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Here we show how filter selection
for epi-fluorescence microscopes can extend their capabilities.
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| 1. What are epi-fluorescence
microscopes? |
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Fluorophores
The phenomenon of fluorescence occurs when a specific wavelength
of light (excitation light) is absorbed by a substance, which
gives off light when it releases energy as it returns to its
ground state from the excited state. Many fluorescent substances
(called fluorophores) have been developed for different uses,
with excitation wavelengths ranging from the ultraviolet to
the infrared. However, emission wavelengths are usually longer
than the wavelength of the excitation energy which causes
them.
Epi-fluorescence microscopy
 Epi-fluorescence microscopy refers to
microscopes that are designed to observe the aforementioned
fluorophores. Optical systems contain a light source for excitation
(usually mercury lamps) and a filter block to separate the
excitation light from the fluorescence. There are many filter
blocks available for different kinds of fluorophores, which
may be switched any time for continuous viewing of specimens
stained with multiple kinds of fluorophores
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| 2. The role of filters in
epi-fluorescence microscopy |
 As shown in Figure 1, filter blocks are
constructed from 2 types of filters and 1 dichroic mirror.
Selecting appropriate filters and mirrors for each use allows
researchers to attain a high signal to noise (S/N) ratio
between the fluorescence and background light. The functions
of these optical elements are described below.
Excitation filter (EX)
The excitation filter is the optical element that passes
only the wavelength of light necessary for excitation from
the excitation light source (usually a mercury lamp) to
the fluorophore. As shown in Figure 2, only the excitation
wavelength passes through the filter, usually a "band pass
filter".
Dichroic mirror (DM)
 The dichroic mirror is the optical element
that separates the excitation light from the fluorescence.
Dichroic mirrors are special mirrors that reflect only a
specific wavelength of light, allowing all other wavelengths
to pass through (Figure 3). Dichroic mirrors used in epi-fluorescence
microscope filter blocks are placed in a 45?incidence angle
to light, creating a "stop band"of reflected light and a
"pass band"of transmitted light. Light passing through the
excitation filter is reflected 90?toward the objective and
the specimen. Finally, light emanating from the specimen
is passed through and directed toward the observer (or high-sensitivity
camera).
Barrier filters (BA) or emission (EM) filters
 Barrier filters are optical elements
that separate fluorescence emanating from the fluorophore
from other background light. As shown in Figure 4, the barrier
filter transmits light of the fluorescence wavelength which
passes through the dichroic mirror while blocking all other
light leaking from the excitation lamp (reflected from the
specimen or optical elements). This is necessary because
the strength of the fluorescent light is weaker than the
excitation light by a factor of more than 100,000:1. Most
barrier filters are positioned at a slight angle to allow
better fluorescent imaging by suppressing ghost images.
Nikon's noise terminator
 Nikon's epi-fluorescence microscopes have
a proprietary Nikon optical element called a “noise terminator?(on
models TE2000 and later). As shown in Figure 1, the noise
terminator allows efficient processing of light transmitted
through the dichroic mirror by preventing the excitation
light from scattering within the filter block and leaking
into the observed image. This helps to eliminate background
light noise and more effective fluorescent images.
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| 3.
Selecting fluorescent filters and mirrors |
The following steps should be followed when selecting fluorescent
filters and mirrors
Fluorophore (fluorescent substance) wavelength
Ascertain the wavelength of the fluorophore in use. This information
is noted in catalogs, but the wavelength properties of fluorophores
also change slightly depending on solution conditions such
as salinity, pH and intracellular conditions. Therefore, we
recommend using a fluorometer to measure both excitation and
fluorescence wavelengths. Then, use this information to select
the most appropriate optical elements.
Dichroic mirror selection
 The selected dichroic mirror must effectively
separate the wavelengths of the excitation light and the fluorescent
light. Specifically, a dichroic mirror whose transmittance
cutoff lies between the fluophoreÅfs excitation wavelength
and its fluorescence wavelength must be found by comparing
the mirrorÅfs characteristic curve with the fluorophoreÅfs wavelength
when the mirror is placed on a 45? incidence angle.
If the fluophore's excitation wavelength and its fluorescence
wavelength are close and the Stoke's shift (Note 1) is small,
then a mirror with a smaller cutoff wavelength should be chosen
to allow as much fluorescence signal to pass as possible.
Barrier filter selection
Select a filter that allows fluorescence wavelengths from
the specimen to pass. Usually, long pass filters that transmit
long wavelengths are chosen over band pass filters. However,
a band pass filter that does not transmit these longer wavelengths
is often used as a barrier filter when separating wavelengths
from a multiple-stained specimen or when using a camera sensitive
to longer wavelengths.
Excitation filter selection
 Select a filter that allows excitation
wavelengths to pass smoothly. Especially when a mercury lamp
is used as the light source, efficient excitation is achieved
by incorporating the line spectrum of the mercury lamp into
the wavelengths. Allowing wavelengths other than the excitation
wavelength of the target fluorophore to pass will both increase
background light and damage the specimen. As shown in Figure
7, excitation efficiency is low for a wavelength of 440 nm.
Combinations of barrier filters
and excitation filters
The ideal combination of barrier filters and
excitation filters is one that lets no light
pass when combined. The fluorescence emitted
is very weak, so any light that leaks through
the filters will reduce image quality.
Obtaining bright fluorescence
images
Sometimes it is recommended to remove the
ND filter in the excitation optics and increase
the strength of the excitation light source.
However, increasing the strength of the excitation
light source will bleach the fluorophore quickly
and damage the specimen, as well as increasing
autofluorescence (Note 2) in the cell. For
these reasons, the light source should be
kept as weak as possible and the observation
optics made as efficient as possible in picking
up the fluorescence signal (by widening the
objective aperture, increasing the wavelength
band of the fluorescence filter, using a more
sensitive camera, etc.). |
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Note 1: Stoke's shift refers to the energy difference between
excitation energy and fluorescent energy arising from partial
loss of energy as heat as electrons fall back from the excited
state to their base state in fluorescing materials. (From
Iwanami Shoten's Physical and Chemical Dictionary)
Note 2: Autofluorescence refers to the presence of fluorescing
substances in substances other than those generally defined
as fluorescent. For example, cell components such as NAADPH
or riboflavin give off relatively strong fluorescence in the
short wavelength range (ultraviolet to visible) in unstained
cells.
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| 4.
Reducing bleaching |
You can take the following steps to reduce bleaching.
Use an anti-bleaching agent
Anti-bleaching agents such as P-phenylenediamine can be used
for FITC, and n-propyl gallete can be used for rhodamine.
Use ND filters
Use neutral density or ND filters on the microscope
to reduce light levels to the minimum needed for observation.
At times this may require setting the filter to 100%, while
taking steps not to split the light path as much as possible.
Use a bleaching-resistant fluorophore
Use the most bleach-resistant fluorophore available for each
testing purpose. |
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