94
Fluorescence with diaphragm module HC RF: As
a BG 38 filter is not integrated here, it must be
built into the filter magazine (65.13) if needed.
The light path can be blocked by pulling out the
diaphragm module part way. Intensity can be
increased by interposing the illumination
telescope (“booster”, not illustrated).
Light trap
To avoid stray light from the underneath of the
specimen: remove the condenser and put the
light trap (Fig. 64) in its place. Alternatively, a
black metal plate can be pushed into the stage.
Possible errors
Weak fluorescence, weak image intensity due
to:
Incorrectly stored, too old or faded specimens;
fast specimen fading (e.g. with FITC); inspecific
filter combination, numerical aperture of
objectives too low; eyepiece magnification too
high; spent lamp; room too bright.
Low contrast image due to:
Excitation bandwidth too great; inspecific
staining; fluorescing inclusion medium; auto-
fluorescence of the objective or immersion oil.
With double fluochroming, green and red image
details visible at the same time due to:
Filter cubes unsuitable for selective
observation.
Inhomogeneous illumination due to:
Incorrect lamp centration or flickering lamp.
Brightening of image background or red back-
ground due to:
Absence of BG 38 red attenuation filter in light
path.
Metal staining
The case is different for reflecting objects, such
as in the immunogold technique (IGS). Here the
POL filter system (crossed polarizers for
contrast enhancement) is used for incident light
polarization instead of a fluorescence filter
cube, and contrast, resolving power and depth
of field can be influenced with the aperture
diaphragm.
Fig. 64
Light trap for fluorescence microscopy (instead of the
condenser). Instead of the light trap, a dark metal or plastic
strip can be used, which is inserted between the upper and
lower part of the x/y stage under the specimen.
