PRINCIPLES OF

FLUORESCENCE
MICROSCOPE
 INTRODUCTION
Is a type of microscope that uses fluorescence or
phosphorescence instead of light absorption and
reflection like other microscopes.
 fluorescence is a property of some substance that
when illuminated with light usually ‘u.v’ they emit
rays of longer wave length.
Phosphorescence is an induced light that persist
after the stimulatory light is cut off
Fluorochromes these are organic dyes which
fluoresce when subjected to short wave radiation and
which have affinity for certain substances.
The Fluorescence Microscope
•exposes specimen to ultraviolet, violet, or
blue light
•specimens usually stained with
fluorochromes
•shows a bright image of the object
resulting from the fluorescent light
emitted by the specimen
 Classification of fluorescence
 primary(natural/auto-fluorescence)
- ability of some substance to fluoresce
internally
Secondary( induced) fluoroscence
- produced after the reaction of a non auto-
fluoroscent substance with fluoronce dye.
 Dyes(fluorochrome)
Common dyes used for staining;
 auramine- O
 acridine orange
 rhodamine-B
 thioflavin-S
 thiazo- yellow
 g.primulin
 morine.
 How it operates
In fluorescence microscope a fluorochrome
stained specimen is illuminated with a visible
u.v light rays emitted from exciter filter then
passed through a condenser lens; light rays of
longer wave length within the spectrum of
visible light are given off and through the
barrier filter this come to the eye to form the
image and are seen fluorescene(glowing)
against a dark back ground.
 Preparation and Staining of
Specimens
Why do we stain specimens?
•increases visibility of specimen
•accentuates specific morphological
features
•preserves specimens
 Reagents for staining
Auramine-phenol solution

 Auramine O 25 g
 Ethanol 3000 ml
 Phenol 250 g
 Distilled water 5300 ml
 Staining procedure
Place the slides on a staining rack, with the smeared
side facing up, the slides not touching each other
Flood the slides with freshly filtered auramine-
phenol. Let stand for 7-10 min.
Wash well with running water
Decolorize with acid-alcohol for 1-2 min.
Wash as before with water and slope the slides to
air dry
Counter stain with 0.1% KMNO4 for 30 seconds
 Fluorescence Microscopy
Advantages
Covers an increased area of smear field
An increased contrast between stained bacilli &
background
Heating not required
Immersion oil not needed
May be appropriate in the diag. of HIV + assoc.TB
With low bacillary content
Early diagnosis of pulm.TB - Low bacillary content
 Fluorescence Microscopy
Disadvantages

Handling & maintenance of optical equipment

require advanced skill
Periodic replacement of bulbs

Continuous supply of power.

High cost of Microscope and maintenance

•Thank you