Center for Microscopy and Image Anaylsis
Introduction to light
microscopy
(concepts
in imaging and
overview)
Microscopy with light
Components of a light
microscope
1. Light source
2. Objective
3. Sample or specimen holder
4. Focusing mechanism
5. Lens for focusing light on
specimen
6. Eyepiece / Camera
Interaction of light with tissue
matter
Samples
mouse
cells
Fundamentals of light
microscopy
Compound microscope:
Microscope composed of an
objective and an additional lens
(eyepiece, occular, tube lens)
Magnification:
What is the maximal
magnification?
Mfinal = Mobjecive x Moccular
Is there a limit in useful
magnification?
=> Why is there a limit in resolution?
Diffraction at an aperture
or substrate
Disturbance of the electric field of
a planar wavefront by diffraction
upon passage through an
aperture
A mixture of particles diffracts an
incident planar wavefront
inversely proportional to the size
of particles
Plane wave Grating Lens
Object plane Back focal plane Image plane
Plane wave Grating Lens
Object plane Back focal plane Image plane
Plane wave Grating Lens
Object plane Back focal plane Image plane
Image formation in the
microscope
Diffracted light from a periodic
specimen produces a diffraction
pattern of the object in the back
focal plane
Not interacting incident light is
transmitted undeviated and
produce the evenly illuminated
image plane
Diffraction spots in the back focal
plane correspond to constructive
interference of waves differing in
1, 2, …. wavelengths.
Image formation in the image
plane is by interference of
undeviated and deviated waves
Diffraction image of a point
source of light
The image of a self-luminous point
in a microscope is a pattern
created by interference in the
image plane
The pattern is a central bright spot
surrounded by a series of rings
The central spot contains ≈ 84%
of light
The image is called: Airy disk
(after Sir George Airy (1801 –
1892))
http://www.cambridgeincolour.com
Resolution and aperture
angle
The objective aperture must
capture light from a wide angle for
maximum resolution (diffracted or
emitted light)
α α
NA = n sin α
α: half angle of the cone of
specimen light accepted by the
objective
n: refractive index of medium
between lens and specimen
Resolution limits
0.61 × λ
𝑑𝑥𝑦 =
𝑁𝐴
𝑛 × λ
𝑑𝑧 =
𝑁𝐴2
These formula are used for the
calculation of resolution in
widefield microscopy.
In other techniques like confocal
laser scanning, multiphoton
microscopy, etc other formula are
used.
Fluorescence in
microscopy
DNA DNA DNA
Bax Bax Bax
Mitochondria Mitochondria Mitochondria
Cytochrome C Cytochrome C Cytochrome C
Fluorescence in
microscopy
Advantages:
Very high contrast resulting in
high sensitivity
Tagging of specific entities
possible
Excitation / emission allows for
various variants of microscopy
techniques
Jablonski scheme
CONFOCAL LASERSCANNING MICROSCOPY: TRUE 3D MICROSCOPY
Regular widefield fluorescence Problem: out of focus light
Reduced contrast from out of focus light
http://smokingdesigners.com/34-stunning-depth-field-photographs/
CONFOCAL LASER SCANNING MICROSCOPY
Excitation of fluorescence in sample: Notes:
Sample is excited by a laser focused to a
point
CONFOCAL LASER SCANNING MICROSCOPY
Emission of fluorescence from sample: Notes:
Sample is excited by a laser focused to a
Point
Emitted fluorescent from focus is
focused to a point and then reaches a
detector measuring the incoming
fluorescent light.
A computer records the amount of
emitted light and computes an image
point by point over time
CONFOCAL LASER SCANNING MICROSCOPY
Excluding out of focus light: Notes:
Sample is excited by a laser focused to a
Point
Emitted fluorescent from focus is
focused to a point and then reaches a
detector measuring the incoming
fluorescent light.
A computer records the amount of
emitted light and computes an image
point by point over time
Emitted fluorescent from out-of-focus is
also out-of- focus at pinhole and largely
excluded from detector by the presence
of the pinhole
CONFOCAL LASER SCANNING MICROSCOPY
Excluding out of focus light: Notes:
Sample is excited by a laser focused to a
Point
Emitted fluorescent from focus is
focused to a point and then reaches a
detector measuring the incoming
fluorescent light.
A computer records the amount of
emitted light and computes an image
point by point over time
Emitted fluorescent from out-of-focus is
also out-of- focus at pinhole and largely
excluded from detector by the presence
of the pinhole
Comparison of widefield
and confocal microscopy
Confocal microscopy has a very
high signal to noise ratio
𝑛 × λ (prominent in thick samples)
𝑑𝑧 =
𝑁𝐴2
Confocal microscopy allows well
resolved 3D imaging (without any
image processing)
Image acquired with a
widefield microscope
2 2
0.88em n 2 PH
dz
2 NA
n n NA
2
Image acquired with a
confocal microscope
Temporal resolution –
Nipkow disk (spinning disk
– tandem) scanning
microscopy
http://zeiss-campus.magnet.fsu.edu/tutorials
Multiphoton
microscopy
Imaging deep into tissue
Multiphoton
microscopy
Imaging in scattering tissue
All fluorescent photons
provide useful signals.
Helmchen and Denk, Nature Methods
2005
Multiphoton microscopy
Deep tissue two-photon
microscopy
Helmchen and Denk, Nature
Methods 2005
Huisken J , Stainier D Y R Development 2009;136:1963-1975
Light sheet microscopy
Light sheet microscopy
Development, September 1, 2012vol. 139 no. 17 3242-3247
Superresolution
microscopy
Structured
illumination
microscopy
José María Mateos
Super resolution
microscopy
Beyond the diffraction limit
The common feature:
switching fluorophores on and off
sequentially in time and space
so that the signals can be
recorded consecutively below
the diffraction limit
SUPER RESOLUTION MICROSCOPY
Thank you
Literatur
Fundamentals of light microscopy and electronic imaging, Douglas B.
Murphy; Wiley-Liss, 2001
ISBN 0-471-25391-X (Sehr verständliches Buch mit allem nötigen
Grundlagenwissen zu Lichtmikroskopie)
Light Microscopy in Biology – A practical approach, A. J. Lacey; Oxford
University Press, 2004 (Einfache Beschreibung der Lichtmikroskopie mit
praktischen Übungen und Anleitungen)
Light and Electron Microscopy, E. M. Slayter, H. S. Slayter; Cambridge
University Press, 1992 (Detailierte und oft mathematische Beschreibung der Licht
und Elektronenmikroskopie. Gutes Referenzwerk)
http://microscopy.fsu.edu/primer/index.html (Ausführliche und vorzügliche
Beschreibung der Lichtmikroskopie mit Demonstrationen, sehr empfehlenswert)
