Semrock Master Catalog 2018

Fluorophores
Single-band
Sets
Multiband
Sets
Cubes Laser
Sets
NLO
Filters
Individual
Filters
Dichroic
Beamsplitters
Tunable
Filters
Introduction to Fluorescence Filters
Optical fluorescence occurs when a molecule absorbs light at wavelengths within its absorption band, and
then nearly instantaneously emits light at longer wavelengths within its emission band. For analytical purposes,
strongly fluorescing molecules known as fluorophores are specifically attached to biological molecules and
other targets of interest to enable identification, quantification, and even real-time observation of biological and
chemical activity. Fluorescence is widely used in biotechnology and analytical applications due to its extraordinary
sensitivity, high specificity, and simplicity.
Most fluorescence instruments, including fluorescence microscopes, are based on optical filters.
A typical system has three basic filters: an excitation filter (or exciter), a dichroic beamsplitter (or
dichromatic mirror), and an emission filter (or barrier filter). The exciter is typically a bandpass
filter that passes only the wavelengths absorbed by the fluorophore, thus minimizing excitation of
other sources of fluorescence and blocking excitation light in the fluorescence emission band. The
dichroic is an edge filter used at an oblique angle of incidence (typically 45°) to efficiently reflect light
Filter Transmission
TECHNICAL NOTE
excitation
filter
sample
absorption
Wavelength of Light
dichroic
beamsplitter
emission
filter
sample
fluorescence
in the excitation band and to transmit light in the emission band. The
emitter is typically a bandpass filter that passes only the wavelengths emitted by the
fluorophore and blocks all undesired light outside this band – especially the excitation
light. By blocking unwanted excitation energy (including UV and IR) or sample and system
autofluorescence, optical filters ensure the darkest background.
An appropriate combination of optical filters, making up a filter set, enables the
visualization of a given fluorophore. See pages 10-13 for a listing of popular fluorophores
and corresponding filter sets that can be used to image these fluorophores. A filter set
needs to be optimized not only for imaging of distinct fluorophores but also designed to
image a given fluorophore under different experimental conditions.
Most of Semrock filter sets are a balance between high-brightness and
high-contrast. These filter sets are the best choice of filters under standard
imaging conditions. However, when the signal level from a sample is low,
sets with wider passbands of the excitation and emission filters enable
maximum signal collection efficiency. Studies such as imaging of single
molecules typically utilize a filter set with a wide passband or a long pass
emission filter. In studies utilizing such filter sets, it is required to maintain
very low background autofluorescence signal by means of appropriate
sample preparation protocols. However, since the wide passbands of such
filter sets occupy a large spectral bandwidth, such filters are not preferred
in multiplexing assays when imaging of several fluorophores is required.
Filter sets with narrower passbands are preferred options when imaging a
sample labeled with multiple fluorophores. Such filter sets reduce crosstalk
between multiple fluorophores. Narrower passbands allow only the
strongest portion of the fluorophore emission spectrum to be transmitted,
reduce autofluorescence noise and thus improve the signal-to-noise ratio
in high background autofluorescence samples. Such filter sets are ideal for
samples with ample fluorescent signal level.
In most fluorescence instruments, the best performance is obtained with
thin-film interference filters, which are comprised of multiple alternating thin
layers of transparent materials with different indices of refraction on a glass
substrate. The complex layer structure determines the spectrum of light
transmission by a filter. Thin-film filters are simple to use, inexpensive, and
provide excellent optical performance: high transmission over an arbitrarily
determined bandwidth, steep edges, and high blocking of undesired light
over the widest possible wavelength range.
FITC-5050A “High Brightness” Filter Set
100
90
80
70
145%
60
50
100%
71%
40
30
Brightness Contrast
20
10
0
400 450 500 550 600 650
Wavelength (nm)
Advances in thin-film filter technology pioneered by Semrock, and embodied in all BrightLine filters, permit even higher
performance while resolving the longevity and handling issues that can plague filters made with older soft-coating
technology. This advanced technology is so flexible that users have a choice between the highest-performance BrightLine
filter sets and the best-value BrightLine Basic
filter sets.
Transmission (%)
Transmission (%)
100
90
80
70
60
50
40
30
20
10
FITC-2024B “High Contrast” Filter Set
100%
0
400 450 500 550 600
Wavelength (nm)
57%
Brightness Contrast
650
140%
More
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