Nikon Eclipse LV100DA-U Instructions Manual page 71

Selecting the barrier filter (BA filter)
A barrier filter transmits only fluorescent lights emitted by the specimen but blocks the excitation
lights. This filter makes it possible to observe the fluorescent image without unnecessary light (that
is, on a dark background).
There are two types of barrier filters: LP filters (long-pass filters), which block all lights that are
shorter than a certain boundary wavelength and allow all lights to pass that are longer than the
boundary wavelength, and BP filters (band-pass filters), which allow only lights in a certain
bandwidth to pass. Please use a proper filter depending on the purpose.
• LP filters (long-pass filters)
A long pass filter transmits lights that have longer
wavelength than a certain wavelength but blocks lights
that have shorter wavelength. The boundary wavelength is
called the cut-on wavelength.
1) An excitation light is a light that is irradiated to the
specimen. The fluorophore in the specimen absorbs the
excitation light energy. As a result, fluorescent lights
are emitted from the fluorophore instead. When a
specimen is labeled with a fluorophore that emits
fluorescent lights of very close wavelengths to the
excitation light, select a barrier filter with the shortest
cut-on wavelength permitted by performance requirements for efficient fluorescent microscopy.
A longer cut-on wavelength tends to result in a more complete separation between an excitation
light and fluorescent lights, rendering a darker background of the fluorescent image. With the
recent advancement in filter performance, however, filters with shorter cut-on wavelengths can
be used for this purpose and they are used more often than before.
2) An LP filter is used for a specimen labeled with multiple wavelengths where fluorescent images
for all the wavelengths are desired.
However, the usual combination of a dichroic mirror, an excitation light filter, and a barrier
filter of LP filter type, may not be sufficient to excite a fluorophore that emits fluorescent lights
of longer wavelength (for example, the TRIC when the specimen is labeled with the FITC and
the TRITC), making the fluorescent image for the TRITC very dark. In a case like this, a multi-
band filter is recommended.
• BP filters (band-pass filters)
A BP filter transmits lights of a certain bandwidth.
This type of filter is used to observe a fluorescent image only
emitted by a certain fluorophore when the specimen is labeled
with multiple fluorophore. (For example, when a specimen is
labeled with the FITC and the TRITC and you wish to
observe a fluorescent image only emitted by the FITC, use a
filter of BA520-560.)
However, you cannot distinguish the autofluorescence from
the other fluorescences in the image transmitted through the
BP filter because the image will only be of one color (green,
in the above example).
When you wish to distinguish the autofluorescence by a
subtle difference of hue, an LP filter is more useful.
III. Operation of Each Part
Fluorescent
bandwidth of the FITC
LP520
Fluorescent
bandwidth
of the TRITC
Wavelength
Both the FITC fluorescent image and
the TRITC fluorescent image are visible.
Fluorescent
bandwidth of the FITC
BA520-560
(BP type)
Fluorescent
bandwidth
of the TRITC
Wavelength
Only the fluorescent image
emitted by the FITC is visible.
69