly
negative contributes to cytofluorimetric detections. It became
immediately evident that biological tissues can be a spontaneous
source of autofluorescence and that this can interfere with the
analytical results. Fluorescence analysis allows cells to be irradiated
by a beam of light of a certain frequency and high intensity
(depending on the type of excitation source) which, interacting
with the various biological components, can generate (in addition
to diffused and refracted light) also a spontaneous fluorescence
emission called autofluorescence.
In general, fluorescence can be distinguished into primary
(spontaneous, or autofluorescence, or natural fluorescence) and
secondary (or induced) fluorescence. The latter in turn can be
further classified into numerous “subtypes” depending on the
physicochemical treatments used to generate the emission.
Autofluorescence can be more or less high, depending on the
cell type under examination, and is generally produced by the
photoluminescence of some amino acids and therefore by the pro-
teins where they are contained in. In particular, tryptophan and
tyrosine are fairly luminescent especially when excited in ultraviolet
light. Since these amino acids are commonly present in proteins,
they can be considered the main components responsible for the
typical autofluorescence of many biological tissues observed in
ultraviolet light under a fluorescence microscope. Many other com-
ponents (or substances) physiologically or even pathologically pres-
ent in cells can increase the basic autofluorescence of certain cell
types. Many vitamins, lipids, enzymes, and coenzymes are often
characterized by specific fluorescence emissions. As far as lipids are
concerned, there are, for example, porphyrins which represent an
important family of substances naturally present in some tissues
(especially in the hematopoietic compartment); the increase in
porphyrin content under some pathological situations and the
consequent increase in the typical fluorescence intensity can be
used for diagnostic purposes. Lipids also have the peculiar charac-
teristic of being similar to many vitamins and lipofuscins (often very
fluorescent) that they are able to accumulate within cells or tissues
in the form of micro-drops or liposomes.
Other biological components of some tissues, such as elastin
and collagen, show a high level of spontaneous emission when
observed under a fluorescence microscope. In these cases, however,
since the localization of this substance is mostly in the extracellular
matrix, the incidence of this phenomenon is quite modest as far as
single cell (or nuclei) analyses are concerned. In addition, the
autofluorescence of cells or tissues can increase as a result of the
6 Giuliano Mazzini and Marco Danova

presence of non-physiological substances, among which are some

types of drugs such as some antibiotics. Virtually all anthracyclines
are so fluorescent that they can be considered as “fluorochromes,”
and some antibiotics (described below) represent a particular group
of luminescent molecules. These drugs specifically bind DNA so
that their clinical use, even at low doses, allows their accumulation
in the cell nuclei: as a consequence, when analyzed by FC or
observed under a microscope, the nuclei have a much higher spon-
taneous fluorescence. Autofluorescence in cytometry can be a diag-
nostic tool as it has been shown to increase in some tumor tissues
compared to the corresponding normal tissue. So, in particular
situations, autofluorescence can be considered as a positive occur-
rence; however, in general, spontaneous fluorescence represents a
negative aspect in many cytometric approaches, especially in static
cytometry, but also, though to a lesser extent, in FC. In static
cytometry, the sample is often observed under excitation light
with the contribution of the biological matrix surrounding the
cells; on the contrary, in FC, the cells are isolated from the tissue
milieu and even single nuclei are often processed. However, one
should always be aware of the sample autofluorescence and have an
approximate estimate of its level, to predict the possible error
incidence.
The fluorescence that mainly interest the field of cytometry are
the so-called extrinsic or induced fluorescence. All the lumines-
cence artificially induced (by means of physicochemical/chemical
treatments) in a sample belong to this class. Historically, some of
the first fluorochromization processes in histochemistry involved
the transformation of weakly luminescent substances (e.g., the
catecholamines) into compounds with stronger emission. Other
treatments transformed a substrate into a fluorescent product
(e.g., enzyme histochemistry) and still others, on the basis of
intermediate reactions, finally bound a fluorochrome (e.g., the
fluorescent Feulgen reaction). The current labeling techniques for
cytometry are essentially attributable to two types of procedures:
(a) those that lead to the binding of a fluorochrome to the
biological molecule, based on specific chemical-physical interac-
tions, and (b) those that bind a fluorochrome to the target antigen
through an immunochemical reaction.
In recent years, the progress of FC was dependent on the
technological improvement but also on the evolution of the stain-
ing procedures, among which were the manufacture and commer-
cial availability of monoclonal antibodies: thanks to their use, FC
became a routine analytical technique in many research and clinical
laboratories, in particular for leukocyte immunophenotyping.
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 7

4 DNA Probes

As already recalled, the most important chapter on fluorescent

markers undoubtedly deals with DNA cytochemistry.
The Feulgen reaction [7] was the very first attempt to quantify
a cellular component of fundamental importance for a wide range
of biomedical applications. Few years later many contributes [4, 16]
were focused on the automation of this determination (by means of
both static and flow cytometry). The first step was the modification
of the original method, replacing the conventional Shiff’ reagent
with alternative fluorochromes (which paints the nuclei
red-magenta and was used for the first absorption measurements)
[26–29]. The results were not particularly “brilliant” due to the
complex and time-consuming method that required acid hydrolysis
with many washes and centrifugations, which resulted in a high cell
loss. Nonetheless, the first applications of FC were based on the
Feulgen reaction modified with acriflavine, 2,5-bis(benzoxazol-2-
yl)thiophene (BBT), and auramine. In the meantime, a new fluo-
rescent probe acridine orange (AO) was proposed, whose meta-
chromatic property was already exploited in other histochemical
applications [38]. Together with its peculiar chemical-physical
characteristics, AO appeared to be (at least initially) the probe of
choice for the analysis of nucleic acids by FC [39]. In fact, it has an
absorption spectrum favorable for its excitation with the argon ion
laser (488 or 514 nm) which was (and still is) the typical excitation
source of the most popular instruments. AO has also a good quan-
tum yield and, above all, it provides two different information at the
same time: its peculiar characteristic is in fact to intercalate into the
DNA double helix, and in this situation (molecules spaced between
them) it emits green fluorescence, while it can also bind externally
the single RNA helix and in this other condition (molecules close
one to each other) emits red fluorescence. This peculiarity was
initially exploited in numerous applications in the hemato-
oncology field, where the DNA/RNA ratio is a powerful marker
in the study and classification of some leukemias. From the point of
view of general applications, however, this method has numerous
drawbacks, mainly related to its crucial binding mechanism that
requires extremely controlled and not always exactly reproducible
reaction conditions.

4.1 G-C Base- Other fluorochromes able to bind to DNA with different mecha-
Specific nism were later proposed under the name of “fluorescent antibio-
Fluorochromes tics.” Among these mithramycin (MM), cromomycin, and
olivomycin all react with a similar mechanism that involves the
formation of a complex with guanine (in the presence of magne-
sium ions) and are therefore base-specific probes, with a preference
for G-C pairs. Their excitation maximum is in the blue
8 Giuliano Mazzini and Marco Danova

(410–430 nm) and emission in the green-yellow (500–600 nm).

They can therefore be best excited with lamp sources of both
mercury and xenon vapor while maintaining a good emission
even when excited with argon lasers (both in blue and green).
Thanks to their base specificity, they should not bind to RNA;
therefore, they guarantee an accurate DNA determination, without
the need to enzymatic RNA digestion. In fact, MM in particular has
been used for this purpose in combination with EB [40]. Exploiting
the energy transfer between MM and EB, it is possible to excite
MM in the blue region (at 420 nm where EB does not absorb) to
obtain red fluorescence from EB, which does not receive light
directly from the lamp but receives energy from the excited MM
molecules. This phenomenon occurs only if the molecules of the
donor-acceptor pair are in close proximity, as it occurs for MM and
EB when they are bound to DNA. The collected fluorescence signal
is therefore dependent on the amount of DNA only, without the
interference of RNA.
These G-C fluorochromes in combination with other A-T-
specific dyes can provide information on DNA base composition.
Based on this strategy, a method has been applied to the differenti-
ation of single chromosomes using chromomycin in combination
with Hoechst, as well as on the discrimination of classes of bacteria
with DAPI. In more recent years, another fluorochrome belonging
to this family and derived from actinomycin-D, called 7-amino
actinomycin D (7-AD), was proposed for the analysis of DNA in
combination with other probes [24]. The physicochemical charac-
teristics of 7-AD, and in particular its deep red emission, allow a
good separation of its fluorescence even in the presence of the
popular yellow-orange probes. Since 7-AD can penetrate through
the membrane of unfixed cells, it has been proposed for the DNA
multiparametric determination in unfixed cells previously immuno-
labeled for different antigenic markers using FITC- and phycoery-
thrin (PE)-conjugated antibodies [25].

4.2 A-T Base- Some very important fluorochromes for the role they have played in
Specific FC applications belong to this class. DAPI and its analogue DIPI
Fluorochromes have contributed to the development of a family of “European
cytometers,” substantially derived from the fluorescence micro-
scope and thus using lamps as excitation sources [19, 40]. Instead,
another family of instruments born in the USA exploited the power
of the new argon ion lasers in place of lamps. Both types of instru-
ments had ideal fluorochromes fitting their different excitation
performances: FITC fits the argon line at 488 nm while DAPI is
optimally excited by the UV band of the mercury lamp. Its maxi-
mum absorption is in the ultraviolet where mercury has a very
powerful emission line (at 365 nm). DAPI has a high quantum
efficiency that, combined with the high excitation energy, leads to
an extraordinary intensity of fluorescence emission. These findings
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 9

ensued an excellent result, in terms of coefficient of variation of the

DNA data, as reported in the literature about the DAPI-DNA
analysis carried out with lamp instruments.
Other A-T-specific fluorochromes are those developed by
Hoechst and named as HO followed by a number [41]. Among
others the HO33258 e HO33342 became very popular in FC; in
particular, HO33342 is now the probe of choice for cell viability
studies [42, 43], as it can cross the cell membrane and stain the
nucleus in supravital conditions (see below Subheading 7.1). Two
other fluorochromes from the A-T family are quinacrine (QC,
already well known for its use in chromosome banding) and the
more recently proposed LC585 [44]. This latter has interaction
characteristics similar to HO3342 (therefore with the possibility to
be used as viability probe) but with excitation spectrum in the
visible light range.

