Page 30 - Textbook of Pathology, 6th Edition

14 Filters. A variety of filters are used between the source of
light and objective: first, heat absorbing filter; second, red-light
stop filter; and third exciter filter to allow the passage of light
of only the desired wavelength. On passing through the
specimen, light of both exciting and fluorescence wavelength
collects. Exciter light is removed by another filter called barrier
filter between the objective and the observer to protect the
observer’s eyes so that only fluorescent light reaches the eyes
of observer.
SECTION I
Condenser. Dark-ground condenser is used in fluorescence
microscope so that no direct light falls into the object and
instead gives dark contrast background to the fluorescence.

TECHNIQUES. There are two types of fluorescence
techniques both of which are performed on cryostat sections
of fresh unfixed tissue: direct and indirect.
In the direct technique, first introduced by Coons (1941)
who did the original work on immunofluorescence, antibody
against antigen is directly conjugated with the fluorochrome
and then examined under fluorescence microscope.
In the indirect technique, also called sandwich technique,
there is interaction between tissue antigen and specific anti-
body, followed by a step of washing and then addition of
fluorochrome for completion of reaction. Indirect
Figure 2.5 Binocular light microscope (Model E 400, Nikon, Japan).
Courtesy: Towa Optics (India) Pvt. Ltd., New Delhi. immunofluorescence technique is applied to detect auto-
antibodies in patient’s serum.
rays of light vibrate in a single plane at right angle to each APPLICATIONS. Immunofluorescence methods are applied
other. Two discs made up of prism are placed in the path of for the following purposes:
light, one below the object known as polariser and another 1. Detection of autoantibodies in the serum e.g. smooth muscle
placed in the body tube which is known as analyser. The lower antibodies (SMA), antinuclear antibodies (ANA),
General Pathology and Basic Techniques
disc is rotated to make the light plane polarised. .
antimitochondrial antibody (AMA), thyroid microsomal
antibody etc.
IMMUNOFLUORESCENCE
2. In renal diseases for detection of deposits of immuno-
Immunofluorescence technique is employed to localise globulins, complement and fibrin in various types of
antigenic molecules on the cells by microscopic examination. glomerular diseases by frozen section as discussed in
This is done by using specific antibody against the antigenic Chapter 22.
molecule forming antigen-antibody complex at the specific 3. In skin diseases to detect deposits of immunoglobulin by
antigenic site which is made visible by employing a frozen section, particularly at the dermo-epidermal junction
fluorochrome which has the property to absorb radiation in and in upper dermis e.g. in various bullous dermatosis
the form of ultraviolet light so as to be within the visible (Chapter 26).
spectrum of light in microscopic examination. 4. For study of mononuclear cell surface markers using mono-
The immunofluorescent method has the following clonal antibodies.
essential components:
5. For specific diagnosis of infective disorders e.g. viral
FLUORESCENCE MICROSCOPE. Fluorescence microscopy hepatitis.
is based on the principle that the exciting radiation from
ultraviolet light of shorter wavelength (360 nm) or blue light
(wavelength 400 nm) causes fluorescence of certain substances ELECTRON MICROSCOPY
and thereafter re-emits light of a longer wavelength. Electron microscope (EM) first developed in 1930s in Germany
Some substances fluoresce naturally; this is termed
primary fluorescence or autofluorescence though UV light is has undergone modifications so as to add extensive new
knowledge to our understanding the structure and function
required for visualising them better e.g. vitamin A, porphyrin, of normal and diseased cells at the level of cell organelles.
chlorophyll. However, more recently, widespread use of diagnostic
Secondary fluorescence is more commonly employed and
is the production of fluorescence on addition of dyes or chemi- immunohistochemistry in surgical pathology has restricted
the application of EM to the following areas of diagnostic
cals called fluorochromes.
pathology:
Source of light. Mercury vapour and xenon gas lamps are 1. In renal pathology in conjunction with light microscopy
used as source of light for fluorescence microscopy. and immunofluorescence (Chapter 22).