Operation Manual
48
4.5 Fluorescence Photomicrography
For the basic procedure and key points of photomicrography, see the manuals provided with the
photomicrographic equipment
Since the specimen colour may fade effort must be made on minimizing the exposure of the specimen to
irradiation both before, and during exposure.
Select the area of interest without using fluorescence, use phase contrast or interference contrast.
Select an appropriate filter combination for the specimen (whether single, dual or triplepass filter sets are
used, exposure times for acquiring video or photographic images will increase for dual and triplepass
filter sets when compared to singlepass filter sets).
The magnification of the image at the film plane affects the intensity of the image; the latter is inversely
proportional to the magnification. To minimize the magnification required to fill the picture with the
desired object, the format chosen should be as small as considered practical and hence have shorter
exposure times.
Exposure time varies for each objective and projection lens combination even if the total magnifications
are the same. The objective with higher numerical aperture (N.A.) should be chosen over increasing the
magnification by projection lens. (The numerical aperture of the objective increases with increase in
magnification and higher the numerical aperture, the brighter the image).
Excessively bright excitation light will cause the specimen to fade, adjust the brightness by inserting a
neutral density filter in the light path as these filters give a constant transmission over a wide range of
wavelengths.
Focus collector lens give brighter or more even illumination.
Avoid bleaching of the specimen by blocking the excitation light when not viewing or photographing the
specimen.
4.6 Video Fluorescence Microscopy
Electronic photomicrography utilizes electronic detection devices to acquire images, inserting an IR
(infrared) barrier filter in the light path before the detection device may produce better results by avoiding
erroneous readings caused by infrared light.