Chapter 3
Individual Operations
121
Indiv
idua
l
Op
eratio
n
s
Barrier filter (BA filter)
Barrier filters allow only fluorescent light emitted by the specimen to pass, blocking excitation light. This allows the
fluorescent image to be viewed without excess illumination (dark background).
There are two types of barrier filters: LP filters that block all light below a certain wavelength but pass all light of longer
wavelengths, and BP filters that pass light of a certain waveband and block all other light. Use the filter type appropriate
for your intended purpose.
LP filter (long-pass filter)
LP filters block all light below a certain wavelength but pass all
light of longer wavelengths. The border wavelength is called the
cut-on wavelength.
(1) For specimens labeled with a fluorophore in which the
fluorescent waveband and excitation waveband (light that
the specimen absorbs in order to emit fluorescent light) are
very close, selecting a barrier filter with the shortest cut-on
wavelength permitted by the performance requirements will
result in most efficient fluorescent microscopy. If the cut-on
wavelength is long, excitation light and fluorescent light will
be entirely distinct, tending to darken the background of
fluorescent images. However, recent developments in filter
performance have resulted in increased use of filters of
short cut-on wavelengths.
(2) For multiple-labeled specimens, use an LP filter for
microscopy of fluorescent images of all fluorophores. Note
that a combination involving an ordinary dichroic mirror, an
excitation filter, and an LP-filter-type barrier filter will be
incapable of excited fluorophores that emit long-wavelength
fluorescent light – for example, TRITC in the case of FITC
and TRITC. This will result in very dark TRITC fluorescent
images. For such cases, Nikon recommends using
multiband filters.
Long-pass filter
(Both FITC and TRITC images are visible.)
BP filter (band pass filter)
Band pass filters pass only light of a certain wavelength range,
blocking all other light.
BP filters are used for microscopy of fluorescent images
involving a specific fluorophore in multiple-labeled specimens.
(For example, in a double-labeled specimen of FITC and TRITC,
the BA520-560 filter enables microscopy of just the FITC
fluorescent image.)
However, BP filters will not separate auto fluorescence, if any
(because the fluorescent image in the above combination is
green only). LP filters are better suited for making fine
separation of auto fluorescence based on slight color
differences.
Band pass filter
(Only the FITC fluorescent image is visible)
Waveband
emitted by
FITC
LP520
S
pectral
transmitt
ance
Wavelength
Waveband
emitted by
TRITC
Waveband
emitted by
FITC
BA520-560 (BP type)
S
pectral
transmitt
ance
Wavelength
Waveband
emitted by
TRITC
Summary of Contents for ECLIPSE Ni-E
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