4.3 Intercalating Although acridine derivatives, and AO in particular, are the most
Dyes famous intercalating fluorochromes, in the history of DNA cyto-
chemistry [39], the family of phenanthridine derivatives EB and PI
has made the most relevant contribution to FC, from the practical
point of view [33]. EB has been extensively studied from a physi-
cochemical point of view and its interactions with DNA described
in detail even at the ultrastructural level. Its analog, PI, had a wider
methodological application, thanks to its spectrofluorometric char-
acteristics that make it more suitable, especially for multiparametric
FC. Phenanthridines have the peculiar characteristic of being
almost non-fluorescent as free molecules in the solvent while
increasing considerably their quantum efficiency when intercalated
in double-stranded DNA. The explanation for this phenomenon
lies in the change of microenvironment that intercalated molecules
acquire, compared to those free in the surrounding dye solution.
The latter, after light excitation, from the higher electronic level can
disperse much of the absorbed energy, exchanging it with the
surrounding water molecules (polar and very mobile); thus, their
energy is not emitted as fluorescence. On the contrary, the inter-
calated molecules become electronically shielded from each other
and especially from the external water dipoles. In this state, much of
the energy absorbed by excitation is returned into the form of
fluorescence. It means that only the intercalated molecules contrib-
ute to the generation of the fluorescence signal emitted by the
nucleus. This peculiarity, together with its spectrofluorometric
characteristics, made PI the probe of choice for DNA quantitation
by FC. Regarding the spectral characteristics, it should be noted
that PI has a wide absorption band in the visible, with best absorp-
tion in the blue/green region and another band in the UV. It is
therefore widely used both with laser-based instruments, where it is
typically excited by the 488 nm argon line, and with lamp instru-
ments where the 546 nm mercury line is exploited. The emission
10 Giuliano Mazzini and Marco Danova

spectrum is fairly located in the red, with a maximum around

610 nm: this allows it to be widely used in multiparametric FC in
combination, usually, with FITC and PE, although with a discrete
spectral overlap with the latter. Although other fluorochromes with
narrow red emission have been recently proposed (to minimize
cross-talking between probes), PI maintain its wide popularity as a
marker of choice for DNA. Phenanthridines have a single draw-
back, as their binding mechanism does not allow an absolute speci-
ficity toward DNA: they can, in fact, bind also double-stranded
RNA (typically t-RNA). The high-resolution DNA measurement
with EB or PI therefore requires a pretreatment for the enzymatic
digestion of RNA. To overcome this event and thus avoid the use of
enzyme, a specific methodology was proposed [19] that involves
the use of a mixture of MM and EB in equimolar combination and
exploit the principle of energy transfer from one to the other. After
dual staining, the double-stranded DNA will bind EB
(by intercalation) and MM (by interaction with the G-C base
pairs), while RNA will have only EB intercalated (because MM
does not bind to RNA). Using an excitation light beam around
430 nm (e.g., by a mercury lamp), only MM will be excited,
because EB does not absorb light at this wavelength. However,
given the proximity (less than 100 A
) of the molecules of MM and
EB in DNA, the former can transfer directly the absorbed energy to
the molecules of EB that finally emit its typical fluorescence: the
intensity of this fluorescence emission is thus proportional to the
DNA amount.

5 RNA Probes

As far as RNA is concerned, its typical probe is AO, already

described in the previous section. Thanks to the very intense meta-
chromatic effect, AO can act as a versatile bifunctional probe able to
bind both DNA and RNA with two different emission signals.
Intercalated inside the double-stranded DNA may fluoresce
green, while externally bounded to RNA turn its fluorescence to
red [39]. Before the advent of the so-calledproliferation markers,
now widely used for studies of cell proliferation, the interest was in
fact focused on RNA as a cellular component (of second level after
DNA) to “monitor” cell proliferation. Many contributions refer
the importance of assessing the ratio DNA/RNA performed by FC
via AO labeling, especially in the field of oncohematology [22].
Another more tedious method was based on the use of phe-
nanthridines (as mentioned above) with a reverse procedure to
what is done for DNA. Following digestion with DNase, the resid-
ual fluorescence refers to RNA.
Another RNA-specific probe, already widely used as a conven-
tional histochemical dye, is pyronin Y (PY). Together with methyl
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 11

green, it has given rise to one of the first biparametric methods in

absorption microphotometry. In cytometry, this method was then
modified by combining PY with Hoechst, which replaced methyl
green as a DNA fluorochrome [25]. The results obtained with PY
have never been without criticism and its routine use has found the
same obstacles already mentioned for AO.
A specific clinical application of RNA probes was dealing with
the counting of reticulocytes in peripheral blood. The RNA resi-
dues in the cytoplasm of immature erythrocytes were in fact labeled
with different fluorochromes including DiOC1, thioflavin T, and
thiazole orange in addition to PY. Among these, the method most
frequently applied in the literature is the one using thiazole orange
that, despite not being exclusively specific for RNA (as it binds also
DNA), has shown good sensitivity in the automatic counting of the
anucleated reticulocytes in peripheral blood [25].

6 Protein Probes

The most popular protein-specific fluorescent probe is surely fluo-

rescein isothiocyanate (FITC), which was also one of the first
fluorochromes used in fluorescence microscopy. This probe has
practically promoted the amazing development of immunofluores-
cence techniques [22]. Like most of the protein-specific dyes, FITC
establishes a covalent bond between the thiocyanate radical and the
primary amine groups spread everywhere in the biological tissues.
Thanks to the chemical stability of the complex, all the necessary
washing procedures can be performed, in both conventional cyto-
chemistry and immunocytochemistry, without loss of the reaction
products: careful washing is necessary because free and bound
FITC are equally fluorescent, thus being crucial to eliminate
unbound FITC to make a proper quantitative analysis. The spec-
trofluorometric characteristics of FDITC are ideal for an excitation
with the 488 nm line of the argon laser, being the absorption
maximum around 490 nm, while the emission spectrum is in the
green region, with a maximum at 520 nm. FITC has metachro-
matic properties similar to those described for AO, i.e., it changes
its spectroscopic structure depending on its concentration. For
many years, FITC has been used as the classical probe for total
cellular proteins, thanks also to the biparametric method in combi-
nation with PI, for simultaneous DNA determination [33].
Other historical protein markers are ortho-phthalaldehyde and
fluorescamine, which are excited by UV light and emit blue-white
fluorescence [44]. They were not widely applied since the required
UV excitation generates a significant level of background fluores-
cence and also because more efficient fluorochromes became soon
available.
12 Giuliano Mazzini and Marco Danova

An important group of fluorochromes with a FITC-like reac-

tion mechanism is the rhodamine family. Among these, sulforoda-
mine 101 (SR101) and tetramethylrhodamine-isothiocyanate
(TRITC) have been the most widely used in FC [25]. Important
was then the setting of a method that, using SR101 together with
DAPI, with a single UV excitation, allows measuring the blue
fluorescence of DAPI and the red emission of SR101. This method
has been extensively used with mercury lamp cytometers and is still
in use in some laboratories. The results are generally very good, and
often the CV of the DNA is even smaller than in single-stained
DAPI samples (it is not easy to explain this phenomenon, and one
can only speculate that the presence of SR101 prevents the nonspe-
cific interaction of DAPI outside DNA). Even more surprising are
the SR101 data (which follow those of FITC on the same samples)
taking into account the crosstalk between the two emission spectra.
Some coumarin derivatives such as 7-amino-coumarin
(AMCA) and N-(7-dimethylamino-4-methylcoumarinyl) malei-
mide (DACM) are very interesting for their spectrofluorometric
properties: high absorption in UV, emission in the first part of the
visible light that allows obtaining a brilliant white-blue fluores-
cence. The amine-reactive AMCA was rather widely used in the
field of immunofluorescence as AMCA conjugates became com-
mercially available and proved to be useful for multiparametric
analyses in combination with other green- and red-emitting probes
[45]. Another coumarin derivative, DACM, has similar fluores-
cence properties as AMCA, but a different reaction mechanism
being specific for the protein sulfhydryl (-SH) groups
[46]. DACM was initially applied to detect SH-rich proteins in
the skin [47, 48] and to investigate the thiol-to-disulfide transition
in nuclear protamines during mouse sperm maturation
[49]. Thanks to their spectral characteristics, DACM and PI were
used as a dye pair for studying DNA and proteins in nuclear
chromatin by fluorescence resonance energy transfer [50]. This
couple of probes were also applied in lamp-based FC, thanks to
the high performance UV excitation of this type of
instruments [51].
To complete the list of protein probes, there are the so-called
“phyco” derivatives. These organic complexes, called phycobilipro-
teins, are molecules of natural origin (from marine algae or cyano-
bacteria) and exhibit very interesting photophysical properties that
make them suitable for FC application and, in particular, for immu-
nofluorescence labeling [51–55]. In nature, phycobiliproteins are
components of the photosynthetic light-harvesting antenna com-
plexes of red algae and cyanobacteria; they absorb the sun’s energy
and efficiently transfer it to chlorophyll pigments by fluorescence
resonance energy transfer, for the photosynthetic reactions. Phyco-
biliproteins are characterized by a high extinction coefficient (which
is fundamental for a suitable fluorescence emission) and are
Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 13

commercially available as three fluorochromes: B-phycoerythrin

(B-PE), R-phycoerythrin (R-PE), and allophycocyanin (ApC).
Phycobiliproteins are characterized by a high molecular weight,
resulting from the very complex organic structure, which can
include up to 34 chromophore groups per molecule. PEs reach a
molecular weight around 240 kDa, while ApC is about 104 kDa;
they have common quite broad absorption spectra in the visible
band beyond 450 nm. In particular, R-PE has a specific absorption
peak around 490 nm, which makes this fluorochrome particularly
suitable to be excited with the 488 nm line of the argon laser. B-PE
has its excitation maximum more shifted toward the green and is
therefore better excited in this spectral region. In contrast, the
excitation peak of ApC is shifted to the red region with a maximum
around 650 nm. As mentioned above the peculiarity of these fluor-
ochromes is to be very efficient in terms of fluorescence emission, as
a result of the high specific absorption of photons and an equally
high fluorescence quantum yield. The absorption is characterized
by the molar extinction coefficient, which represents the statistical
probability that the molecule has the ability to absorb a photon of a
given wavelength. This value, relative to the wavelength of 488 nm,
is, for example, for R-PE, equal to 2  106, while it is only 86  103
in the case of FITC. For ApC this value is 7  105 at 650 nm. It is
therefore evident that the ability of these fluorochromes to absorb
light is very high as compared to FITC. As far as quantum yield is
concerned, from the theoretical value of 1, PE has a value of 0.98
and ApC of 0.68, while FITC, which is known to be a brilliant
fluorochrome, is only 0.5. Thus, it is quite clear that phycobilipro-
teins are definitely the fluorochromes of choice, in terms of sensi-
tivity (i.e., for the measurement of small amount of antigens/
proteins) [25].
From the application point of view, these fluorochromes have
been very successful, having become, in a few years, the first coun-
terparts to FITC in FC. Actually, in FC the single-parameter immu-
nofluorescence analysis may be performed using just FITC as probe
of choice, while FITC may be combined with PE in dual-parameter
measurements, with the further addition of ApC in case of triple
labeling. In this latter case, a dual-laser excitation system must be
available to properly excite ApC in the red region.
In the case of single-laser blue excitation, the third probe must
be a “tandem” fluorochrome. This belongs to the family of syn-
thetic probes developed by joining together two molecules with
complementary photophysical properties, to exploit the principle
of energy transfer from one (the donor) to the other (the acceptor).
They have various commercial names and generally use PE, as a
donor coupled to another chromophore, which receives the energy
from this latter and finally emits a more red-shifted fluorescence.
They were specifically developed to solve the problem of the
so-called multilabeling immunofluorescence based on the use of a
14 Giuliano Mazzini and Marco Danova

single blue excitation, for the measurement of three signals: green

(FITC), yellow (PE), and red (tandem) [53].
Cyanines (known with the trade names Cy2–7) belong to a new
family of protein probes: they are also synthetic fluorochromes
designed to mimic the characteristics (viz., the advantages) of the
natural phycobiliproteins. They have a specific high light-
absorption coefficient (~ 2  103) and a good fluorescence quan-
tum efficiency (0.1  0.3). Compared to the natural parental
molecules, cyanines have narrower absorption-emission spectra,
which offer better emission selectivity and less mutual reabsorption,
when used in multiparametric analysis. The most interesting and
promising are those that are excited in the red (exploiting the lines
of the He-Ne laser or the solid-state lasers) and emit in the far red,
up to over 800 nm [44].

