Semrock Master Catalog 2018
Semrock Master Catalog 2018
Semrock Master Catalog 2018
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Fluorophores<br />
Single-band<br />
Sets<br />
Multiband<br />
Sets<br />
Cubes Laser<br />
Sets<br />
NLO<br />
Filters<br />
Individual<br />
Filters<br />
Dichroic<br />
Beamsplitters<br />
Tunable<br />
Filters<br />
Introduction to Fluorescence Filters<br />
Optical fluorescence occurs when a molecule absorbs light at wavelengths within its absorption band, and<br />
then nearly instantaneously emits light at longer wavelengths within its emission band. For analytical purposes,<br />
strongly fluorescing molecules known as fluorophores are specifically attached to biological molecules and<br />
other targets of interest to enable identification, quantification, and even real-time observation of biological and<br />
chemical activity. Fluorescence is widely used in biotechnology and analytical applications due to its extraordinary<br />
sensitivity, high specificity, and simplicity.<br />
Most fluorescence instruments, including fluorescence microscopes, are based on optical filters.<br />
A typical system has three basic filters: an excitation filter (or exciter), a dichroic beamsplitter (or<br />
dichromatic mirror), and an emission filter (or barrier filter). The exciter is typically a bandpass<br />
filter that passes only the wavelengths absorbed by the fluorophore, thus minimizing excitation of<br />
other sources of fluorescence and blocking excitation light in the fluorescence emission band. The<br />
dichroic is an edge filter used at an oblique angle of incidence (typically 45°) to efficiently reflect light<br />
Filter Transmission<br />
TECHNICAL NOTE<br />
excitation<br />
filter<br />
sample<br />
absorption<br />
Wavelength of Light<br />
dichroic<br />
beamsplitter<br />
emission<br />
filter<br />
sample<br />
fluorescence<br />
in the excitation band and to transmit light in the emission band. The<br />
emitter is typically a bandpass filter that passes only the wavelengths emitted by the<br />
fluorophore and blocks all undesired light outside this band – especially the excitation<br />
light. By blocking unwanted excitation energy (including UV and IR) or sample and system<br />
autofluorescence, optical filters ensure the darkest background.<br />
An appropriate combination of optical filters, making up a filter set, enables the<br />
visualization of a given fluorophore. See pages 10-13 for a listing of popular fluorophores<br />
and corresponding filter sets that can be used to image these fluorophores. A filter set<br />
needs to be optimized not only for imaging of distinct fluorophores but also designed to<br />
image a given fluorophore under different experimental conditions.<br />
Most of <strong>Semrock</strong> filter sets are a balance between high-brightness and<br />
high-contrast. These filter sets are the best choice of filters under standard<br />
imaging conditions. However, when the signal level from a sample is low,<br />
sets with wider passbands of the excitation and emission filters enable<br />
maximum signal collection efficiency. Studies such as imaging of single<br />
molecules typically utilize a filter set with a wide passband or a long pass<br />
emission filter. In studies utilizing such filter sets, it is required to maintain<br />
very low background autofluorescence signal by means of appropriate<br />
sample preparation protocols. However, since the wide passbands of such<br />
filter sets occupy a large spectral bandwidth, such filters are not preferred<br />
in multiplexing assays when imaging of several fluorophores is required.<br />
Filter sets with narrower passbands are preferred options when imaging a<br />
sample labeled with multiple fluorophores. Such filter sets reduce crosstalk<br />
between multiple fluorophores. Narrower passbands allow only the<br />
strongest portion of the fluorophore emission spectrum to be transmitted,<br />
reduce autofluorescence noise and thus improve the signal-to-noise ratio<br />
in high background autofluorescence samples. Such filter sets are ideal for<br />
samples with ample fluorescent signal level.<br />
In most fluorescence instruments, the best performance is obtained with<br />
thin-film interference filters, which are comprised of multiple alternating thin<br />
layers of transparent materials with different indices of refraction on a glass<br />
substrate. The complex layer structure determines the spectrum of light<br />
transmission by a filter. Thin-film filters are simple to use, inexpensive, and<br />
provide excellent optical performance: high transmission over an arbitrarily<br />
determined bandwidth, steep edges, and high blocking of undesired light<br />
over the widest possible wavelength range.<br />
FITC-5050A “High Brightness” Filter Set<br />
100<br />
90<br />
80<br />
70<br />
145%<br />
60<br />
50<br />
100%<br />
71%<br />
40<br />
30<br />
Brightness Contrast<br />
20<br />
10<br />
0<br />
400 450 500 550 600 650<br />
Wavelength (nm)<br />
Advances in thin-film filter technology pioneered by <strong>Semrock</strong>, and embodied in all BrightLine filters, permit even higher<br />
performance while resolving the longevity and handling issues that can plague filters made with older soft-coating<br />
technology. This advanced technology is so flexible that users have a choice between the highest-performance BrightLine<br />
filter sets and the best-value BrightLine Basic <br />
filter sets.<br />
Transmission (%)<br />
Transmission (%)<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
FITC-2024B “High Contrast” Filter Set<br />
100%<br />
0<br />
400 450 500 550 600<br />
Wavelength (nm)<br />
57%<br />
Brightness Contrast<br />
650<br />
140%<br />
More<br />
14