TIRF filters
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We have a new TIRF system (Nikon) that we are getting to know. One of the things we are trying to sort out is the filters to use in the cube. We are exciting with one of 4 laser lines from a laser launch through a single fiber. Mention has been made by Nikon of having a cleanup filter in the exciter position of hte cube. Why would you do that if you have discrete laser lines from diode lasers? I would think you would take out the exciter filter completely. Dave
Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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I remove the excitation filters from the fluorescence filter cubes on our home-built TIRF microscope that uses only DPSS lasers. I've examined the spectral characteristics of these lasers with an Ocean Optics (no commercial interest) fiber optic spectrometer and all of them emit a sharp line centered on the main wavelength (532 nm, 473 nm, 405 nm, etc.) with a FWHM that is typically about 5 nm or less. I don't see the purpose in putting in a "clean up" filter. If anything, this kind of filter cuts down on the amount of light reaching the sample. If you find out why those filters are there, please let us know.
John Oreopoulos On 20-Mar-09, at 2:32 PM, David Knecht wrote: We have a new TIRF system (Nikon) that we are getting to know. One of the things we are trying to sort out is the filters to use in the cube. We are exciting with one of 4 laser lines from a laser launch through a single fiber. Mention has been made by Nikon of having a cleanup filter in the exciter position of hte cube. Why would you do that if you have discrete laser lines from diode lasers? I would think you would take out the exciter filter completely. Dave |
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In reply to this post by Knecht, David
I'm not the best person to comment on this as most of what I'm about to
relate I know secondhand, but the Vale lab has done a fair amount of testing of different filter combinations for TIRF imaging with the Nikon system, both in their lab and at the Physiology course at Woods Hole. The conclusion is that having a laser line clean up excitation filter reduces background quite a bit. The guess is that this is because there is some scattered laser light that is not part of the TIRF illumination beam that is eliminated by the clean up filter because it hits the filter at an angle. It is also possible that the laser is emitting some light at wavelengths other than the lasing line that the filter is cleaning up (I heard this as a justification for cleanup filters on Ar-ion lasers a decade ago). They have also found that a laser line blocking notch filter on the emission side of the cube also helps reduce the background. Finally, getting flat dichroics is very important for good TIRF imaging and surprisingly tricky. The spring that holds the dichroics in place in the Nikon cubes can bend the dichroic and give rise to astigmatism in the TIRF beam. Chroma is working on some tricks to get around this - for a while they have made 1.5mm thick dichroics that don't need the spring to be held in place and can be glued in place for minimal bending. They are working on another iteration of this now using 2mm substates and special cubes, but I'm not sure on the timing. Hopefully that sheds some light on TIRF filter choices. Kurt David Knecht wrote: > We have a new TIRF system (Nikon) that we are getting to know. One of > the things we are trying to sort out is the filters to use in the > cube. We are exciting with one of 4 laser lines from a laser launch > through a single fiber. Mention has been made by Nikon of having a > cleanup filter in the exciter position of hte cube. Why would you do > that if you have discrete laser lines from diode lasers? I would > think you would take out the exciter filter completely. Dave > > Dr. David Knecht > Department of Molecular and Cell Biology > Co-head Flow Cytometry and Confocal Microscopy Facility > U-3125 > 91 N. Eagleville Rd. > University of Connecticut > Storrs, CT 06269 > 860-486-2200 > 860-486-4331 (fax) > > -- Kurt Thorn, PhD Director, Nikon Imaging Center University of California San Francisco UCSF MC 2140 Genentech Hall Room S252 600 16th St. San Francisco, CA 94158-2517 http://nic.ucsf.edu phone 415.514.9709 fax 415.514.4300 |
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In reply to this post by Knecht, David
Here we do not use excitation filters for our TIRF. Perhaps it may improve the
quality. We'll test it. The remark of Kurt about the quality of the dichroic is absolutely right. Together with Nikon people here in Amsterdam, we experiemented with different dichroics to see the effect on the TIRF image. The effects are shocking. The only way to get a good dichroic mounted in a cube is by buying 25 filtercubed with dichroics and test them. Select the best and throw away the other 24. You can test by projecting the excitation spot on the ceiling. If you have a nice round spot, your dichroic is fine. Do not touch it anymore. Taking the dichroic out and in the cube changes the pattern on the ceiling. Note that not only the dichroic that reflects the excitation influences the wavefront, but also other filters and dichroics in the excitation path (in our case a second dichroic for TIRF detection) influences the TIRF quality. To my opinion the quality of a TIRF-objective is not important (Zeiss, Nikon etc. are all the same). The dichroic is the limiting factor!! So, if I would again buy a TIRF system, I would select the manufacurer that also provides selected, high quality dichroics, mounted in a cube!!! Kind regards, Erik Manders |
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We just replaced our Chroma filters iwth Semrock laser dichroics and so far (2/2) they have both given sharp spots where the Chroma's were diffuse. They give a flatness spec that others don't seem to provide. The metal Nikon filter holders also made a big difference over the standard plastic ones.
Related: If you want to do multiple wavelengths of TIRF, what are the merits of a multi-pass dichroic and emission filter vs. trying to use separate cubes. So far the separate cube approach leads to the spot in slightly different places on the ceiling with slightly different focus. I am not sure how off this can be and still get good TIRF. Are you using an emission filter wheel or just cubes? Semrock is about to come out with a quad dichroic TIRF dichroic, but I suspect that will necessitate an emission filter wheel to eliminate bleedthrough of fluors. Dave
On Mar 21, 2009, at 3:30 PM, Erik Manders wrote:
Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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In reply to this post by Knecht, David
Some advice for the TIRF dichroics:
1) Buy the dichroics from the microscope maufacturer specifically for TIRF and send them back whe they are not fit for the job, this is more expensive and might take longer, but usually less trouble 2) If you cannot get the ones you need, agree with the company which provides them that they take them back if they should ot work, this is usually possible. AHF Aalysentechnik in Germany (no commercial interest) supply dichroics for Olympus in Europe and know about the problems with the TIRF dichroics. 3) Do not put pressure on them during mounting |
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