7 Cell Function Probes

7.1 Viability Probes The fluorochromes described so far are the most historical and did
contribute to the progress of the microfluorometric techniques,
having been developed to specifically and stoichiometrically bind
different cellular components to obtain their quantitative determi-
nation. Thanks to histochemists’ efforts, perfectly controlled and
reproducible conditions of dye concentration, pH, reaction time,
and temperature have been defined to ensure a perfect correlation
between fluorescence intensity and probe content, as the funda-
mental concept of any cytometric techniques is the stoichiometry of
the probe toward the cellular component to be quantified.
More recently, however, it has become interesting to use some
fluorescent probes not for “quantitative” investigations but to
obtain information on specific cellular features or functions.
Evaluating cell viability is crucial in biology and is a basic
finding in many experimental studies. Cell viability is mainly
assessed through the integrity (impermeability) of the plasma mem-
brane. Most of the fluorochromes described above are unable to
penetrate the intact cell membrane due to their chemical structure
and high molecular mass. However, there are some of them able to
do that. Among these, a specific probe has become very popular for
this property. It belongs to the “Hoechst” series of DNA-binding
fluorochromes characterized by the initials HO, which has become
the progenitor of a family of markers used for this purpose. Specifi-
cally, the HO33342 under appropriate (physiological) incubation
conditions it is easily internalized in all (both living and dead) cells.
In living cells, however, a metabolic pump is active, capable of
expelling most of the internalized fluorochrome [43], whereas in
dead cells this pump is off and all the fluorochrome molecules that
had crossed the membrane are kept inside: due to the DNA speci-
ficity of HO33342, the nuclei of dead cells will thus have a higher
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 15

fluorescence than those of living cells. In addition, the metachro-

masia of HO33342 makes a further decisive contribution to the
differentiation between life and death: at low concentration its
fluorescence emission is typically white/blue, whereas in conditions
of higher concentration the emission turns toward the yellow.
Thanks to these two coexisting phenomena (i.e., concentration
and metachromatic effect), it is simple to discriminate living from
dead cells, both by fluorescence microscope and FC.
HO33342 can also be used in combination with another
“functionally” different fluorescent probe, namely, PI that cannot
penetrate the intact membrane of living cells and therefore cannot
intercalate into nuclear DNA. Using a mixture HO33342/PI on a
cell sample, an impressive rainbow-color result is obtained that
characterizes the different cell viability status: viable intact cells
are only labeled by HO33342 and show a faint blue fluorescence;
mildly damaged cells may be only strongly labeled by HO and
fluoresce brilliant white/blue; more damaged cells are stained by
both dyes and exhibit a brilliant pink (white plus red) fluorescence;
and dead cells are strongly stained by both probes and fluoresce
deeply red.
Other DNA-specific fluorochromes can be used in microscopy
and FC as viability probes: 7-AAD is capable of crossing the cell
membrane of all cells and accumulate more in the nuclei of the dead
ones. On the contrary, LDS 751 like PI is not able to enter intact
viable cells but can instead stain only the nuclei of dead cells. The
emission spectra are in the deep red and therefore better than PI;
these fluorochromes are suitable for multiparametric analyses with
other green-emitting cell markers [56].

7.2 Proliferation Cell proliferation, among the cellular functions, is of particular

Markers interest in the biomedical field. The nuclear DNA content is cer-
tainly the most significant marker to estimate cell proliferation in
normal cell populations as well as in tumors [57, 58], and the
cytometric evaluation of DNA histograms is one of the most widely
studied biological indicators.
A limitation of this methodological approach, however, is the
fact that DNA content is a “static” probe as it just shows the cell
distribution through the cell cycle, at the time of sample collection.
The alternative may be the “active” study of cell proliferation
through the labeling of S-phase cells, which involves the use of
DNA precursors such as tritiated thymidine (3H-TdR) or bromo-
deoxyuridine (BrdU) [59–62].
The cyto-autoradiographic method based on the incorporation
of 3H-TdR is a classic approach to the study of proliferative kinet-
ics, and in recent years there has been a progressive validation of the
so-called labeling index, in the clinical field [36]. Obviously, this
method has some disadvantages including the need to use radioac-
tive material and the time-consuming procedure that makes it
16 Giuliano Mazzini and Marco Danova

unsuitable for the clinical studies that require rapid answers. The
availability of the new DNA precursor, BrdU, opened new horizons
in cell proliferation studies. BrdU tagging is comparable to that of
3
H-TdR without the disadvantages of radioactivity and with a
number of advantages related to its labeling with specific monoclo-
nal antibody, which may be detected by microscopy and
FC. Following the administration of BrdU in vivo and the analysis
by dual-parameter flow cytometry of the BrdU labeling and DNA
content, it is possible to measure the S-phase cell fraction and to
assess dynamic proliferation parameters, such as the duration of the
S phase (or DNA synthesis time, TS) and the potential doubling
time of the normal or tumor cell population (Tpot). This approach
has been the first example of DNA multiparametric analysis where
the classical “DNA content” can be associated with other kinetic
parameters that can increase the intrinsic value of the data [63–66].
Later on, other markers of potential diagnostic or prognostic
importance (surface antigens, intracytoplasmic antigens, nuclear or
nucleolar antigens, hormonal receptors, molecules related to par-
ticular cell functions, etc.) have been proposed to be simultaneously
correlated to the DNA content. This approach allows a quantitative
and statistically significant analysis of the cellular components in
direct correlation with the cell cycle, resulting in a more accurate
assessment of the potential clinical significance of these parameters.
The multiparametric FC evaluation of the expression of anti-
gens (such as the one recognized by the Ki-67 antibody, PCNA/
cyclin, and other cell cycle-related antigens) allows a precise assess-
ment of the proliferative status in various clinical diseases [67–
75]. Again, the quantification of other nuclear or nucleolar anti-
gens (e.g., p105) may be useful for a more objective assessment of
the degree of anaplasia. Some products of cellular oncogenes have
proved promising as markers of tumor aggressiveness, while few
glycoproteins localized on the cytoplasmic membrane have been
shown to be related to the process of anticancer drug
resistance [64].

7.3 Other Probes (for A large family of fluorescent tracers has become commercially
Mitochondrial available, in recent years, to study specific cellular conditions related
Intermembrane to both the plasma membrane function and the cytoplasmic orga-
Potential, pH, Ca++ nelles. These tracers are often complex molecules characterized by
Content, and Others) distinctive commercial acronyms [44].
Among the cationic lipophilic markers, JC1 is widely used
especially in confocal microscopy but also in FC for studies of
mitochondrial function. In viable mitochondria with high inter-
membrane potential (ΔΨm), the probe is concentrated inside the
organelles in the form of aggregates and metachromatically emits
red fluorescence; in mitochondria with low ΔΨm, the dye remains
in a less concentrated status and its fluorescence emission is typically
green. Rhodamine 123 (Rh123) is also a viable fluorochrome quite
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 17

specific to mitochondria. Its non-fluorescent derivative, dihydror-

hodamine 123 (DHR), is able to penetrate viable cells where in
presence of oxidative radicals (especially H2O2) it may be converted
to Rh123: the resulting green fluorescence allows easy monitoring
of the fraction of cells in which reactive oxygen species are present
[25]. The cationic rhodamine derivative, tetramethylrhodamine
(TMRE), is able to accumulate inside mitochondria according to
their membrane potential, and the level of emitted fluorescence
allows monitoring the mitochondrial functional status. Informa-
tion on the density (or mitochondrial mass) can be obtained with
two probes whose fluorescence is independent from the potential
and/or the energy metabolism: nonyl acridine orange (NAO) and
MitoTracker Green passively accumulate inside the mitochondria,
and the emitted fluorescence by the cells is a function of the
mitochondrial mass. However, it should be kept in mind that all
these mitochondrial markers do not bind stoichiometrically the
target and may therefore provide pseudo/quantitative information
only. Other markers such as chloromethyl-X-rosamine (CMXRos)
and MitoTracker Red give information on metabolic mitochondrial
activity; the latter, in particular, turns from a non-fluorescent to a
fluorescent form induced by intracellular oxidative processes.
For the evaluation of intracellular pH, there are several probes
such as 20 ,70 -Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein
(BCECF) and seminaphtorhodafluor-1 (SNARF-1) that are called
ratiometric probes because they show different emission spectra as a
function of pH [44].
In many cell biology researches, there is the need to know the
concentration levels of the Ca++ ion that is involved in many pro-
cesses such as muscle contraction, movement, secretion, etc. For
this purpose several markers have been proposed: Indo 1 that is
excitable in UV and Fluo-3, Fluo-4, Oregon Green, and Fura that
are excitable at 488 nm, thus being of choice for argon laser-based
FC [24, 25].

