Size of a bleach point
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear listers, When using the Bleach Point feature in the Leica confocal systems (I'm sure it also exists in the other brands), I don't know how to measure the surface of the bleached area. I figure out the wavelength of the laser beam and the NA of the lens affect the size of that area, so would it be correct to use the resolution formula to calculate it? Or am I mixing up things here? Thank you very much, Xavi. ___________________________________ *Xavier Sanjuan* Advanced Light Microscopy Unit Parc de Recerca Biomèdica de Barcelona Doctor Aiguader, 88 08003 Barcelona - Spain Tel: + 34 93 316 0195 (ext 1195 dins el PRBB) Fax: + 34 93 316 09 01 E-mail: [hidden email] Web: http://www.crg.eu/en/core/programmes-groups/advanced-light-microscopy-unit |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Xavi, I tend to use the PSF generator in Imagej / Fiji. http://bigwww.epfl.ch/algorithms/psfgenerator/ You can take the full width at half maximal to indicate the bleach area. In xy and z. I tend to think of bleaching a volume rather than an area. I'm sure this isn't perfect, as the energy isn't equally spread within the PSF. If I'm teaching this, I use the plot profile on live view of a PSF, fix the axes ranges on the graph, and run through the z stack to show the energy distribution in different z slices. Or save a small (65x65x65 pixel) PSF with 50 or 100 nm xyz pixels, and use ICY 3D viewer with a colour map (LUT) like Parula. I always need to fix the metadata in ICY to the correct XYZ dimensions. cheers Dale ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of SANJUAN SAMARRA, XAVIER <[hidden email]> Sent: 23 April 2020 23:21 To: [hidden email] <[hidden email]> Subject: Size of a bleach point ***** To join, leave or search the confocal microscopy listserv, go to: https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7C%7C2bfc157e40d44d87276308d7e7d4e4c2%7C1faf88fea9984c5b93c9210a11d9a5c2%7C0%7C0%7C637232773874476244&sdata=dKKqbAwczgxRhBmOmjGPHC6IjoXxuLINOUlBB6Ff3%2Bo%3D&reserved=0 Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=02%7C01%7C%7C2bfc157e40d44d87276308d7e7d4e4c2%7C1faf88fea9984c5b93c9210a11d9a5c2%7C0%7C0%7C637232773874476244&sdata=PooWLcpsZuKH92MexbQHDnHBrTddXfUCkyfce7WkRaI%3D&reserved=0 and include the link in your posting. ***** Dear listers, When using the Bleach Point feature in the Leica confocal systems (I'm sure it also exists in the other brands), I don't know how to measure the surface of the bleached area. I figure out the wavelength of the laser beam and the NA of the lens affect the size of that area, so would it be correct to use the resolution formula to calculate it? Or am I mixing up things here? Thank you very much, Xavi. ___________________________________ *Xavier Sanjuan* Advanced Light Microscopy Unit Parc de Recerca Biomèdica de Barcelona Doctor Aiguader, 88 08003 Barcelona - Spain Tel: + 34 93 316 0195 (ext 1195 dins el PRBB) Fax: + 34 93 316 09 01 E-mail: [hidden email] Web: https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.crg.eu%2Fen%2Fcore%2Fprogrammes-groups%2Fadvanced-light-microscopy-unit&data=02%7C01%7C%7C2bfc157e40d44d87276308d7e7d4e4c2%7C1faf88fea9984c5b93c9210a11d9a5c2%7C0%7C0%7C637232773874476244&sdata=juEpNzsBmonT8Y5j8OpQGz1q%2BZlOI6VR5x8NA8kytpM%3D&reserved=0 |
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In reply to this post by xavier Sanjuan
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Xavi, For fastest photobleaching, generally best to use maximum power for as brief a time as possible. The more photons, the bigger the spot. Oxygen radicals and dye radicals diffuse short distances relative to PSF (nanometers ... though activated tyramide radicals have a diffusion radius of ~100 nm, and can be restrained by increasing viscosity or additives ... cytoplasm is higher viscosity and density than water ... so: radicals diffusion radius might be suppressed in live cells that are [over]expressing catalase). A(nother) good PSF calculator is available free at SVI https://svi.nl/NyquistCalculator Microscopy Nyquist rate and PSF calculator. I liked this recent publication on the photophysics and photochemistry of fluorescence - and generalization across the UV-Visible fluorophores: Aleksandr Barulin and Jeroome Wenger 2020 Ultraviolet Photostability Improvement for Autofluorescence Correlation Spectroscopy on Label-Free Proteins J. Phys. Chem. Lett. 2020, 11, 2027−2035 https://dx.doi.org/10.1021/acs.jpclett.0c00209 From abstract: "Our results show that the underlying photochemical concepts initially derived for organic visible fluorescent dyes are quite general." George p.s. tyramide references: * Chen ... Belmont 2018 J Cell Biol https://doi.org/10.1083/jcb.201807108 * Phil Moens, pers. comm. [1993?] (ex-NEN, before acquisition by PerkinElmer and Phenoptics division spinout to Akoya). On 4/23/2020 6:21 PM, SANJUAN SAMARRA, XAVIER wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Dear listers, > > When using the Bleach Point feature in the Leica confocal systems (I'm sure > it also exists in the other brands), I don't know how to measure the > surface of the bleached area. I figure out the wavelength of the laser beam > and the NA of the lens affect the size of that area, so would it be correct > to use the resolution formula to calculate it? Or am I mixing up things > here? > > Thank you very much, > > Xavi. > > ___________________________________ > > *Xavier Sanjuan* > Advanced Light Microscopy Unit > > Parc de Recerca Biomèdica de Barcelona > Doctor Aiguader, 88 > 08003 Barcelona - Spain > Tel: + 34 93 316 0195 (ext 1195 dins el PRBB) > Fax: + 34 93 316 09 01 > E-mail: [hidden email] > Web: > http://www.crg.eu/en/core/programmes-groups/advanced-light-microscopy-unit |
