Semrock Master Catalog 2018

BrightLine ® FRET Single-band Sets
TECHNICAL NOTE
Optical Filter Configurations for FRET
The classical approach to FRET measurements involves changing filter cubes (see Figure (a) and page 22 for filters and more
information on FRET). For example, in the acceptor-photobleaching method, a donor-specific cube is first used to collect the
emission from the donor (e.g., CFP). Then a filter cube for the acceptor is used to visualize and photobleach the acceptor (e.g.,
YFP). Intensity measurements of the donor before and then again after photobleaching the acceptor are used to calculate
FRET efficiency. Steep spectral edges of the filters ensure that only the acceptor is photobleached and minimize the signal
contamination due to bleedthrough in multiply labeled FRET samples. This technique suffers from several drawbacks, including:
slow speed (changing filter cubes takes typically a second or more) and imaging artifacts (due to the movement of the filter
turret and other vibrations).
The most popular approach to FRET imaging, shown in Figure (b), is often called the FRET-cube method. A single-band exciter
and a single-edge dichroic beamsplitter, each specific to the donor, are placed in a cube in the microscope turret, and a filter
wheel with single-band emission filters is used to select the emission from either the donor or the acceptor. Filter wheels cause
minimal vibrations and have much faster switching times (as low as tens of ms) compared to filter turrets, and are therefore
better suited for live-cell FRET applications.
Fluorophores
Single-band
Sets
Multiband
Sets
(a) (b) (c)
Many researchers prefer to utilize a Sedat filter set
configuration (see page 33 for filters). This approach
provides additional flexibility in the visualization of the
sample as well as to perform control experiments – such as
finding regions or samples labeled with only the donor or
acceptor and collection of pure spectral contributions from
each. The added flexibility also enables the donor
photobleaching method for calculation of FRET efficiency.
Cubes
However, the most demanding FRET applications, such as
live-cell imaging and imaging of single molecules may require
“simultaneous” imaging of the emission signal from both the donor and the acceptor. Figure (c) shows a configuration for
simultaneous imaging, in which an image-splitting dichroic beamsplitter (see page 64) placed in the emission channel of the
microscope is used to separate the signals from the donor and the acceptor and steer them onto two different CCDs or two
distinct regions of the same CCD. Since there are no moving parts, motion-based imaging artifacts are also eliminated.
Laser
Sets
TECHNICAL NOTE
Individual
Filters
NLO
Filters
Leading Competitor’s Quad-band “Pinkel” Filter Set
Semrock BrightLine Quad-band “Pinkel” Filter Set
Multicolor Fluorescence: Four Times Brighter and Twice the Contrast
Comparison images of Rat Mesangial Cells: labeled with Hoechst, Alexa 488, MitoTracker Red and Cy5 using the Semrock
Quad-band DA/FI/TR/Cy5-4X-B “Pinkel” filter set on an Olympus BX61WI-DSU Spinning Disk Confocal Microscope.
Images courtesy of Mike Davidson – Molecular Expressions.
DA/FI/TR/Cy5-4X-A
Dichroic
Beamsplitters
Transmission (%)
DA/FI/TR/Cy5-4X-B “Pinkel” Set Spectra
This four color, quad-band, filter set is designed for high speed, sequential imaging of DAPI, FITC,
TRITC, and Cy5. The complete, 6-filter set is comprised of 1 quad-band dichroic beamsplitter, 1 quadband
emitter, and 4 single-band exciters. All six filters are ‘no burn out’ hard-coated in design that will
provide consistent, high performance. This set is also available in a ‘Sedat’ version, that replaces the
single quad-band emitter with four single-band emitters. Both our Sedat and Pinkel sets are designed
for the single-band filters to be installed in filter wheels.
Tunable
Filters
100
90
80
70
60
50
40
30
20
10
0
350 400 450 500 550 600 650 700 750
Wavelength (nm)
800
23
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