8 Quantum Dots

Most of the fluorochromes described so far belong to the family of

organic compounds. Many of them are synthetic products designed
and produced by the chemical industry, but some are instead of
natural origin. The latter, playing the role of “light detectors” in
nature, are characterized by high absorption/emission efficiency of
light. All fluorochromes of organic nature are characterized by
molecules with a complex chemical structure rich in aromatic
rings and double bonds [76–79].
This structural complexity is related to a great variety of energy
levels that the molecule can assume which in turn allows the com-
plexity of their spectrofluorometric characteristics. In other words,
18 Giuliano Mazzini and Marco Danova

their absorption/emission spectra are very broad, practically

extended over most of the frequencies of the visible spectrum.
There is also frequently a significant overlap between absorption
and emission spectra (very short Stokes shift). All these are unde-
sirable characteristics in FC especially in the case of multiparametric
tests: broadband fluorescence emission obviously entails the risk of
overlapping between the emission spectra of the different probes
and may cause possible errors in their quantitative measurements.
To try to eliminate (or reduce) these drawbacks, a new family of
fluorochromes called nanocrystals or QDs has recently been devel-
oped. Unlike all the above described fluorochromes, they are inor-
ganic compounds derived from research on semiconductor
materials: their development derives in fact from the enormous
advances in the world of microelectronics and nanotechnology.
QDs are currently composed mainly of cadmium/selenide and
cadmium/telluride compounds. Compared to the large organic
molecules of traditional fluorochromes, their crystal size is in the
order of a few nanometers. Their optical characteristics are a func-
tion of their size and not of their chemical nature. In particular,
with the same composition but increasing the crystal size, the
emission of fluorescence shifts to the right in the visible spectrum.
So, a QD of 2 nm in diameter can have an emission spectrum in the
violet, whereas a crystal of 12 nm has an emission spectrum in the
red. Very peculiarly instead, their absorption spectrum is more or
less constant and mainly located in the first part of the visible (from
ultraviolet to blue). For these spectrofluorometric characteristics,
they proved to be of choice for multiparametric applications in FC
[80]. Due to their exceptionally large Stokes shifts (up to 400 nm),
these probes can contribute to the setting of very large analytical
panels with small spectral overlaps. This allows multiparametric FC
analyses even with a single excitation without the need of complex
compensation procedures [45].
As mentioned before, these probes are inorganic crystals of very
small size that do not have chemical functional groups or radicals
that could make them chemically reactive. Being chemically inert,
they cannot practically be conjugated to antibodies or specific cell
targets, hence the need to coat them with organic polymers to make
them suitable for conjugation chemistry. This organic coating
increases their solubility in water and enriches them with functional
groups (such as NH2, NH3) for subsequent conjugation to anti-
bodies, streptavidin, or nucleic acids. Their final size thus becomes
close to that of fluorochromes commonly used to label antibodies,
such as PE. The QDs’ quantum efficiency is similar to that of PE or
APC and sometimes even higher. Unlike the generally used
fluorochromes that have a rather limited emission range (in the
green-to-red spectrum), QDs are extremely versatile in terms of
spectrofluorometric properties.
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 19

There is a wide range of products characterized by commercial

labels (QD 450, QD 500, QD 510, . . .QD 630) that indicate their
emission peak, practically over the entire spectrum from blue
to red.
Theoretically, QDs are the ideal fluorescent markers and appar-
ently without any bugs or limitations; obviously, all these merits
have a price and QDs are currently more expensive than the classic
fluorochromes. They are still subject to development especially in
the coating: those of the latest generation have a shell modified
with long polyethylene glycol (PEG) chains rich in amino groups
that allow QDs to more easily conjugate to antibodies [81, 82].

9 Mass Cytometry

We have seen in previous sections how the increasing demand for

new markers has contributed to the design of more sophisticated
panels for multiparametric analyses, especially in the field of clinical
diagnostics. Nevertheless, the use of conventional fluorescent
probes is limited by the mutual crosstalk, which limits the number
of those that can be simultaneously used on a single sample. The
instrumental strategy of using multiple excitation sources (lasers)
only partially allows the number of markers to be increased. It is
also possible to divide the sample into several aliquots, subjected to
different analytical panels of probes, and finally sequentially ana-
lyzed. This obviously increases the costs and, primarily, the time
required for the analyses.
A new FC approach has recently been proposed [83–86] using
a fully innovative labeling strategy. It uses heavy metal isotopes
instead of fluorochromes. The detection of these probes obviously
cannot be performed by an optical mode (through spectrofluorom-
etry) but is made by emulating what happens in mass spectrometry
by “time-of-flight” detection (FC is thus replaced by cytometry by
time-of-flight, CyTOF).
The mass isotopes used as probes are not naturally present in
biological samples and are therefore used to tag specific reagents
(e.g., antibodies) similar to what is done with fluorochromes. Bio-
chemical research has made available several binding processes for
specific custom-labeling recipes.
The sample preparation is very similar to that required for the
more traditional FC mode, but the analytical strategy is completely
different. The labeled sample (the cell suspension) once introduced
into the system is nebulized and further completely ionized. Clouds
of molecules and particles are then further disrupted at the atomic
level. The sequence of atomic particles generated by each single cell
is analyzed by CyTOF, which allows associating a specific set of
markers to each single cell. The data collection, handling, and
display software are completely similar to the ones of the
20 Giuliano Mazzini and Marco Danova

conventional cytometers. The three-dimensional representation of

complex multiparametric panels characterizing the immunopheno-
type analyses is now routine in many clinical laboratories [87–89].
The great commercial boost driving the field of clinical diag-
nostics has already made a very wide range of specific markers
available [90, 91]. The first field of interest is certainly that of
monoclonal antibodies. It is therefore foreseeable in the short
period a sort of war between the two fronts: “fluorescence versus
mass.” The panels with conventional fluorescent probes have
become increasingly sophisticated and efficient but still require
operator adjustments (compensation) on the acquired data. On
the contrary, the panels with mass markers should not have this
problem.
Obviously, both analytical approaches (fluorescence vs mass)
have specific advantages and limitations:
1. Fluorescence FC allows the analysis of thousands of cells in very
short times and reliable results in many fields of clinical diag-
nostics. Classical FC is nowadays the routine analytical tech-
nique in research centers and hospitals. The instrumentation
and the related methodology, after more than 40 years of
development and progress, have reached absolute levels of
performances and reliability. The analytical power covers a
very wide range of particle sizes, from whole cells to bacteria,
to subcellular constituents such as the microvesicles. Even the
various cellular functions have been considered for specific
analytical approaches. The cells analyzed can also be separated
(with flow sorters) into subpopulations identified with specific
markers to be subjected to further molecular investigations.
Some instruments (image in flow) even allow storing image
files for subsequent correlation between quantitative fluores-
cence data and detailed morphology of the relevant cells. This
technology has a great diagnostic power as it combines the
information of the microscope with those from the FC.
2. The CyTOF boasts as a special feature the possibility to per-
form multi-parameter analysis (in a compensation-free mode)
with dozens of markers in simultaneous analysis. Of great
strength is the detection without mutual probe crosstalk and
the consequent ability to measure a very high number of mar-
kers (actually 40–100) on a single sample aliquot. This
approach seems therefore to be of choice for the complex
panels of immunophenotype analyses that are nowadays very
popular (and powerful) in various fields of immunohematolo-
gical diseases. However, one of the main limitations is the need
to destroy the cells to be measured and the consequent loss of
morphometric information. Of course in the wide panel of
results, the classical scatter parameters are missed (worldwide
used in classical CF analysis) which must be surrogated by other
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 21

markers. The analysis time is longer than in FC because the

sample undergoes a series of destructive treatments to get from
cells to clouds of atoms.
In summary, CyTOF could be defined as a “high-content
analysis,” compared to conventional CF, as it allows acquiring a
very high number of information on a more limited number of
cells. In the decade to come, we will see if CyTOF may replace FC
or if, more likely, these two approaches will integrate.

10 Concluding Remarks

For over half a century, fluorescence has been the milestone for all
quantitative analyses both at chemical/biochemical level (in tubes,
vials, cuvettes) and in cytometry (in both sections, cell smears, and
suspensions). In particular, FC (especially thanks to immunofluo-
rescence) promoted the development of multiparametric analytical
panels that are now routinely used in many areas of clinical diag-
nostics. Due to the continuous development of new fluorescent
molecules, a wide variety of markers have become available for the
determination of both cell components and cellular functions. The
fluorescent markers of the latest generation have chemical/physical
characteristics that can ensure quantitative multiparametric analysis
with high sensitivity, efficiency, and reliability.
New horizons in cytometry are now emerging, following the
development of a new analytical strategy based on the use of mass
isotope markers. These probes seem to represent the ideal way for
the setting of high-performance multiparametric analyses. How-
ever, we will have to wait a few years of application to understand if
the costs/benefits of this new analytical strategy will really be able
to replace the classic “old fluorescence.”

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Another Random Scribd Document
with Unrelated Content
pahempi seikka, sellainen, jota hän ei ensin aavistanutkaan, oli se,
että osa niitä pahansuopia tietoja, jotka Rudolf hänestä sai, oli
lähtenyt Ernestin suusta. Tuo pikkulurjus ymmärsi kyllä Christophen
ja Rudolfin erilaisen arvon: ei ollut epäilemistäkään, että hän tunsi
Christophen ylemmyyden ja että hän ehkä oli suopeakin hänelle
hänen vilpittömyytensä tähden, joskin hiukan ivallisella tavalla. Mutta
hän piti hyvän huolen siitä, että voi käyttää tuota vilpittömyyttä
hyväksensä; ja samalla, kun hän halveksi Rudolfin mataluutta, kiskoi
hän siitäkin hävyttömästi itselleen hyötyä. Ernest mairitteli Rudolfin
turhamaisuutta ja kateutta, otti nöyrästi vastaan hänen
haukkumisensa, varusti hänet tuoreilla tiedoilla kaupungin julkisista
juoruista, varsinkin Christophea koskevista, — ja niistä onki hän aina
ihmeellisen tarkat tiedot. Hän saavutti tarkoituksensa; ja vaikka
Rudolf olikin kovin saita, antoi hän Ernestin kyniä itseään, samoin
kuin Christophekin.

Siten kiskoi Ernest heitä molempia ja pilkkasi samalla mitalla heitä

kumpaakin. Ja molemmat he pitivät hänestä.

Kaikesta ovelista tempuistaan huolimatta oli Ernest kurjassa tilassa

nyt äitinsä luokse ilmestyessään. Hän tuli Münchenistä, jossa hän oli
saanut ja melkein heti tapansa mukaan menettänyt paikkansa.
Hänen oli täytynyt kulkea jalkaisin suurin osa matkaa, pahimpana
sade-aikana ja nukkuen Herra tiesi missä. Hän oli loan vallassa,
vaatteet rikki, kuin mikä kerjäläinen, ja hän yski surkeasti; sillä hän
oli matkalla saanut pahan keuhkokatarrin. Louisa tyrmistyi, ja
Christophe juoksi liikutettuna hänen luokseen heti, kun he näkivät
hänen astuvan sisään. Ernestin oli helppo heruttaa kyyneleitä, eikä
hän jättänyt tätä vaikuttavaa keinoa käyttämättä; kohtauksesta tuli
yleinen heltyminen: he itkivät kaikki kolme sylitysten.

