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Overview of Optical Filters
Introduction
Turner Designs fluorometers require an
excitation and an emission filter; the Model 10-AU also uses a reference
filter. Optical filters are chosen to be optimal for each application,
cost effective, and durable. Filters are used to selectively pass a
portion of the ultraviolet or visible spectrum.
In combination with a light source, the
excitation filter allows only light which excites the molecule of interest
to strike the sample. The emission filter allows the fluorescence from
the sample to pass to the detector and blocks stray light from the light
source or interfering components in the sample. The reference filter
is used in the reference path of the 10-AU series and is a factor in
determining the basic operating level of the instrument.
Filters can be used alone or in combination
to select the desired spectral band. Optical filters obey the Bouguer-Lambert
Law, which states that the spectral transmittance of two or more optical
filters used simultaneously is equal to the product of the spectral
transmittance of each filter.(1)
Filters with four types of spectral
characteristics are used in Turner Designs fluorometers: broadband,
narrowband, sharpcut, and neutral density.
- A broadband filter can pass
a broad band of light. For instance, a broadband filter may transmit
light from 300 - 400 nm, but block light with wavelengths shorter
than 300 and longer than 400.
- Narrowband filters pass a narrow
band of light (as little as 1 nm). For example, a 436 nm filter with
a bandpass of 10 nm, will pass light from 431 - 441 nm (5 nm on either
side of 436 nm).
- Sharpcut or edge filters can
be used to block light that is longer or shorter than a nominal wavelength.
A 450 nm long-wave filter will allow transmission of light that is
longer than 450 nm, but it will block light that is shorter than 450
nm. A 450 nm short-wave filter will transmit light that is shorter
than 450 nm and block light that is longer than 450 nm.
- A neutral density filter, primarily
used as a reference filter, can be used to decrease the transmitted
light across a very broad spectrum. For instance, a neutral density
filter can be used to decrease the total light transmission by a factor
of 10 or 100.
Three types of optical filters are used
in Turner Designs Fluorometers: 1. Optical Glass, 2. Interference, and
3. Gel Wratten.
1. Optical Glass Filters.
Optical glass filters are made from glass that absorbs specific wavelengths
of the spectrum. They are relatively inexpensive and are very durable
under most conditions. Both bandpass, sharpcut, and neutral density filters
are available in optical glass. However, the choice of filter glasses
is limited. The amount of transmission and band width is dependent on
the glass thickness. The following factors may affect optical glass:
- Thermal shock caused by a rapid temperature
change;
- Solarization caused by prolonged exposure
to ultraviolet light can cause an increase in absorption (decrease
in transmission);
- Exposure to high humidity or corrosive
environments can cause ‘spotting’ or ‘staining’,
which changes the surface, resulting in increased light scattering
off the surface and decreased transmission through the glass.(2)
However, we have found that glass filters
can be used for years or decades under most conditions.
2. Interference Filters.
In terms of spectral characteristics, interference filters can have
broad or narrow bandpasses, or can be sharpcut filters. Interference
filters used in Turner Designs fluorometers are primarily narrow bandpass.
Interference filters are made by coating optical glass with two thin
films of reflecting material separated by an even-order spacer layer.
The central wavelength and bandwidth of the filter can be controlled
by varying the thickness of the spacer layer and/or the number of reflecting
layers. To ensure out-of-band blocking (blocking undesirable wavelengths
of light) an additional blocking component is added. While the additional
blocking eliminates out-of-band light transmission and decreases background
noise, it also decreases the overall light transmission through the
filter which decreases the fluorescent signal. Interference filters
typically permit 10 to 70% light transmission. The minimum specified
transmission depends on the transmitted wavelength and bandwidth.
Interference filters are affected by
temperature. The center wavelength will shift linearly with, and in
the direction of, changes in temperature. For example, the temperature
coefficient for a 400 nm filter is about 0.015 nm/°C. The center wavelength
and maximum transmission of interference filters can drift with age,
especially under conditions of high humidity and variable temperatures.3
Good quality filters are hermetically-sealed to mitigate the affects
of aging. Hermetically sealed filters are guaranteed for one year; we
have found that under good ambient conditions, such as in a laboratory,
the filters show minimum signs of aging after two years or more.
A new interference filter usually has
a uniformly dark side and a uniformly reflective or mirrored side. To
protect the filter from heat and light, the reflective side should always
face the light source. A filter that is affected by age and humidity
will show discoloration around the outside diameter, this discoloration
will move toward the center of the filter with time and additional damage.
A symptom of aging is a significantly decreased maximum transmission
which results in less sensitivity for a fluorescent assay. The recommended
operating conditions for interference filters is -40 degrees C to +70
degrees C, and a maximum temperature change of 5 degrees C/minute.(3)
3. Gel Wratten Filters.
Gel Wratten filters can have broad or narrow bandpasses or can be sharpcut
filters. Gelatin filters are made by dissolving specific organic dyes
into liquid gelatin. The gelatin is coated onto prepared glass and when
it is dry, it is stripped off the glass and coated with lacquer. Each
filter is standardized for spectral transmittance and total transmittance.
At Turner Designs, the gelatin filter is placed between two pieces of
glass or in combination with other filters for use in the fluorometer.
Like dyes in other applications, the spectral characteristics of the
dyes used in filters may change depending on the dye used, age, and
exposure to heat and light. Gelatin filters should be kept cool, dry
and should not be subjected to temperatures higher than 50 degrees C.(1)
Most of the gelatin filters used by Turner Designs have been found to
be stable under test conditions, which include up to two weeks of continuous
exposure to several light sources.
References
- Kodak Filters for Scientific and Technical
Uses, Eastman Kodak Company, 3 ed. 1981.
- Schott Color Filter Glass, Schott
Optical Glass Inc., 1976
- Andover Corporation Optical Filter
Guide, Andover Corporation.
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