Page 51 - Fundamentals of Light Microscopy and Electronic Imaging

34 ILLUMINATORS, FILTERS, AND THE ISOLATION OF SPECIFIC WAVELENGTHS

Finally, inspect the mercury arc spectrum with its continuous spectrum and
superimposed prominent emission lines. Half of the output of this lamp is in the
UV, with one of the most prominent (but invisible) emission lines being located
at 366 nm. This wavelength is commonly used for photoactivation of caged fluo-
rescent compounds, stimulation of UV-excitable fluorescent dyes, and conversion
of colchicine to its photoisomer, lumicolchicine. This line and the 405 nm violet
line can be visualized using the optical bench setup by holding a piece of fluo-
rescent white paper in the proper location in the spectrum in a darkened room. A
suitable piece of paper can be found using a handheld near-UV black light to
select for sheets that exhibit brilliant bluish white fluorescence due to the pres-
ence of phenolic compounds in the paper. The 405 nm violet line and the 366 nm
near-UV line suddenly leap into view when the white card is inserted into the blue
end of the spectrum.

ILLUMINATOR ALIGNMENT AND BULB REPLACEMENT

Microscope illuminators consist of a lamp housing with a lamp and concave reflector, a
focusable collector lens, an electrical socket for holding the bulb, and an external power
supply. The socket and power cord, in particular, deserve attention. Oxidized metal sur-
faces of the socket electrodes and the copper conducting wire in an arc lamp should be
cleaned with an emery cloth each time the lamp is changed to assure good electrical
contact. The socket’s power cord should never be crimped (as occurs when the illumi-
nator is shoved against a wall) as this action loosens wires, which can lead to inconven-
ient and expensive repair. The bulb, rear reflector, and front collector lens should be kept
clean of all dirt, lint, and finger oils. At the time of changing a bulb and after the collec-
tor lens and metal housing have been removed, move the illuminator’s adjustment
screws with a screwdriver to observe their effect on the location of the bulb and the
reflector. Some arc lamp housings only contain adjustment screws for the rear reflector,
whereas others contain additional screws for adjusting the bulb itself. Arc lamp illumi-
nators should be maintained in an upright position during use to preserve the life of the
bulb. Never ignite an arc lamp when it is outside its protective metal housing!
After a bulb is changed and aligned, the image of the arc or filament should be
focused in the front aperture plane of the condenser using the illuminator’s collector
lens focusing dial, which is located on the illuminator housing. On some microscopes it
may be possible to place a lens tissue across the front of the condenser aperture or to
stop down the condenser diaphragm in order to see the image of the focused filament.
Alternatively, the focused image of the filament or arc may be viewed at its conjugate
location at the objective back aperture using an eyepiece telescope or Bertrand lens. In
this case, the light should be turned down to a minimum or attenuated with appropriate
neutral density filters. To see the image clearly, it may be necessary to remove a ground
glass diffusing screen, whose function is to remove the pattern of the filament from the
image of the specimen.
Alignment of a new bulb is especially critical for mercury or xenon arc lamps, such
as those mounted in epi-illuminators used for fluorescence microscopy, because the arc
in the lamp is small ( 1 mm) and the arc’s image at the condenser aperture must be