Christophe antoi Ernestille huoneensa; hankittiin sängynlämmitin,

ja sairas, joka näytti tuossa paikassa heittävän henkensä, sijoitettiin
vuoteeseen. Louisa ja Christophe istuivat hänen pääpuolessaan
vuorotellen öisin häntä vaalimassa. Tarvittiin lääkäri, lääkettä,
lämmittää kamari hyvin, erikoista ruokaa.

Sitten täytyi hänet hommata uusiin vaatteisiin kiireestä kantaan:

aluspuvut, kengät, päällysasu, kaikki oli hankittava kokonaan
uudestaan. Ernest antoi hoidella itseään. Louisa ja Christophe iskivät
ankarasti suontansa saadakseen jotain säästymään näihin menoihin.
Heidän raha-asiansa olivat nykyään sangen vaikeat: muutto uuteen
paikkaan, kalliimpi huoneisto kuin entinen, joskin yhtä epämukava,
Christophella entistä vähemmän tunteja ja kuitenkin menot
lisääntyneet. He tuskin saivat pysymään asiat tasapainossa. Heidän
täytyi ponnistaa kaikki voimansa. Christophe olisi kyllä saattanut
kääntyä Rudolfin puoleen, joka oli paremmissa varoissa kuin hän;
mutta sitä hän ei tahtonut tehdä: hänestä oli kunnianasia pitää itse
huoli veljestänsä. Hän luuli sen velvollisuudekseen, koska hän oli
veljeksistä vanhin, — ja koska hän oli Christophe. Häpeästä
suunniltaan täytyi hänen nyt lähteä itse hyväksymään eräs tarjous,
jonka hän oli pari viikkoa sitten vihoissaan hyljännyt, nimittäin erään
rikkaan ja maineettoman amatöörin ehdotus, jonka jälleen muuan
välittäjä oli esittänyt Christophelle: tuo amatööri tahtoi ostaa
Christophelta jonkin sävellyksen julkaistakseen sen omalla nimellään.
Louisa taas sitoutui korjailemaan kaiket päivät ihmisten
liinavaatteita. Hän ja Christophe salasivat toisiltaan nämä
uhrautumishommansa. He valehtelivat toisilleen, mistä olivat saaneet
rahaa, kun toivat sitä kotiin.
 Taudista toipuessaan tunnusti Ernest eräänä päivänä istuen
kuurussa takkavalkean ääressä ja yskien rajusti pitkän aikaa, että
hänellä oli eräitä velkojakin. Ne velat maksettiin. Kukaan ei häntä
niistä soimannut. Sellainen ei olisi ollut kaunista sairasta ja
tuhlaajapoikaa kohtaan, joka tuli katuvaisena kotiin. Sillä
koettelemukset näyttivät Ernestin muuttaneen. Hän puhui kyyneleet
silmissä entisistä harha-askeleistaan. Ja Louisa syleili häntä ja pyysi
häntä olemaan enää niitä ajattelematta. Ernest oli mielistelevä
olento: hän oli aina osannut mairia äitiään hellyydellä; Christophe oli
ennen ollut siitä hänelle hiukan kateellinenkin. Mutta nyt oli hänestä
luonnollista, että nuorempi ja heikompi veli sai enemmän
rakkauttakin osakseen. Hän itse piti Ernestiä paremminkin poikanaan
kuin veljenään, vaikka heidän ikäeronsa olikin vähäinen. Ernest
käyttäytyi sangen kunnioittavaisesti häntä kohtaan; hän vihjaili
joskus, minkälaisen taakan Christophe oli ottanut hartioilleen, puhui
hänen rahallisista uhrauksistaan…; mutta silloin ei Christophe
antanut hänen jatkaa, ja Ernest alistui kiittämään ainoastaan
nöyrällä ja liikutetulla katseella. Hän myönteli Christophen neuvot
oikeiksi; hän näytti voivan alkaa uutta elämää, pystyvän ryhtymään
parannuttuaan vakavasti työhön.

Ernest parani; mutta toipumisaika oli pitkä. Lääkäri selitti, että

hänen kovin väärinkäytetty terveytensä tarvitsi vielä varovaista
hoitelua. Ernest jäi siis yhä edelleen kotiin äidin luokse, jäi osille
Christophen vuoteesta, söi hyvällä ruokahalulla veljensä ansaitsemaa
leipää ja pieniä herkkuruokia, joita Louisa koetti kaikin keinoin
hänelle hankkia. Hän ei ollut lähdöstä tietääkseenkään. Eivätkä
Louisa ja Christophe siitä hänelle suinkaan puhuneet. He olivat liian
onnellisia, kun olivat jälleen löytäneet rakastetun pojan ja veljen.
 Vähitellen alkoi Christophe viettäessään illat pitkät Ernestin kanssa
jutella hänelle välittömämmin omia asioitaan. Hänen teki mieli avata
sydäntänsä jollekulle. Ernest oli älykäs; hänellä oli nopea tajunta ja
hän ymmärsi — tai näytti ymmärtävän — kaiken puolella sanalla.
Hänen kanssaan oli ilo jutella. Kuitenkaan ei Christophe uskaltanut
virkkaa vielä mitään siitä, mikä oli lähinnä hänen sydäntään:
rakkaudestaan. Sillä häntä hillitsi jonkinlainen kainous. Ernest, joka
tiesi jo kaikki, ei ollut tietävinään mitään.

Eräänä päivänä lähti Ernest, nyt täysin terveenä, kävelemään

iltapuolella auringon paisteessa pitkin Rheinin rantaa. Vähän matkaa
kaupungista huomasi hän eräässä hälisevässä ravintolassa, jonne
kaupungista tultiin sunnuntaisin tanssimaan ja kallistamaan pikaria,
Christophen Aadan ja Myrrhan seurassa samassa pöydässä. Tytöt
pitivät kovaa ääntä. Christophe näki puolestaan Ernestin ja punastui.
Ernest tekeytyi hienotunteiseksi, ja meni ohitse tulematta heidän
luokseen.

Christophe oli kovin hämillään tästä kohtauksesta: se sai

tuntemaan hänet paremmin, millaisessa seurassa hän oleskeli; ja
hänestä oli tuskallista, että veli hänet näki siellä: ei ainoastaan siksi,
että hän arveli tästä lähtien menettävänsä oikeutensa tuomita
Ernestin käytöstä, vaan myöskin sen tähden, että hänellä oli
vanhimman veljen velvollisuuksista sangen korkea, yksinkertainen ja
hieman arkamainen käsitys, sellainen, että se olisi monista muista
tuntunut suorastaan naurettavalta: hän ajatteli, että jos hän löi
laimin velvollisuutensa niinkuin hän teki, alensi hän itseään omissa
silmissään.

Kun Christophe ja Ernest tulivat molemmat illalla yhteiseen

kamariinsa, odotti Christophe, että Ernest vihjailisi jollakin tavalla
tuohon päivän tapahtumaan, mutta Ernest oli viisaasti vaiti, ja
hänkin odotti. Silloin, riisutuessaan, päätti Christophe puhua hänelle
rakkaudestaan. Christophe oli niin hämmennyksissään, ettei hän
tohtinut katsoa Ernestiin; ja arkuudessaan turvautui hän jöröön ja
katkonaiseen puhetapaan. Ernest ei auttanut häntä sulavuuteen; hän
oli vaiti, eikä katsonut myöskään puolestaan Christopheen, mutta
näki hänet kuitenkin: häneltä ei jäänyt huomaamatta, miten paljon
Christophen kömpelöissä ja avuttomissa sanoissa oli koomillista.
Christophe tuskin uskalsi hiiskua Aadan nimeä; ja kuva, jonka hän
Aadasta antoi, olisi sopinut mihin rakastettavaan naiseen tahansa.
Mutta hän puhui rakkaudestaan; ja heittäytyen vähitellen hellyyden
valtaan, joka täytti hänen sydämensä, hän sanoi, mikä onni on
rakastaa, kuinka ihminen on viheliäinen ennen kuin hän on nähnyt
tuon valonsa yössä, ja ettei elämä ole mitään ilman pyhää ja syvää
rakkautta. Toinen kuunteli vakavana; hän vastasi hienovaistoisesti
eikä tehnyt minkäänlaisia kysymyksiä; mutta heltynyt kädenpuristus
osoitti muka, että hän oli samaa mieltä kuin Christophe. He purkivat
toisilleen ajatuksiaan rakkaudesta ja elämästä. Christophe oli
onnellinen, että häntä oli niin ymmärretty. He syleilivät toisiaan
veljellisesti ennenkuin asettuivat nukkumaan.

Christophelle tuli sitten tavaksi uskoa rakkausasioitaan Ernestille,

jonka vaiteliaisuus oli hänet aivan rauhoittanut, mutta teki sen
kuitenkin hyvin arkatuntoisesti eikä paljastanut mitään liikaa. Hän
antoi Ernestin huomata kautta rantain huolensa Aadaan nähden;
mutta koskaan ei hän Aadaa syyttänyt: hän soimasi ainoastaan
itseään, ja kyyneleet silmissä hän julisti, ettei hän voisi elää jos
Aadan kadottaisi.

Hän ei unohtanut puhua Aadalle myöskään Ernestistä: hän kiitti

Ernestin älyä ja kauneutta.
 Ernest ei pyytänyt Christophea esittelemään itseään Aadalle;
mutta hän sulkeutui alakuloisesti kamariin ja kieltäytyi lähtemästä
sieltä mihinkään, sanoen, ettei hän tuntenut kaupungissa ketään.
Christophe moitti itseään siitä, että hän lähti sunnuntaisin maalle
kävelyretkille Aadan kanssa ja jätti veljensä yksin kotiin. Kuitenkin
tuntui hänestä kiusalliselta, ettei hän olisi saanut olla yksin
ystävättärensä kanssa; mutta hän syytti itseään itsekkäisyydestä ja
ehdotti viimein, että Ernest tulisi heidän mukaansa.

Esittely tapahtui Aadan ovella, hänen asuintalonsa sisäportailla.

Ernest ja Aada tervehtivät toisiaan jäykän kohteliaasti. Aada tuli
sitten ulos, ja hänen perästään hänen eroittamaton ystävättärensä
Myrrha. Kun viimemainittu näki Ernestin, puhkesi hänen huuliltaan
hämmästyksen huudahdus. Ernest hymyili, lähestyi ja syleili Myrrhaa,
joka näytti pitävän sitä aivan luonnollisena asiana.

— Kuinka! Tehän tunnette toisenne? kysyi Christophe ällistyneenä.

— Erinomaisesti! vastasi Myrrha nauraen.

— Milloinka te olette tutustuneet?'

— Kauan sitten!

— Ja sinä tiesit sen? kysyi Christophe Aadalta? Miksi et sitä minulle

sanonut?

