2013 University of Virginia's W.M. Keck FRET Workshop: Basic Filter Considerations for FRET Imaging
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The document outlines essential considerations for designing filter sets in fluorescence microscopy, focusing on components like excitation filters, dichroic beamsplitters, and emission filters. It discusses filter performance metrics, such as transmission and blocking capabilities, and their impact on applications like FRET and TIRF microscopy. Additionally, it highlights the complexities in FRET imaging and the specific requirements for laser applications.
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2013 University of Virginia's W.M. Keck FRET Workshop: Basic Filter Considerations for FRET Imaging
- 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