Nikon eclipse lv150 Instruction Manual page 43

Selecting a barrier (BA) filter
The barrier filter transmits only the fluorescence emitted by the sample, blocking the excitation
light. This enables observation of fluorescence images having less unnecessary light (darker
background).
BA filters are available in two types: long-pass (LP) and band-pass (BP). The LP filter blocks all
the light of shorter wavelength than a given value. The BP filter transmits light in a given
wavelength range. Use the suitable types in accordance with your purposes.
• Long-pass (LP) filter
The LP filter blocks all the light of shorter wavelength
than a given value, called the cut-on wavelength.
1) Some sample may be stained with a fluorescent color
for which the fluorescence waveband and the
excitation waveband (the light that the sample absorbs
to emit fluorescence) are very close to each other.
Then, fluorescence microscopy generally will be more
efficient by selecting a filter for which the cut-on
wavelength is as short as feasible.
A longer cut-on wavelength tends to result in a more
complete separation between excitation light and
fluorescent light, rendering a darker background of the fluorescence image. With the recent
advancement in filter performance, however, shorter cut-on wavelengths are used more often
than before.
2) LP filters are used for samples stained in multiple colors where fluorescence images for all the
colors are desired.
However, the usual combination of a dichroic mirror, an excitation mirror, and a barrier filter of
LP filter type, may not be sufficient to excite a stain that emits fluorescence of longer
wavelength (for example, TRIC when the sample is stained with FITC and TRITC), making the
fluorescence image for TRITC very dark. In a case like this, a multi-band filter is recommended.
• Band-pass (BP) filter
The BP filter transmits light of a certain wavelength range.
This type of filter is used for samples stained in multiple
colors where fluorescence images due to a certain stain are
desired. (For example, in the case of a dual-stained sample,
say FITC and TRITC, and fluorescence images due only to
FITC are desired, then BA520-560 should be selected.)
If a BP filter is used, however, any self-fluorescence
cannot be discriminate (because the fluorescence image
will be green all over for the above combination).
The LP filter is more useful when you wish to discriminate
self-fluorescence by a subtle difference in hue.
III. Operation of Each Part
Fluorescence
waveband for FITC
LP520
Fluorescence
waveband
for TRITC
Wavelength
Both fluorescence images due to FITC
and TRITC are seen
Fluorescence
waveband for FITC
BA520-560
(BP type)
Fluorescence
waveband
for TRITC
Wavelength
Only fluorescence image due to
FITC is seen
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