This was part of an in-person, live presentation at the 2012 annual University of Virginia's Keck FRET workshop. The presentation covered basic optical filter considerations for FRET microscopy, including the basics of how optical interference filters work. Filter set combinations for different approaches to FRET imaging are proposed.

1. Basic Filter Considerations for
FRET Imaging
Jeff Carmichael
Chroma Technology Corp.
Rockingham, VT. USA

2. Topics for today:
• Basic filter set design:
 Exciter/dichroic/emitter
• Filter performance:
 Transmission
 “Blocking” or attenuation
 Transition Slope
• Image registration
• FRET
• Epi-fluorescence widefield FRET
• Acceptor photobleaching
• Epi-laser/confocal/TIRF FRET
• MPE FRET

3. Types Of Filters Used In Fluorescence Microscopy:
1) Excitation Filter
Filters out all but the desired wavelengths of excitation light which are emitted from
a light source (such as an Hg lamp) in order to effectively excite a particular
fluorochrome. The excitation filter bears the burden of attenuating a very broad
spectrum at a very high level. Used at 0 degrees AOI. Named using CWL and
bandwidth at FWHM. ET470/40x = 470nm CWL, 40nm FWHM.

4. 1) Excitation Filter
Measured absorbance data, expressed in Optical Density units
5-9 orders of magnitude is typical difference in radiance between light source
and fluorescence signal in epi-fluorescence and confocal microscopy
OD 1.0 = 10%T, OD 6.0 = 0.0001%T

5. 2) Dichroic Beamsplitter
The dichroic beamsplitter (also called dichroic mirror or dichromatic beamsplitter) is
a thin piece of coated substrate, typically set at a 45 degree angle to the optical
path of the microscope. The coating reflects a particular range of wavelengths
(typically excitation) and transmits a different range of wavelengths (typically
emission). A longpass dichroic reflects excitation wavelengths to the sample, and
transmits the longer wavelength fluorescence emission to the detector. A
shortpass dichroic reflects longer wavelengths, while transmitting shorter.

6. Dichroics do not necessarily reflect “everything below” the cut-on and transmit
“everything above”. The “cut-on” wavelength of any filter (including ex. and em.
filter) is the wavelength at which transmission is 50% of maximum transmission.
All of the following are approx. “600nm” dichroics:

7. 3) Emission Filter
The emission filter (also called the barrier filter) bears the burden of blocking or
attenuating the excitation light sufficiently, thereby allowing the detection of weak
fluorescence signals. This is its primary function. Secondary to this function is
maximizing the level of transmission of the desired wavelengths. Used at 0
degrees AOI. Named using CWL and bandwidth at FWHM. ET525/50m =
525nm CWL, 50nm FWHM.

8. Emission filter “blocking” of wavelengths transmitted by the excitation filter
OD 1.0 = 10%T, OD 6.0 = 0.0001%T

9. Transmission differences between filter coating type:
D-type soft coatings
HQ-type soft coatings (more cavities, i.e. more coating layers)
ET-type hard coatings (sputter-coated)

10. Blocking differences between filter coatings:
D-type soft coatings
HQ-type soft coatings (more cavities, i.e. more coating layers)
ET-type hard coatings (sputter-coated)

11. Why not place filters closer together spectrally?

12. Insufficient blocking
OD 1.0 = 10%T, OD 6.0 = 0.0001%T

13. Not all wavelengths are created equal….depending on
the light source

14. With a mercury lamp or metal-halide source, blocking at 546nm
is much more important than blocking at 496nm…

15. Fluorescence Resonance
Energy Transfer (FRET)
Simple concept:
Excite the Donor and
Look for Emission at the
Acceptor…

16. Not so Simple in Practice:
-Direct excitation of acceptor
at donor wavelengths
-Emission of donor into acceptor
band
-Emission of acceptor into donor
band
-Bleaching/photo-damage
-Motion and registration
-Autofluorescence

17. EGFP to TagRFP FRET with three filter cubes:
Donor filter set:
• EGFP excitation/EGFP emission

18. EGFP to TagRFP FRET with three filter cubes:
FRET filter set:
• EGFP excitation/TagRFP emission

19. EGFP to TagRFP FRET with three filter cubes:
Acceptor filter set:
• TagRFP excitation/TagRFP emission

20. Sputter-coated multi-band filter set with:
 polychroic mirror for EGFP/TagRFP FRET
individual excitation filters in a filter wheel
individual emission filters in a filter wheel or an emission-splitting system
such as a DualView or OptoSplit

21. EGFP to TagRFP FRET:
• Same polychroic in stationary filter cube
• Donor Channel filters (selectable in external filter wheels)

22. EGFP to TagRFP FRET:
• Same polychroic in stationary filter cube
• FRET Channel filters (selectable in external filter wheels)

23. EGFP to TagRFP FRET:
• Same polychroic in stationary filter cube
• Acceptor Channel filters (selectable in external filter wheels)

24. EGFP to TagRFP FRET:
• Acceptor photobleaching set; choose Acceptor excitation bandpass
far removed from Donor excitation spectra

25. Filters for use in laser applications, including TIRF:
• Dichroics must be flatter to prevent distortion of reflected lasers – this is critical
for TIRF applications
• More bandwidth available for fluorescence transmission
• TIRF requires additional blocking compared with other laser imaging applications because
of “total internal reflection” of lasers returning back through microscope objective

26. Shortpass dichroic and emission filter for multi-photon excitation (MPE)
for use In a filter cube in the microscope turret:
• Shortpass “2p” emission filter provides extra blocking of NIR ex. wavelengths (OD8)
• Shortpass “2p” dichroic provides extended reflection to =/>1100nm and extended
transmission from 400-750nm

27. Emission-splitting filter set for Non-Descanned Detector (NDD)
used with MPE:
• “2p” emission filters providing OD8 blocking of NIR wavelengths
• similar set would be used in Optosplit and DualView image-splitting

28. Thanks….
Questions?
Jeff: jcarmichael@chroma.com
Or apps@chroma.com