Semrock Master Catalog 2018

Fluorophores
Single-band
Sets
Multiband
Sets
Cubes Laser
Sets
BrightLine ® Dichroic Super-resolution / TIRF Laser
Dichroic Beamsplitters
TECHNICAL NOTE
incident
wavefront
reflected
wavefront
Choosing Dichroic Beamsplitters with Flatness/
RWE Appropriate to the Microscopy Method
Wavefront distortion can degrade image quality by reducing contrast or
compromising resolution. In several microscopy applications, reducing
wavefront distortion is critical to achieving the microscopy method. Specifying
and selecting optical filters that minimize wavefront aberration is important to
maximize or enable optical system performance. This article elucidates how to
select optical filters for high performance microscopy, and provides guidance on
choosing Semrock catalog filters for wavefront distortion performance required
for applications.
reflected flatness
wavefront
Both standard and advanced microscopy methods require error certain minimum
standards of flatness on dichroic beamsplitters. For example, super-resolution
and TIRF microscopy cannot be achieved if the flatness of the critical dichroic
beamsplitters is worse than required. This Tech Note introduces the topic of how to
choose Semrock dichroic beamsplitters appropriate to the microscopy method.
incident
wavefront
reflected
wavefront
flatness
reflected
wavefront
error
Figure [1]: For light incident at an angle upon a dichroic
beamsplitter, deviations from flatness cause distortions in
the reflected wavefront.
Optical filters are generally composed of multi-layered, thin-film coatings on plane, parallel glass substrates. There is variability in substrate
flatness, and, additionally, the substrate may slightly bend after coating. For transmitted light, such bending has little effect on transmitted
wavefront Total error TWE (TWE) other than a = slight displacement Tiltof the beam axis. However, + for reflected Spherical light, especially light + incident at Irregularity
nonperpendicular
angles, deviations from flatness TWE (see Contribution
Figure 1) have two effects on reflected TWE wavefront Contribution error (RWE): (1) the focal TWE plane Contribution
may
shift position, or (2) the beam may acquire optical aberrations (e.g., astigmatism).
2 μm
Figure [2]: Image sequence demonstrating the effect of substrate bending on image
quality. A lower radius of curvature means greater substrate bending. As the radius
of curvature decreases (and bending increases), image quality degrades. [Images of
F-actin in bovine pulmonary artery endothelial cells (FluoCells® Prepared Slide #1,
ThermoFisher Scientific, Waltham, MA, USA) as imaged on a BX41 microscope (Olympus
Corporation of the Americas, Center Valley, PA, USA) with a 40×, 0.75NA objective and
Retiga camera (QImaging, Surrey, BC, Canada)]
NLO
Filters
Individual
Filters
Dichroic
Beamsplitters
incident
transmitted
incident transmitted
wavefront
wavefront
wavefront wavefront
Radius of Curvature (m) ~6 ~1275
Microscopy Technique
incident
wavefront
Flatness Need in a Reflected
Excitation Beam
transmitted
wavefront
Widefield Fluorescence Microscopy Non-critical Critical
Total Internal Reflection Fluorescence (TIRF) Microscopy Critical Critical
incident
wavefront
A focal plane shift can be corrected by lens or camera adjustment in some microscopy methods, but in others this shift cannot be
adjusted for and can therefore impede proper function. Also of significant concern, optical aberrations can degrade image quality, as
illustrated in Figure [2], and can compromise the microscopy method. In determining suitable flatness need for a given application, the
most important parameter is often the diameter of the beam striking the dichroic beamsplitter surface. Table 2 shows the Semrock
product best suited to the application, for maximum diameter values. For more specific information, and for other beam diameter
value and microscopy examples, the Semrock White Paper on this topic [1] provides additional information on RWE, TWE, and
microscopy methods, as well as guidance from a system designer’s perspective.
Because of potential degradation of imaging quality, it is important to determine when the flatness of dichroic beamsplitter is
critical to a microscopy method. Table 1 lists examples of popular microscopy techniques in which it is critical to have dichroics of
high flatness. As noted in this table, high flatness requirements can apply to both the illumination (excitation) and the detection
(emission) light paths.
Flatness Need in a Reflected
Emission Beam
transmitted
wavefront
Stochastic Switching (PALM, STORM, etc.) Critical Critical
Tunable
Filters
Stimulated Emission Depletion (STED) – Pulsed Microscopy Critical Non-critical
Confocal Single-point Scanning Microscopy Critical Non-critical
Combining multiple laser beams Critical Not applicable
More
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Table 1: A list of popular standard and advanced microscopy methods, categorized as to criticality of dependence on reflected wavefront flatness.
(continued)