— Minä nyt muka tuntisin Myrrhan kaikki rakastajat, virkkoi Aada

kohauttaen olkapäitään.

Myrrha sai puheen jälleen vilkastumaan ollen muka suuttuvinaan.

Christophen tiedot supistuivat siihen. Hän oli tullut surulliseksi.
Hänestä tuntui, että Ernest, Myrrha ja Aada olivat olleet hänelle
vilpillisiä, vaikka hän ei totta sanoen voinut syyttää heitä mistään
valheesta; mutta olihan vaikea uskoa, että Myrrha, jolla ei ollut
mitään salaisuutta Aadalle, olisi peitellyt tätäkään asiaa häneltä,
enempää kuin sitäkään, etteivät Ernest ja Aada olisi tunteneet
toisiaan jo aikaisemmin. Christophe piti heitä silmällä. Mutta Ernest
ja Aada puhuivat keskenään vain jonkun jokapäiväisen sanan ja koko
kävelyretken aikana ei Ernest sitten välittänyt muusta kuin
Myrrhasta. Aada puolestaan puheli ainoastaan Christophelle; ja hän
oli Christophea kohtaan paljoa rakastettavampi kuin tavallisesti.

Siitä alkaen tuli Ernest mukaan kaikille heidän kävelyretkilleen.

Christophe olisi mielellään tahtonut hänestä päästä, mutta hän ei
uskaltanut sitä sanoa. Ei silti, että hänellä olisi ollut muita syitä
erottaa veljeään seurasta kuin se, että hän häpesi pitää häntä
huvittelu-toverinaan. Christophe ei epäillyt mitään eikä Ernest
antanutkaan hänelle siihen aihetta. Hän näytti kiintyneen Myrrhaan,
ja Aadaa kohtaan käyttäytyi hän kohteliaasti ja syrjään vetäytyvästi,
jopa melkein sopimattoman hienotunteisesti; näytti kuin hän olisi
tahtonut antaa hieman veljensä rakastajattarellekin sitä kunnioitusta,
jota hän tunsi Christophea itseään kohtaan. Aada ei sitä
kummastunut, ja hän piti tarkan vaarin itsestään.

He tekivät yhdessä pitkiä kävelyretkiä. Veljekset kulkivat edeltä;

Aada ja Myrrha seurasivat heitä, nauraen ja supatellen, parin
askeleen päässä. He pysähtyivät usein pitkäksi aikaa juttelemaan
toistensa kanssa, keskellä maantietä. Christophe ja Ernest
seisahtuivat myöskin heitä odottamaan. Viimein tuli Christophe
kärsimättömäksi ja lähti menemään; mutta pian hän kääntyi
harmissaan takaisin, kuin kuuli Ernestin nauravan ja puhuvan jotakin
noiden kahden lavertelijan kanssa. Hän olisi tahtonut tietää, mitä he
keskustelivat; mutta kun hän pääsi heidän luoksensa lakkasi heidän
pakinansa.

— Mitä salavehkeitä teillä aina on? kysyi hän. Hänelle vastattiin

jotain leikillistä. Nuo kolme vetivät yhtä köyttä kuin varkaat.

Christophe riiteli eräänä sunnuntai-aamuna Aadan kanssa. Sitten

mököttivät he toisilleen koko alkupuolen huvimatkaa. Aada ei ollut
arvokkaan ja loukatun näköinen niinkuin hän tavallisesti sellaisissa
tapauksissa menetteli, kostaen siten, että koetti olla niin
sietämättömän ikävä kuin mahdollista. Tällä kertaa hän sensijaan ei
ollut muka tietääkseen koko Christophesta, vaan pakisi kävelyretkellä
toisten toverusten kanssa ja oli erinomaisella tuulella heidän
seurassaan. Tuntui kuin hän pohjaltaan olisi oikein toivonut tätä
riitaa.

Christophe olisi hyvin mielellänsä tahtonut tehdä jälleen sovinnon;

hän oli rakastuneempi kuin koskaan ennen. Hänen hellyyteensä yhtyi
kiitollisuus kaikesta, mitä hyvää heidän rakkautensa oli hänelle
tuonut, ja hän suri, että he tuhlasivat aikaa tällaisilla älyttömillä
riidoilla, — ja pelkäsikin jotain merkillistä: se oli salaperäistä
tunnetta, että tämä rakkaus oli loppuva. Hän katseli alakuloisena
Aadan kauniita kasvoja, Aadan, joka ei nyt ollut häntä
näkevinäänkään, vaan naureskeli toisten kanssa; nuo kasvot
herättivät hänessä niin paljon kalliita muistoja, hyvän rakkauden ja
läheisen yhdessäolon muistoja; ja niissä hurmaavissa kasvoissa oli
joskus, — (samoin tälläkin hetkellä) — niin paljon hyvää ja puhtaan
hymyilevää, että Christophe kummasteli mielessään, miksi heidän
välinsä eivät olleet paremmat, miksi he turmelivat tahallaan ilonsa,
miksi Aada koetti itsepintaisesti unohtaa heidän onnelliset hetkensä,
väittää ne olemattomiksi, ja taistella kaikkea vastaan, mikä hänessä
oli hyvää ja kunniallista; — mitä omituista tyydytystä Aadalle
tuottikaan häiritä ja tahrata heidän tunteittensa puhtautta, vaikkapa
ainoastaan ajatuksilla. Christophe kaipasi suunnattomasti saada
uskoa siihen olentoon, jota hän rakasti, ja hän koetti nyt viimeisen
kerran rauhoittaa itseään kuvitelmilla. Hän syytti itseään: hän oli
muka väärässä; hänen tuntonsa kolkutti, kun hän ajatteli, millaiseksi
hän syytti Aadaa mielessään; ja hän oli muka itse liian tyly.

Christophe lähestyi Aadaa, hän koetti päästä puheisiin hänen

kanssaan: Aada ei vastannut hänelle muuta kuin pari kuivaa sanaa:
hän ei tahtonut laisinkaan tehdä hänen kanssaan sovintoa.
Christophe toivoi sitä ehdottomasti, hän kuiskasi Aadan korvaan ja
pyysi häntä hiukan puheilleen, syrjemmällä muista. Aada seurasi
häntä nyrpeän näköisenä. Kun he olivat jääneet jonkun askeleen
päähän toisista eivätkä Myrrha ja Ernest voineet heitä enää nähdä,
tarttui Christophe rajusti Aadan käsiin, hän pyysi häneltä anteeksi,
polvistui hänen eteensä, metsässä, keskellä lakastuneita lehtiä.
Christophe sanoi, ettei hän voinut elää näin, riidassa Aadan kanssa;
hän ei voinut enää nauttia koko kävelyretkestä, ei kauniista päivästä,
hän ei voinut nauttia mistään, ei enää edes hengittääkään, kun tiesi
Aadan häntä vihaavan; hän kaipasi niin suuresti Aadan rakkautta.
Niin, hän oli usein väärämielinen, väkivaltainen, epämiellyttävä; hän
pyysi Aadaa antamaan hänelle anteeksi: syynä siihen kaikkeen oli
hänen rakkautensa; hän ei voinut sietää Aadassa mitään
keskinkertaista, mitään joka ei ollut Aadan ja heidän kauniin
yhdessäolonsa muistojen arvoista. Christophe palautti ne muistot
Aadan mieleen, kertoi hänelle heidän ensimmäisen kohtauksensa,
ensimmäiset yhdessä vietetyt päivät; hän sanoi, että hän rakasti
Aadaa yhä niinkuin ennen, että hän rakastaisi häntä aina. Älköön
Aada jättäkö häntä! Aada oli hänelle kaikki kaikessa… Aada kuunteli
häntä, hymyillen, hämmentyneenä, melkein heltyneenä. Hän katseli
Christophea jälleen suloisin silmin, katsein, jotka ilmaisivat, että
rakkaus eli ja etteivät he enää olleet toisilleen suutuksissa. He
suutelivat toisiaan ja painuivat kädet tiukasti toistensa ympärillä
edelleen lehdettömään metsään. Christophe oli Aadasta kiltti, ja hän
oli kiitollinen hänelle noista hellistä sanoista; mutta hän ei silti
luopunut pahankurisista oikuistaan, jotka hän oli saanut päähänsä.
Kumminkin hän nyt epäröi hiukan, hän ei ollut aivan luja
päätöksessään. Ja sittenkään ei hän jättänyt tekemättä, mitä oli
ajatellut. Minkä tähden? Kuka sen voi selittää?… Senkö vuoksi, että
hän oli varmasti päättänyt sen tehdä?… Kuka tietää? Hänestä tuntui
ehkä omintakeisemmalta ja makeammalta pettää ystäväänsä juuri
sinä päivänä, näyttääkseen hänelle ja itselleen, että hän oli vapaa.
Hän ei ajatellut, että hän voisi kadottaa Christophen: sitä ei hän olisi
tahtonut. Hän luuli olevansa hänestä varmempi kuin koskaan ennen.

Seurue oli tullut aholle. Siitä haarautui kaksi eri tietä! Christophe
lähti menemään toista niistä. Ernest väitti, että toinen veisi
suorempaan sille kukkulalle, jonne he nyt aikoivat mennä. Aada oli
samaa mieltä hänen kanssaan. Christophe tunsi tien hyvin, sillä hän
oli kulkenut sitä usein, ja väitti, että nuo toiset olivat väärässä. He
pysyivät jyrkästi mielipiteessään, ja silloin sovittiin, että koetettaisiin;
ja kumpikin puoli löi vetoa saapuvansa omaa tietänsä ensin perille.
Aada lähti Ernestin kanssa toista polkua, Myrrha seurasi Christophea;
hän oli muka aivan vakuutettu että Christophe oli oikeassa; ja siihen
hän lisäsi:

"Kuten aina." Christophe oli käsittänyt vedon vakavasti; ja kun hän

ei koskaan halunnut joutua tappiolle, kulki hän nopein askelin, niin,
liian nopeasti Myrrhan mielestä, jolla ei ollut laisinkaan niin kiire kuin
Christophella.

— Älä hätäile, hyvä ystävä, sanoi hän Christophelle pilkallisesti ja

tyynesti; kyllä me joka tapauksessa joudumme ennen.

Christophelle tuli jokin epäilys:

— Se on totta, sanoi hän; ehkä kuljemme liian nopeasti: se ei

kuulu vetoon.

Hän hiljensi askeleitaan.

— Mutta minä tunnen heidät, jatkoi Christophe; olen varma, että

he juoksevat ennättääkseen sinne ennen meitä.

Myrrha purskahti nauruun:

— Ei ei, älä ole milläsikään siitä!

