Operation Manual

44
4. MICROSCOPY
Epi-Fluorescence Micoscopy
4.1 Selecting fluorescence filters:
For best results, select excitation and emission filters with center wavelengths as close to the absorption
and emission peaks as possible.
Note: The center wavelength is situated at the midpoint of the bandwidth. It is not necessarily the peak
transmission wavelength although with a symmetrical band the center wavelength and the peak
wavelength are equal.
4.2 Selecting excitation filters (EX)
Excitation filters selectively pass the light within the range of wavelengths needed to cause the specimen
to fluoresce and filter out other light.
To maximize the brightness of the desired fluorescence (the signal) relative to brightness of the
background (the noise), one can choose excitation and emission filters with wide bandwidths.
However, this may result in unacceptable overlap of the emission signal with the excitation signal,
resulting in poor resolution and wide bandwidth which also leads to a high level of self-fluorescence and
severe fading.
To minimize spectral overlap, one can instead choose excitation and emission filters that are narrow in
bandwidth and are spectrally well separated to increase signal isolation. This will conversely yield a dark
image. Since little excitation light reaches the specimen, self-fluorescence and fading are minimal.
4.3 Selecting barrier filters (BA)
The barrier filter prevents the excitation light from reaching the observers eye. Its transmission should be
as low as possible in the range of light used for excitation and as high as possible within the spectral
range of the emission from the specimen.
Barrier filters may be Long pass (LP) or Band pass (BP).