Semrock Master Catalog 2018

VersaChrome ® Tunable Filters
TECHNICAL NOTE
Spectral Imaging with VersaChrome ® Filters
Conventional spectral imaging systems are generally not able to offer the key advantages of thin-film interference filters,
i.e., high transmission combined with steep spectral edges and high out-of-band blocking. Now with VersaChrome filters,
these advantages can be realized in simple spectral imaging systems for applications ranging from fluorescence microscopy
to hyperspectral imaging.
To demonstrate spectral imaging in a fluorescence microscope, a “lambda stack” of images (corresponding to a nearly
continuous series of emission wavelengths) was acquired of a sample labeled with three spectrally overlapping fluorophores
using a Semrock VersaChrome tunable filter (TBP01-617/14) placed in the emission channel of a standard upright
microscope. Figure 1 shows six of the 61 images taken at 1 nm intervals, and Figure 2 shows measured intensity spectra
taken from parts of the image where only a single fluorophore is present. The nucleus labeled with SYTOX ®
Orange can
be easily discriminated from the other cellular structures (Fig. 1). However, since the F-actin and mitochondria are labeled
with fluorophores that are highly overlapping (Alexa Fluor 568 and MitoTracker ®
Red, respectively), linear unmixing is
necessary to discern the corresponding cellular constituents. Images deconvolved with linear unmixing are shown in Figure 3.
It is important to note that the spectral properties of these tunable filters are almost identical for both s- and p-polarizations
of light – a feature that cannot be easily obtained using liquid-crystal and acousto-optic tunable filters. Polarization
independence is highly desirable for spectral imaging systems, and yet polarization limitations of current tunable filters
account for a loss of at least half of the signal in most instruments. Therefore VersaChrome filters not only enhance the
throughput in spectral imaging but they also greatly simplify the complexity of instrumentation.
1.0
0.9
0.8
0.7
0.6
620 nm 610 nm 600 nm
0.5
Nucleus: SYTOX Orange F-Actin: Alexa Fluor 568
0.4
SYTOX Orange
0.3
Alexa Fluor 568
MitoTracker-Red
0.2
Background
0.1
0.0
590 nm 580 nm 570 nm
570 580 590 600 610 620
Wavelength (nm)
Mitochondria: MitoTracker Red Composite image
Figure 1 Figure 2 Figure 3
Normalized Intensity
Fluorophores
Single-band
Sets
Multiband
Sets
Cubes
Laser
Sets
MyLight TM
Interested in seeing how a Semrock standard
filter behaves at a particular angle of
incidence, state of polarization
or cone half angle of illumination?
Simply click the
Click for MyLight Tool
button located above the spectral graph
and the MyLight window will access our
theoretical design data and allow you to see
spectral shifts in filter performance under
varying illumination conditions. You can also
expand (or contract) the displayed spectral
range and assess filter performance in real
time that previously required you to contact
us and iterate towards an answer. MyLight
data can be downloaded as an ASCII file and
the graphs printed or saved as PDFs.
NLO
Filters
Dichroic
Beamsplitters
Tunable
Filters
Individual
Filters
79
More