Myrrha riippui Christophen käsipuolessa, hän painautui aivan

Christophea vasten. Hän oli hiukan lyhyempi kuin Christophe ja
katseli kävellessään ylös häneen, älykkäin ja hyväilevin silmin. Hän
oli tosiaan sievä ja kiehtova. Christophe tuskin tunsi häntä nyt:
kukaan ei ollut alttiimpi hetkellisille muutoksille kuin Myrrha;
tavallisesti olivat hänen kasvonsa kelmeät ja pöhöiset; mutta ei
tarvittu muuta kuin pieni kiihdytys, jokin iloinen ajatus tai halu
miellyttää, niin tuo vanhamainen ilme katosi ja Myrrhan poskille
syttyi puna, rypyt silmäluomista, silmien alta ja ympäriltä hävisivät,
hänen katseensa syttyi, ja hänen koko muotonsa sai sellaisen
nuoruuden, elämän ja älykkyyden sävyn, ettei Aadan piirteissä ollut
moisesta merkkiäkään. Christophe hämmästyi Myrrhassa
huomaamaansa muutosta, hän käänsi katseensa pois välttäen
Myrrhan silmiä: hän oli hieman hämmennyksissään, näin kahden
kesken Myrrhan kanssa. Myrrhan läsnäolo vaivasi häntä; hän ei
kuunnellut, mitä tyttö sanoi, eikä vastannut hänelle, tai hän vastasi
aivan päin honkia: hän ajatteli, tahtoi ajatella pelkästään Aadaa. Hän
muisteli miten kauniisti Aada oli juuri äsken häneen katsellut,
muisteli hänen hymyään, hänen suudelmaansa; ja hänen sydämensä
täytti ylenpalttinen rakkaus. Myrrha koetti puhua hänelle, kuinka
kaunis metsä oli: nuo pienet ja hienot oksat, taustanaan kuulas
taivas; niin, kaikki oli kaunista: pilvi oli haihtunut, Christophe oli
saanut Aadan takaisin; hänen oli onnistunut sulattaa jää heidän
väliltään, he rakastivat jälleen toisiaan; olivatpa toisiaan lähellä tai
kaukana ja erillään, olivat he kuitenkin aivan yhtä. Christophe
huokaisi helpoituksesta; kuinka keveä ilma nyt oli! Aada oli tullut
hänelle takaisin… Kaikki muistutti hänelle Aadasta… Sää oli hiukan
kostea: eiköhän Aadalla ollut kylmät… Kauniita puita verhosi kuura:
mikä vahinko, ettei Aada niitä puita nähnyt!… Mutta Christophe
muisti lyödyn vedon, ja hän kiirehti taas askeleitaan; hän varoi
tarkoin, ettei vain eksyisi tieltä. Ja hän huudahti riemuissaan
päästyään perille:

— Me jouduttiin ensimmäisinä.

Hän heilutti iloisesti hattuaan. Myrrha katseli häneen hymyillen.

Kukkula, jonka laella he nyt seisoivat, oli pitkä kalliojyrkänne

keskellä metsää. Tuota huippua ympäröivät pähkinä- ja
vaivaistammipensaat, ja sieltä näkyi kauas: metsäisiä rinteitä
punasinervien ja usvan harsoamien kuusenlatvojen takaa, sekä
Rhein-virta, joka kiemurteli nauhana sinertävässä laaksossa. Ei
linnunviserrystä kuulunut. Ei ääntä, ei tuulen henkäystä. Oli
liikkumaton ja hartaan-tyyni talvinen päivä, joka lämmitteli viluissaan
värähdellen auringon turtuneessa valossa. Silloin tällöin kajahti
kaukaa laaksosta junan lyhyt vihellys. Christophe seisoi jyrkänteen
reunalla ja katseli maisemaa. Myrrha tarkasteli Christophea.

Christophe kääntyi Myrrhan puoleen, selvästi oikein hyvällä

tuulella.

— Kas niin, ne laiskurit, virkkoi hän; johan sanoin sen heille!… No,
mitäpä muuta, täytyy heitä odottaa…

Hän heittäytyi loikomaan auringonpaisteeseen, pengermälle, joka

oli rakoillut kuivuudesta.

— Aivan niin, odotetaan, sanoi Myrrha ottaen hatun pois päästään.

Myrrhan äänessä oli jotain niin pilkallista, että Christophe

kohottausi ylös ja katsoi häneen.

— No, mitä nyt? kysyi Myrrha rauhallisesti.

— Mitä sinä sanoit?

— Minä sanoin, että odotetaan vaan. Ei kannattanut, kuten näet,

juoksuttaa minua niin kiireesti.

— Se on totta.

He odottivat, loikoen molemmat rosoisella pengermällä. Myrrha

hyräili jotakin laulua. Christophe hyräili myöskin muutamia säkeitä.
Mutta hän keskeytti vähän väliä kuunnellen korva tarkkana:

— Nyt he taitavat tulla, olin kuulevinani. Myrrha lauloi yhä vaan.

— Ole nyt hiljaa, hetkinen. Myrrha keskeytti.

 — Ei, se ei ollut mitään. Myrrha alkoi taas laulaa. Christophe ei
pysynyt enää paikallaan:

— Ehkäpä he ovat eksyneet.

— Eksyneetkö? Ei täällä voi eksyä. Ernest tuntee kaikki tiet.

Omituinen ajatus pälkähti Christophen päähän.

— Jospa he pääsivät tänne ennen meitä, ja lähtivät takaisin jo

ennenkuin me tulimme?

Myrrha makasi seljällään maassa ja katseli taivaaseen, ja puhkesi

keskellä lauluaan niin hurjaan nauruun, että oli tikahtua. Christophe
pysyi itsepintaisesti mielipiteessään. Hän tahtoi nyt lähteä asemalle,
ja sanoi, että ystävät olivat varmaan jo siellä. Myrrha päätti jättää
kylmän salailunsa.

— Se olisi hyvä keino juuri joutuaksemme heistä erilleen!… Eihän

ole sovittu mitään asemalla kohtaamisesta. Mehän päätimme tavata
toisemme täällä.

Christophe istahti häntä lähelle. Myrrhaa Christophen odottelu

huvitti. Christophe tunsi, että Myrrha tarkasteli häntä ivallisin silmin.
Hän alkoi nyt tosiaan tulla rauhattomaksi, — rauhattomaksi, mihin
Aada ja Ernest olivat joutuneet: hän ei heitä laisinkaan epäillyt. Hän
nousi jälleen ylös. Hän sanoi, että nyt täytyisi mennä takaisin
metsään, etsiä ja huutaa heitä. Myrrhan kurkusta kuului silloin pieni
naurunkurahdus; hän oli ottanut taskustaan neulan ja sakset, alkoi
nyt ratkoa hatustaan irti sulkia, ja ompeli sitten ne siihen kiinni
uudella tavalla: hän näytti aikovan jäädä tähän kaikeksi päivää.
 — Ei, hölmöseni, vastasi hän Christophelle. Jos he tahtoisivat tulla,
luuletko, etteivät he osaisi tänne?

Se iski Christophea suoraan sydämeen. Hän käännähti Myrrhaan

päin; tyttö ei häneen katsellut, vaan jatkoi työtään. Christophe meni
lähemmäksi häntä:

Myrrha! sanoi hän.

— No? äännähti Myrrha keskeyttämättä ompeluaan.

Christophe asettui polvilleen voidakseen tarkastaa häntä

paremmin.

— Myrrha! toisti Christophe.

— No, mikä nyt? kysyi Myrrha nostaen silmänsä työstä ja katsellen

häneen hymyillen. Mikä sinulla on?

Nähdessään, että Christophe oli aivan tyrmistynyt, tuli Myrrha

ilveilevän näköiseksi.

— Myrrha! kysyi Christophe kurkkuun takertuvin äänin, sano, mitä

sinä tästä ajattelet…

Myrrha kohautti olkapäitään, hymyili, ja ryhtyi jälleen työhönsä.

Christophe tarttui hänen käteensä ja tempaisi häneltä pois hatun,

jota hän korjaili:

— Jätä tuo, jätä, ja sano minulle… Myrrha katsoi häntä silmiin ja

odotti. Hän näki Christophen huulten vapisevan.

— Sinä ajattelet, virkkoi Christophe hiljaa, että Ernest ja Aada…?

 Myrrha hymyili:

— Hm, mitäs muuta!

Christophe ponnahti loukkautuneena ylös:

— Ei, ei! Se ei ole mahdollista. Et ajattele sellaista!… Ei! Ei!

Myrrha pisti kätensä hänen olkapäälleen ja vääntelehti naurusta.

— Kuinka sinä olet tuhma, niin tuhma, ystäväni!

Christophe ravisti häntä rajusti:

— Älä naura! Minkä tähden sinä naurat? Sinä et nauraisi, jos se

olisi totta! Sinä pidät Ernestistä…

Myrrha nauroi vain. Ja vetäen Christophea luokseen suuteli hän

häntä. Vasten tahtoaankin Christophe suuteli Myrrhaa. Mutta kun
hän tunsi hänen huultensa kosketusten, noiden huulten, jotka olivat
vielä lämpöiset hänen veljensä suudelmista, riuhtausi hän
taaksepäin, piti Myrrhan päätä kiinni edessään ja kysyi:

— Sinä tiesit sen? Oliko se teidän kesken sovittu?

Myrrha äännähti: "Oli", ja nauroi.

