Introduction to

Fluorescence
Microscopy and
Immunofluorescence

Danny L. Wiedbrauk, Ph.D.

Warde Medical Laboratory
Ann Arbor, MI
 Vocabulary
Photoluminescence: The ability of living or non-
living, organic or inorganic specimens to absorb
and subsequently re-radiate light.
If the light emission persists for up to a few seconds
after the excitation light is withdrawn, the
phenomenon is known as phosphorescence.
Fluorescence: light emission occurs only when the
excitation light is present.
 Background
In the middle of the nineteenth century Stokes
observed that the mineral fluorspar fluoresced when
ultraviolet light was directed upon it.
Stokes coined the word "fluorescence".
Fluorescing light was always of a longer wavelength
than the excitation light (Stokes Law).
FITC
— Excitation 495 nm
— Emission: 520 nm
 Fluorescence Microscopy
The intensity of the fluorescence is very weak in
comparison with the excitation light (10-3 to 10-5).
The emitted light re-radiates spherically in all
directions.
Dark background is required to enhance resolution.
Spectral Overlap
Barrier Filters
 Fluorescence Microscopy
The basic task of the fluorescence microscope is to
permit excitation light to irradiate the specimen and
then to separate the much weaker re-radiating
fluorescent light from the brighter excitation light so
that only the emission light reaches the eye.
The resulting fluorescing areas shine against a dark
background with sufficient contrast to permit
detection.
The darker the background of the non-fluorescing
material, the more efficient the instrument.
Transmitted Light Microscopy
Transmitted Light Microscopy
Because a darkfield condenser is used, the excitation energy
reaching the specimen is relatively low and the fluorescence
intensity is lowered.
Darkfield condenser limits the NA of the objectives and
reduces image brightness.
For maximum intensity, both the condenser and the objective
must be oiled.
Darkfield condensers require accurate centering.
Thick specimens (and slides) are not satisfactory.
At low power, only the center of field of view is lighted.
Epifluorescence
 Epifluorescence
Darkfield condenser is not required; the
objective acts as a condenser.
Centering, focusing, and oiling the condenser is
not needed.
Objectives with the highest NA should be used
to maximize brightness.
Specimen thickness does not interfere with
flourescence intensity.
Fading only occurs in the field of view.
 Light Sources
Ultrahigh pressure mercury lamps
— 50 W, 100 W, 200 W
Xenon high-pressure lamps
— 75 W, 150 W, 450 W
Low-voltage lamps
— 12V 50W, 12V 100 W tungsten halogen lamps
Mercury and Xenon Bulbs
Arc lamp glass envelopes are filled with mercury or
xenon gas at moderately high pressure
Never handle these lamps when they are hot
Avoid applying a mechanical force that might cause
the lamp to explode.
Avoid touching the new lamp with fingers
— Oils from the hands are acidic and may etch the quartz
envelope enough to weaken it.
— Residue from fingerprints can become fused to the exterior
of the bulb when it becomes hot.
If a bulb does explode, use mercury cleanup and
decontamination procedures.
 Bulb Alignment

After installing a new bulb in a mercury or xenon arc

lamphouse, the arc must be carefully aligned and focused
to achieve a homogeneous field of illumination.
The arc itself is very small (about 1 or 2 millimeters in
length), and the image of the arc must be positioned along
the optical axis of the microscope, at the center of the
condenser aperture in the vertical illuminator, to ensure
even illumination.
Poorly aligned light paths can cause false-negative results
in FA testing.
http://www.microscopyu.com/tutorials/java/arclamp/index.html
 Fading
Bleaching
— Irreversible decomposition of the fluorescent molecules

because of light intensity in the presence of molecular O2.

Quenching
— Caused by free-radical oxidation, oxidizing agents or the

presence of salts of heavy metals or halogen compounds.

Fluorescent-Resonance Energy Transfer (FRET)
— The transfer of energy to other so-called acceptor

molecules physically close to the excited fluorophores.

 Antifade
Comments Reference
Reagent
The most effective reagent for FITC. Also
G. D. Johnson & G.
effective for Rhodamine. Should be adjusted
M. Araujo (1981) J.
p-phenylene- to 0.1% p-phenylenediamine in glycerol/PBS
Immunol.
diamine for use. Reagent blackens when subjected to
Methods, 43: 349-
light exposure so it should be stored in a dark
350
place. Skin contact is extremely dangerous.
G. D. Johnson et.
DABCO Highly effective for FITC. Although its effect
al., (1982) J.
(1,4-diazabi- is slightly lower than p-phenylenediamine, it
Immunol.
cyclo-2,2,2- is more resistant to light and features a
Methods, 55: 231-
octane) higher level of safety.
242
H. Giloh & J. W.
The most effective reagent for Rhodamine,
n- Sedat (1982),
also effective for FITC. Should be adjusted to
propylgallate Science, 217:
1% propylgallate in glycerol/PBS for use.
1252-1255
Used to observe chromosome and DNA S. Fujita & T.
specimens stained with propidium iodide, Minamikawa
2-mercapto-
acridine orange, or Chromomysin A3. Should (1990),
ethylamine
be adjusted to 0.1mM 2-mercaptotheylamine Experimental
in Tris-EDTA Medicine, 8: 75-82
 Historical Perspective
Early investigations showed that many substances
autofluoresce when irradiated with ultraviolet light.
In the 1930's Haitinger and others developed the
technique of secondary fluorescence--employing
fluorochrome stains to stain specific tissue components,
bacteria, or other pathogens which do not autofluoresce.
[acridine orange]
In the 1950's Coons and Kaplan used fluorescein-
tagged antibodies to localize antigens in tissues.
[Immunofluorescence]
 Today

Immunofluorescence is used to:

— Identify infectious agents in clinical specimens
— Serotype and identify viruses from cultures
— Quantify antibody levels
— Localize antigenic determinants on cells
— Identify different cell types
Fluorescent Antibody Staining

Direct Fluorescent Indirect Fluorescent

Antibody (DFA) Antibody (IFA)
Direct Specimen Detection
Using Immunofluorescence
Fluorescent Antibody Detection
Allows the laboratory to evaluate specimen
adequacy
— Type of cells present
— Number of cells
Does not require strict cold-chain transport
Increased specificity due to pattern and staining
evaluations.
Faster Turnaround Times

Supports patient cohorting and STAT

testing
Initiate/discontinue antiviral therapies
Reduced use of antibiotics
Reduced hospital stays
 RSV Testing

RSV is thermolabile
DFA methods are significantly more
sensitive than culture
Varicella-Zoster Virus

Cell culture isolation

Shell vials
DFA
Varicella-Zoster Virus

Tube culture 5 - 7 days

Shell vials 2 - 4 days
DFA 1 - 2 hrs
What do we miss?
Not for Every Specimen

PCR for CSF and ocular fluids

Culture other specimens (BAL, tissue)
Herpes Simplex Virus

Culture 1-2 days

EIA (daily batches)
DFA 1-2 hrs
 Bottom Line

Two-step testing for dermal specimens

— DFA (detects 72% of positives)
— Culture DFA-negative specimens
PCR for CSF and ocular fluids
Culture other specimens
Respiratory Viruses

RSV 2-5 days

Influenza A/B 2-10 days
Parainfluenza 2-14 days
Adenovirus 2-10 days
Respiratory Virus Testing

DFA (eliminates >50% of cultures)

Culture DFA-negative specimens
EIA only when necessary
 Breakthrough Rates

Overall, 1-10% of DFA-negative

specimens were culture-positive

RSV: << 1%
Parainfluenza 1,2,3: 1-4%
Influenza A: 7-10%
Adenovirus: ~15%