Fluorescence Microscopy, reflected light gives much better results, so almost all
fluorescence microscope take this configuration.
The configuration of a reflected light fluorescence
(also known as epi-fluorescence) microscope is schematically shown on the left:
The light from source (1) pass through neutral density filter, aperture, field stop
(2,3,4) for Köhler illumination adjustment, reaches excitation
filter(5). After filtering away unwanted wavelength, excitation light goes further and reaches a special and important filter
called beam splitter (6), BSP for short. The excitation light selected by BSP is
the specimen via objective (7) and excites fluorophores within specimen (8). The
fluorescence emission from the specimen comes back through the same path to BSP
again. This time, the light should not be reflected away but passes through
it and reaches emission filter(9).
filtered by emission filter, the emission light is either focused on the front focal plane of
binocular or projected to infinite by tube lens in case of infinite
corrected objectives(10). The final image is further magnified by
binocular(11), detected by your retinal, or sent to a camera port, detected
by devices coupled above the port: CCD camera or
a photographic film camera.
Filters 5, 6, 9 are usually installed together into a cube, called
filter-set. It is the pivotal part of fluorescence microscope and will be
discussed more in next section: filter-set.
In a conventional reflected fluorescence light microscope, the light is usually from mercury arc lamp which provides a mixture of wave length
from UV to red, like the figure on the left depicted. A emission filter is used
to separate desired wavelength. Contrary to common
assumption, the peaks distributed mostly in UV and violet-blue area. For
the frequently used fluorophore FITC, which need excitation at 490 nm, there
is no beak but only
continuous wave at less than 10% intensity. But this seems to be enough for FITC due to its high
molar extinct coefficient and high quantum yielding.