Chapter 2
Individual Operations
53
Chapter 2
Individual
Operations
EX Filter Bandwidth and Fluorescent Image
Narrow
EX filter bandwidth
Wide
Brightness of
fluorescent image
Dark
Bright
Generation of
autofluorescence
Low
High
Degree of color fading
Low
High
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, we recommend using multiband
filters.
Long-pass filter
(Both FITC and TRITC images are visible.)
BP filter (bandpass filter)
Bandpass 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 autofluorescence, if any
(because the fluorescent image in the above combination is
green only). LP filters are better suited for making fine separation
of autofluorescence based on slight color differences.
Band-pass filter
(Only the FITC fluorescent image is visible.)
Waveband
emitted by
FITC
LP520
S
pectral
transmission
Wavelength
Waveband
emitted by
TRITC
Waveband
emitted by
FITC
BA520-560 (BP type)
S
pectral
transmission
Wavelength
Waveband
emitted by
TRITC