coating glass pipettes
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all, I am using a CSU-22 spinning disc for calcium imaging in dendrites and spines of neuronal cells. When doing synaptic stimulation, the glass pipette is positioned pretty close to the imaged region and therefore reflects the excitation light. My question: does anybody have experience with non-reflecting coatings that can be applied to glass pipettes and are of no effect on the tissue? We thought about black nail polish and would like to know if anybody has a betteer suggestion. Thanks a lot, Friedrich Dr. Friedrich W. Johenning NWFZ -Campus Mitte / AG Schmitz Charite University Medicine Berlin Charitéplatz 1 10117 Berlin Tel.: 030 450 528077 Mobil: 0173 2144701 Fax: 030 450 539943 |
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Friedrich Are you sure you are seeing the excitation light reflected by the pipet? If so maybe more stringent filtering might help with your problem? If not, might it rather be the fluorescence emission of your sample that is reflected by the pipet? BTW, as a general question to the list: is there a reason why one couldn't use two inteference filters in series? The ODs should simply add up, shouldn't they? Beat At 15:13 12-03-2008, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Dear all, >I am using a CSU-22 spinning disc for calcium >imaging in dendrites and spines of neuronal >cells. When doing synaptic stimulation, the >glass pipette is positioned pretty close to the >imaged region and therefore reflects the >excitation light. My question: does anybody have >experience with non-reflecting coatings that can >be applied to glass pipettes and are of no >effect on the tissue? We thought about black >nail polish and would like to know if anybody has a betteer suggestion. >Thanks a lot, > >Friedrich > > >Dr. Friedrich W. Johenning >NWFZ -Campus Mitte / AG Schmitz >Charite University Medicine Berlin >Charitéplatz 1 >10117 Berlin >Tel.: 030 450 528077 >Mobil: 0173 2144701 >Fax: 030 450 539943 |
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Beat, You may want to check the Chroma "Handbook of Optical filters...." They discuss some of the properties/issues of various types of fluorescence filters... I also don't see why you couldn't put two such filters in series, except if there are polarization issues. The Optical Density is defined as the Absorbance per unit of length (for conventional filters), and therefore it is the Absorbance, rather than the OD, that should add up, but yes, if a filter passes 10% at wavelength x, two such filters in series should pass 1%. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 == On Mar 12, 2008, at 8:12 AM, Beat Ludin wrote:
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Beat/
Julio,
If you are talking strictly about interference filters
(made with dielectric coatings) the Absorbance of a particular filter is mostly
only a function of the glass substrate the coating is deposited on, as the
filter is working to attenuate incoming light through reflection at the filter's
front surface. If you place two of these in series, you will not see an
additive blocking effect because of reflections bouncing back from the second
filter to the first, to second, and so on, and there is always some
transmittance through the second filter that inhibits the level of blocking you
might otherwise expect to achieve.
If you
were to place a low absorbing piece of glass (such as an NG glass
with ~95%T) in between the two filters, then the slight absorption
properties of that glass would inhibit the reflections and you would achieve (or
nearly so) the additive blocking.
If you
were using absorption glasses only as a blocking component, then
you would get the full benefit of their OD blocking at a given
wavelength.
cheers,
Dan
Dan
Osborn Please
stop by to see us at any of the following events: Photonics
Europe, Palais de la Musique et de Congres, Strasbourg, France April 8 - 10,
2008
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dan - Good point! Thanks for the explanation. I guess, another solution might be to put the filters at a certain distance and at a slight angle to each other, if you can afford the slight spectral change. Beat At 20:19 12-03-2008, you wrote: >Beat/ Julio, > If you are talking strictly about interference filters (made > with dielectric coatings) the Absorbance of a particular filter is > mostly only a function of the glass substrate the coating is > deposited on, as the filter is working to attenuate incoming light > through reflection at the filter's front surface. If you place two > of these in series, you will not see an additive blocking effect > because of reflections bouncing back from the second filter to the > first, to second, and so on, and there is always some transmittance > through the second filter that inhibits the level of blocking you > might otherwise expect to achieve. >If you were to place a low absorbing piece of glass (such as an NG >glass with ~95%T) in between the two filters, then the slight >absorption properties of that glass would inhibit the reflections >and you would achieve (or nearly so) the additive blocking. >If you were using absorption glasses only as a blocking component, >then you would get the full benefit of their OD blocking at a given >wavelength. >cheers, >Dan |
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Beat, You are correct, and you really do not have to worry about any spectral shift of the angled filter(s) as you would only need a very small tilt to successfully "walk" the reflected light out of the light path. One engineer suggested 1-2 degrees of relative off normal angle would do the trick, and that would not appreciably shift the interference coatings performance. Only drawback would be if the scattered light managed to find its way around the filter and into the optical path. Dan Dan Osborn Fluorescence Microscopy Product Manager Omega Optical, Inc. Delta Campus Omega Drive Brattleboro, VT 05301 Phone: Direct line: (802) 251-7305 or Toll Free: (866)-488-1064 Fax: 802-254-3937 Email: [hidden email] < mailto:[hidden email]> www.omegafilters.com <http://www.omegafilters.com> Please stop by to see us at any of the following events:<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" /> Photonics Europe <https://www.omegafilters.com/index.php?page=news_events> , Palais de la Musique et de Congres, Strasbourg, France April 8 - 10, 2008 FOM <https://www.omegafilters.com/index.php?page=news_events> , Osaka, Awaji Island, Japan; April 13 - 16, 2008 ISAC <https://www.omegafilters.com/index.php?page=news_events> , Budapest Sportarena,Budapest, Hungary; May 17 - 21, 2008 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]]On Behalf Of Beat Ludin Sent: Friday, March 14, 2008 5:38 AM To: [hidden email] Subject: Re: interference filters Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dan - Good point! Thanks for the explanation. I guess, another solution might be to put the filters at a certain distance and at a slight angle to each other, if you can afford the slight spectral change. Beat At 20:19 12-03-2008, you wrote: >Beat/ Julio, > If you are talking strictly about interference filters (made > with dielectric coatings) the Absorbance of a particular filter is > mostly only a function of the glass substrate the coating is > deposited on, as the filter is working to attenuate incoming light > through reflection at the filter's front surface. If you place two > of these in series, you will not see an additive blocking effect > because of reflections bouncing back from the second filter to the > first, to second, and so on, and there is always some transmittance > through the second filter that inhibits the level of blocking you > might otherwise expect to achieve. >If you were to place a low absorbing piece of glass (such as an NG >glass with ~95%T) in between the two filters, then the slight >absorption properties of that glass would inhibit the reflections >and you would achieve (or nearly so) the additive blocking. >If you were using absorption glasses only as a blocking component, >then you would get the full benefit of their OD blocking at a given >wavelength. >cheers, >Dan |
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