# Presentation: 107995

## Introduction to Fluorescence Microscopy and Immunofluorescence 

- 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- If the light emission persists for up to a few seconds after the excitation light is withdrawn, the phenomenon is known as phosphorescence.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

## Slide 7

## 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

## Slide 10

## 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.

## Slide 12

## 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.

## Slide 20

## 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

## Slide 23

## Fluorescent Antibody Staining

- Y

- Indirect Fluorescent
- Antibody (IFA)

- Direct Fluorescent
- Antibody (DFA)

## 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.

## Slide 27

## Slide 28

## 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

## Slide 31

## 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?

## Slide 35

## 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

## Slide 38

## 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

## Slide 40

## Slide 41

## Slide 42

## 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%

## Slide 46

## Slide 47