These photographs show
the Desmid Micrasterias thomasiana that have been illuminated
with ultra violet radiation. The red colouration is due to the
algal cell absorbing the short wave radiation and re-emitting
at longer wavelengths which in this case is a vivid red colour.
This type of luminescence is called fluorescence. A high pressure
mercury lamp is used with various exciting and barrier filters
and when reflected light fluorescence is used a dichromatic mirror
reflects 90% of the shorter wavelength while allowing the longer
wavelength to pass unhindered. The light emitted by the arc lamp
might be 360nm which is invisible to human eyes. This short wavelength
excites some of the molecules causing them to vibrate rapidly
which enables them to jump up to a higher energy level. When
the light is eventually re-emitted it is of a longer wavelength.
A barrier filter is then put between the specimen plane and the
eye. The barrier filter stops any of the excitation energy from
passing through to the eye but allows the longer wavelengths
to pass. The image, which in this case was red, is seen against
a very dark background. Filters play an extremely important part
in fluorescence microscopy and choosing the correct filter can
mean the difference between good photographs and poor quality,
with backgrounds too light etc. The correct objectives are of
utmost importance to combat any flaring that might be experienced
with ordinary objectives. Special objectives with the correct
non fluorescing materials are to be preferred along with higher
than normal numerical apertures.
Mercury arc lamps can be extremely dangerous if used incorrectly
and should never be handled when hot or warm. The glass can explode
releasing mercury vapour into the room and also causing blast
injuries to the handler. Always refer to the maker's handbook
for instructions on how and when to change the lamp. |