Christophelta ei päässyt ääntä, hänessä ei näkynyt vihan

merkkiäkään. Hän aukoi suutansa, niinkuin hänen olisi ollut vaikea
hengittää; hän sulki silmänsä, ja pusersi käsin rintaansa: hänen
sydämensä oli pakahtua. Sitten hän heittäytyi maahan, painoi
päänsä käsiinsä, ja häntä täristi raju inhon ja epätoivon puuska,
samanlainen kuin usein ennen lapsena.
 Myrrhan tuli sääli häntä, vaikkei hän yleensä ollutkaan kovin
helläluonteinen; tiedottomasti valtasi hänet äidillinen hellyyden
tunne. Hän kumartui Christophen puoleen, hän puhui hänelle
lohdutellen, hän tahtoi, että hänen olisi pitänyt haistella hänen
hajuvesipulloaan. Mutta Christophe sysäsi sen kauhuissaan luotaan
ja nousi ylös niin rajusti, että Myrrhaa peloitti. Christophella ei ollut
voimaa eikä halua kostaa. Hän katseli Myrrhaa, ja hänen kasvonsa
olivat tuskasta väännyksissä:

— Viheliäinen, sanoi hän menehtyvällä äänellä; sinä et tiedä, mitä

pahaa sinä teet…

Myrrha koetti estää Christophea lähtemästä. Christophe ryntäsi

pois kukkulalta, läpi metsän, syljeskellen inhosta ajatellessaan
moista julkeaa törkeyttä, moisia saastaisia sieluja ja sukurutsaista
jakelua, johon he tahtoivat häntä osalliseksi. Hän itki, hän vapisi,
nyyhkytti inhosta. Hänelle tuli kauhu Aadaa, kaikkea, omaa itseään,
ruumistaan ja sydäntään kohtaan. Halveksimisen myrsky purkausi
hänessä valloilleen: kauan aikaa oli se jo uhkaavana kohonnut;
ennemmin tai myöhemmin täytyi hänessä syntyä vastarinta tuollaista
mataluutta, moisia häpäiseviä sovinnoita ja ummehtunutta ja
myrkyllistä ilmapiiriä vastaan, jossa hän oli elänyt muutamia
kuukausia; mutta hänen kaipuunsa rakastaa ja pettää itseään
näkemästä oikeassa muodossaan sitä olentoa, jota hän rakasti, oli
viivyttänyt tuota auttamatonta purkausta, niin kauan kuin se oli
suinkin mahdollista. Nyt se kuohahti ilmi yhtäkkiä; ja näin oli
parempi. Vapauttava, raitis ja tuiman puhdas tuulenhenkäys,
jääkylmä viima pyyhkäisi tuossa tuokiossa pois hänestä nuo
myrkkyhöyryt. Inho tappoi yhdellä iskulla hänen rakkautensa Aadaa
kohtaan.
 Jos Aada oli luullut vahvistavansa valtaansa Christopheen tällä
teollaan, todisti se vain vielä kerran, miten typerän törkeästi hän
ymmärsi sen olennon, joka häntä rakasti. Mustasukkaisuus, joka
kyllä saattaa kiinnittää toisiinsa tahraisia sydämiä, nosti ainoastaan
kapinaan sellaisen nuoren, ylpeän ja puhtaan luonteen kuin
Christophe. Mutta varsinkin eräs seikka, jota Christophe ei antanut
anteeksi, jota hän ei voinut antaa koskaan anteeksi, se, ettei tämä
Aadan petos johtunut intohimosta eikä varmaankaan edes noista
mielettömistä ja turmeltuneista oikuistakaan, joiden
vastustamattomia puuskia naisen äly ei juuri jaksa estää. Ei, — nyt
Christophe sen ymmärsi, — se johtui Aadassa vaan salaisesta
halusta alentaa häntä, nöyryyttää häntä, rangaista häntä hänen
moraalisen jäykkyytensä tähden, hänen uskonsa tähden, joka oli
Aadalle vihallinen; saada hänet suistumaan alas yleiselle matalalle
tasolle, polkea hänet jalkoihinsa, tuntea oma epäterveellinen
voimansa. Ja kauhuissaan Christophe ajatteli: mikä ihmeellinen halu
onkaan enimmillä ihmisillä tahrata: — tahrata kaikkea, mikä heissä ja
muissa on puhdasta, — noilla tunkiosieluilla, jotka nauttivat
hekkumaa saadessaan kieriskellä loassa, jotka ovat onnellisia, kun
heidän pinnassaan ei ole enää yhtään ainoaa puhdasta paikkaa!…

Aada odotti kaksi päivää, että Christophe tulisi takaisin. Sitten alkoi
hän tulla levottomaksi ja hän lähetti Christophelle suloisen kirjeen,
jossa hän ei vihjaillut vähääkään tapahtuneeseen. Christophe ei
siihen edes vastannut. Hän vihasi Aadaa nyt niin syvästi, ettei
hänellä ollut enää edes sanoja vihaansa ilmaistakseen. Hän oli
pyyhkäissyt Aadan pois elämästään. Aadaa ei ollut hänelle enää
olemassa.
 Christophe oli vapautunut Aadasta, mutta itsestään ei hän ollut
vapautunut. Turhaan koetti hän sitä kuvitella ja päästä takaisin
entiseen puhtaaseen ja voimakkaaseen tyyneyteen. Entisyyteensä ei
ihminen koskaan palaa. Hänen täytyy jatkaa matkaansa; eikä
hyödytä kääntyä takaisin, paitsi katsoakseen niitä paikkoja, joiden
ohitse kulki, niiden majain sauhuja, joiden katon alla nukkui, kuinka
ne haipuvat ilmanrannalla muistojen utuun. Mutta mikään ei vie
meitä nopeammin erillemme entisestä sielustamme kuin lyhyet
intohimon ajat. Tie kääntyy yhtäkkiä, maisema muuttuu uudeksi;
tuntuu kuin joutuisimme sanomaan viimeisen kerran hyvästit sille,
jonka jätämme taaksemme. Christophe ei voinut alistua sellaiseen.
Hän ojensi käsiään menneisyyttä kohti. Hän koetti itsepintaisesti
saada jälleen heräämään eloon entisen sielunsa, yksinäisenä ja
alistuvana. Mutta sitä sielua ei enää ollut olemassa. Intohimo on
vaarattomampi itsessään kuin sen kasaamien raunioiden tähden.
Christophe saattoi kyllä olla enää rakastamatta, hän saattoi halveksia
rakkautta: — lyhyen aikaa, — se oli jo kuitenkin jättänyt häneen
kyntensä merkit; hänen sydämeensä oli jäänyt tyhjyys, joka täytyi
täyttää. Tuon hirvittävän hellyyden ja ilon kaipuun vuoksi, joka
kalvaa elämän onnea maistaneita olentoja, tarvitsi hän jotain muuta
intohimoa, vaikkapa jotakin aivan entiselle vastaista: halveksumisen,
ylpeilevän puhtauden, hyveeseen uskomisen intohimoa. — Ne
tunteet eivät kuitenkaan riittäneet; ne eivät jaksaneet enää
masentaa hänen nälkäänsä; ne eivät olleet muuta kuin tuokion
ravintoa. Hänen elämänsä oli yhtäjaksoisia rajuja vastavaikutuksia,
— hyppyjä yhdestä äärimmäisyydestä toiseen. Milloin tahtoi hän nyt
pakoittaa itsensä epäinhimillisen pidättyväisyyden sääntöihin; hän ei
syönyt enää, joi vain vettä, kiusasi ruumistaan kävelyillä,
ponnistuksilla, valvomalla, kielsi itseltään kaiken ilon. Milloin hän
jälleen vakuutti itselleen, että voima on hänenlaistensa ihmisten
ainoa moraali; ja hän syöksyi tavoittelemaan ainoastaan iloa.
Kummassakin tapauksessa oli hän onneton. Hän ei enää voinut olla
yksin. Se kävi hänelle yhä mahdottomammaksi.

Hänen ainoa pelastuksensa olisi löytää ollut oikeaa ystävyyttä, —

esimerkiksi Rosan: siitä hän olisi saanut turvansa. Mutta noiden
kahden perheen välit olivat nykyään auttamattomasti rikki; ne eivät
enää tavanneetkaan toisiaan. Yhden ainoan kerran oli Christophe
kohdannut Rosan. Nuori tyttö tuli kirkosta. Christophe epäröi,
yrittääkö hänen seuraansa; ja Rosa puolestaan teki hänet
nähdessään sellaisen liikkeen, kuin olisi aikonut tulla häntä
tapaamaan; mutta kun Christophe jo halusi mennä Rosan luokse,
läpi uskovaisten tungoksen, joka laskeutui alas portaita, käänsi Rosa
katseensa hänestä pois; ja kun Christophe tuli häntä lähelle, tervehti
Rosa häntä kylmästi, ja meni sivuitse. Hän tunsi, että nuori tyttö
halveksi häntä perinpohjaisesti ja kylmäsydämisesti. Eikä Christophe
tuntenut, että Rosa rakasti häntä yhä ja olisi tahtonut sen hänelle
sanoa, vaikka hän moittikin sentähden itseään kuin jostakin viasta tai
typeryydestä; hän piti Christophea kunnottomana ja turmeltuneena,
itselleen kaukaisempana kuin koskaan ennen. Sitten kadottivat he
toisensa ainaiseksi. Se oli ehkä hyväksi heille molemmille.
Hyvyydestään huolimatta ei Rosa ollut tarpeeksi eloisa Christophea
ymmärtämään. Vaikka Christophe tarvitsi kiihkeästi hellyyttä ja
kunnioitusta, olisi hän tukehtunut keskinkertaisessa ja suljetussa
elämässä, jossa ei ollut iloa eikä tuskaa, ei ilmaa. He olisivat
kärsineet kumpikin. Olisivat kärsineet siitä, että olisivat tuottaneet —
toisilleen kärsimyksiä. Kova onni, joka heidät eroitti toisistaan, oli siis
ehkä lopultakin hyvä onni, niinkuin usein on, — ja on aina niiden,
jotka ovat väkeviä ja kestävät.
 Mutta tällä hetkellä oli se heille suuri surun syy ja suuri
onnettomuus. Varsinkin Christophelle. Moinen hyveellinen
suvaitsemattomuus, henkinen ahtaus, joka joskus näyttää
kadottavan täydellisesti älyn juuri niiden päästä, joilla sitä on
enimmän, ja hyvyyden niiden sydämestä, jotka ovat parhaita, ärsytti,
loukkasi Christophea, syöksi hänet uhmaan ja entistä vapaampaan
elämään.

Kierrellessään ennen Aadan kanssa ulkoravintoloissa kaupungin

ympärillä oli Christophe tutustunut eräihin reiluihin poikiin —
bohêmeihin, joiden huolettomuus ja tapojen vapaus eivät olleet
hänestä liioin vastenmielisiä. Eräs heistä, nimeltään Friedemann,
muusikko niinkuin Christophe, kolmisenkymmentä vuotta urkurina
elänyt mies, ei ollut henkisyyttä vailla, ja tunsi hyvin ammattinsa,
mutta oli auttamattoman laiska ja olisi mieluummin kuollut nälkään,
jopa janoonkin, kuin ponnistanut hiukan voimiaan päästäkseen
keskolaisuudestaan. Hän lohdutti vetelyydessään itseään
haukkumalla niitä, jotka elämässä touhuavat; ja hänen hieman
kömpelöt pilapuheensa olivat naurattavan sukkeliakin. Hän oli
tovereitaan vapaampi eikä peljännyt singota tyytymättömyyttään
vallassa oleviin henkilöihin, — vaikka hän tekikin sen vielä arasti,
ikäänkuin silmää iskien, salavihjauksilla —; olipa hän kyllin uskalikko
hyljätäkseen valmiit ja yleisesti hyväksytyt mielipiteet musiikista, ja
salasisuisesti iski hän piikkinsä päivän suuruuksiin, jotka olivat
anastamalla siepanneet maineensa. Naiset eivät saaneet myöskään
häneltä armoa. Hän omisti heille leikkiä laskien erään muinaisen
naisia vihaavan munkin lauselman, jonka tuimuudesta Christophe
nautti nykyään hartaammin kuin moni muu:

"Femina mors animae."