Selecting Filters - Nikon Eclipse Ni-E Instruction Manual

Selecting Filters

17.2
A filter cube consists of three types of optical components: an
excitation filter (EX filter), a barrier filter (BA filter), and a dichroic
mirror (DM). Select the filter cube with the desired combination
of optical components to suit the characteristics of the sample
and the fluorophore by referencing the properties of each filter,
and attach the filter cube to the epi-fluorescence cube turret.
You can select a combination of an excitation filter and a barrier
filter even if you are using the same excitation method.
Excitation filters, barrier filters, and dichroic mirrors can be
purchased separately.
Since excitation filters are exposed to strong light during
operations, they will deteriorate over time. Replace the filter at
intervals determined by usage.
Excitation filter (EX filter)
Excitation filters allow selective transmission of light (excitation
light) in the wavelength range required for fluorescent light
emissions from the sample, blocking light of all other
wavelengths. The range of wavelengths allowed to pass through
a filter is referred to as the bandwidth.
The bandwidth range of an excitation filter determines the
brightness of the fluorescent image, the generation of
autofluorescence (fluorescence resulting from substances other
than the fluorophores), and degree of fading. The broader the
bandwidth, the greater the amount of excitation light irradiated
onto the sample, thereby increasing the brightness. However,
this also increases the amount of autofluorescence and causes
faster color fading. Narrow bandwidth reduces the amount of
excitation light striking the sample and causes the image to
appear darker, but reduces autofluorescence and fading. For
samples with pronounced autofluorescence, use excitation
filters with a narrow bandwidth. (Note that this will make the
fluorescent image darker.)
Since excitation filters are exposed to strong light during
operations, they will deteriorate over time. Replace the filter at
intervals determined by usage.
Brightness of
fluorescent image
Generation of
autofluorescence
Degree of color fading
Chapter 3
Individual Operations
EX Filter Bandwidth and Fluorescent Image
Narrow EX filter bandwidth
Dark
Low
Low
71
U V - 2 A
E X 33
0- 38 0
D M 40
0
B A 42
0
Excitation filter
Dichroic mirror
(inside the cube)
Filter cube
EX filter
Bandwidth
0
Wavelength
EX filter bandwidth
Barrier filter
Wide
Bright
High
High