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In reply to this post by xavier Sanjuan
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** For quantitative analysis of recovery after bleaching a spot with a Gaussian laser beam (TEM00), the beam waist typically is used for the radius term. This is the width at which the normalized intensity of the beam drops to 1/e^2. To measure the intensity profile of the beam, you would take an image of the parked beam on a uniform, photostable sample, use ImageJ to get an intensity profile across it and fit a Gaussian to the profile. In principle, you could also do this from the inverted intensity profile of the bleached region in the first image acquired after bleaching, but if recovery is fast, this will be an underestimate of the "true" radius of the spot. The FWHM is approximated by 2.355 x the standard deviation of the Gaussian and the waist is about 1.7 x FWHM. The difference is important for quantitative analysis to obtain the transport characteristics of what you are bleaching because the area of the bleached region is significantly larger using beam waist than using FWHM. Have fun! Kate |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Plexiglas may not scatter & absorb light the same as your biological samples, but with SP2 AOBS and SP5 AOBS we bleached spots inside fluorescent Plexiglas and then went back and imaged Z series of the bleached volumes to see focal plane and cones above and below. Two examples: https://www.flickr.com/photos/mcammer/2605562876/ https://www.flickr.com/photos/mcammer/2608080259/ Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016 [hidden email]<mailto:[hidden email]> http://nyulmc.org/micros http://microscopynotes.com/ Voice direct only, no text or messages: 1-914-309-3270 and 1-646-501-0567 ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Kate Luby-Phelps <[hidden email]> Sent: Friday, April 24, 2020 9:29:14 AM To: [hidden email] Subject: Re: Size of a bleach point [EXTERNAL] ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=YJyaJS5Pwb8Zg6dczRi0rVLxlHeDMwbYM8OyMQbLFzE&s=mJQPNDFWkGLVKGebUbdHiizkMDaZ3HD5Ucv4s-v-x4U&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=YJyaJS5Pwb8Zg6dczRi0rVLxlHeDMwbYM8OyMQbLFzE&s=UrJ19f1ZxYCM3NSacL6C9zMDAd53klqCEjvcEk5MuPo&e= and include the link in your posting. ***** For quantitative analysis of recovery after bleaching a spot with a Gaussian laser beam (TEM00), the beam waist typically is used for the radius term. This is the width at which the normalized intensity of the beam drops to 1/e^2. To measure the intensity profile of the beam, you would take an image of the parked beam on a uniform, photostable sample, use ImageJ to get an intensity profile across it and fit a Gaussian to the profile. In principle, you could also do this from the inverted intensity profile of the bleached region in the first image acquired after bleaching, but if recovery is fast, this will be an underestimate of the "true" radius of the spot. The FWHM is approximated by 2.355 x the standard deviation of the Gaussian and the waist is about 1.7 x FWHM. The difference is important for quantitative analysis to obtain the transport characteristics of what you are bleaching because the area of the bleached region is significantly larger using beam waist than using FWHM. Have fun! Kate |
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In reply to this post by xavier Sanjuan
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** I forgot to say that in the Plexiglas reply that this worked well as a model for cell cultures, but we are having an issue now (ok, in February and maybe again someday...) that as we focus deeper in drosophila embryos using a Bruker galvo miniscanner that there is a lot of scattering. At the surface of a zebrafish embryo we can target membranes specifically, but deeper in drosophila scatters too much for fine targeting. We have tried three different lenses and adjusting the collar of one empirically. This is at 405 and 470 nm. So spot size is very depth and sample dependent. Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016 [hidden email]<mailto:[hidden email]> http://nyulmc.org/micros http://microscopynotes.com/ Voice direct only, no text or messages: 1-914-309-3270 and 1-646-501-0567 ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of SANJUAN SAMARRA, XAVIER <[hidden email]> Sent: Thursday, April 23, 2020 6:21:30 PM To: [hidden email] Subject: Size of a bleach point [EXTERNAL] ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=yP7vq3EcHuDcKVCDZvMEH2V4DrnW01XUvq51lazvtWw&s=DntAv-VWsKnK4897yAOPY2bnxV_76AqObBoVvIDrPDU&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=yP7vq3EcHuDcKVCDZvMEH2V4DrnW01XUvq51lazvtWw&s=8UwNlUtSrvILTUYU-v7t7-F7F1BY90K5BB8Sg6aEefQ&e= and include the link in your posting. ***** Dear listers, When using the Bleach Point feature in the Leica confocal systems (I'm sure it also exists in the other brands), I don't know how to measure the surface of the bleached area. I figure out the wavelength of the laser beam and the NA of the lens affect the size of that area, so would it be correct to use the resolution formula to calculate it? Or am I mixing up things here? Thank you very much, Xavi. ___________________________________ *Xavier Sanjuan* Advanced Light Microscopy Unit Parc de Recerca Biomèdica de Barcelona Doctor Aiguader, 88 08003 Barcelona - Spain Tel: + 34 93 316 0195 (ext 1195 dins el PRBB) Fax: + 34 93 316 09 01 E-mail: [hidden email] Web: https://urldefense.proofpoint.com/v2/url?u=http-3A__www.crg.eu_en_core_programmes-2Dgroups_advanced-2Dlight-2Dmicroscopy-2Dunit&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=yP7vq3EcHuDcKVCDZvMEH2V4DrnW01XUvq51lazvtWw&s=JdJGheTLRjXZDGcKpba4K1HsNjRbLhgZKVRxqIgzx50&e= |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Thanks for all the feedback! I did not want this for a photobleaching experiment, we have been using a multiphoton laser at 830 nm to heat nanoparticles within cells, but I guess this does not affect the calculations! Best, Xavi. ___________________________________ *Xavier Sanjuan* Advanced Light Microscopy Unit Parc de Recerca Biomèdica de Barcelona Doctor Aiguader, 88 08003 Barcelona - Spain Tel: + 34 93 316 0195 (ext 1195 dins el PRBB) Fax: + 34 93 316 09 01 E-mail: [hidden email] Web: http://www.crg.eu/en/core/programmes-groups/advanced-light-microscopy-unit Missatge de Cammer, Michael <[hidden email]> del dia dv., 24 d’abr. 2020 a les 18:10: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > I forgot to say that in the Plexiglas reply that this worked well as a > model for cell cultures, but we are having an issue now (ok, in February > and maybe again someday...) that as we focus deeper in drosophila embryos > using a Bruker galvo miniscanner that there is a lot of scattering. At the > surface of a zebrafish embryo we can target membranes specifically, but > deeper in drosophila scatters too much for fine targeting. We have tried > three different lenses and adjusting the collar of one empirically. This > is at 405 and 470 nm. So spot size is very depth and sample dependent. > > > Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory > > NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY > 10016 > > [hidden email]<mailto:[hidden email]> > http://nyulmc.org/micros http://microscopynotes.com/ > > Voice direct only, no text or messages: 1-914-309-3270 and 1-646-501-0567 > > > > ________________________________ > From: Confocal Microscopy List <[hidden email]> on > behalf of SANJUAN SAMARRA, XAVIER <[hidden email]> > Sent: Thursday, April 23, 2020 6:21:30 PM > To: [hidden email] > Subject: Size of a bleach point > > [EXTERNAL] > > ***** > To join, leave or search the confocal microscopy listserv, go to: > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=yP7vq3EcHuDcKVCDZvMEH2V4DrnW01XUvq51lazvtWw&s=DntAv-VWsKnK4897yAOPY2bnxV_76AqObBoVvIDrPDU&e= > Post images on > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=yP7vq3EcHuDcKVCDZvMEH2V4DrnW01XUvq51lazvtWw&s=8UwNlUtSrvILTUYU-v7t7-F7F1BY90K5BB8Sg6aEefQ&e= > and include the link in your posting. > ***** > > Dear listers, > > When using the Bleach Point feature in the Leica confocal systems (I'm sure > it also exists in the other brands), I don't know how to measure the > surface of the bleached area. I figure out the wavelength of the laser beam > and the NA of the lens affect the size of that area, so would it be correct > to use the resolution formula to calculate it? Or am I mixing up things > here? > > Thank you very much, > > Xavi. > > ___________________________________ > > *Xavier Sanjuan* > Advanced Light Microscopy Unit > > Parc de Recerca Biomèdica de Barcelona > Doctor Aiguader, 88 > 08003 Barcelona - Spain > Tel: + 34 93 316 0195 (ext 1195 dins el PRBB) > Fax: + 34 93 316 09 01 > E-mail: [hidden email] > Web: > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.crg.eu_en_core_programmes-2Dgroups_advanced-2Dlight-2Dmicroscopy-2Dunit&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=yP7vq3EcHuDcKVCDZvMEH2V4DrnW01XUvq51lazvtWw&s=JdJGheTLRjXZDGcKpba4K1HsNjRbLhgZKVRxqIgzx50&e= > |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Xavi, Are you trying to estimate the size of the region that is experiencing a temperature change? Many fluorescent proteins are actually reversibly temperature sensitive in their brightness and a number of folks have shown that it's possible to measure the temperature achieved at a laser focus by comparing against a calibration curve of FP brightness vs temperature. See: https://www.nature.com/articles/ncomms14100 https://www.nature.com/articles/s41592-018-0168-y?WT.feed_name=subjects_experimental-organisms This may not work if you're heating to the point of bleaching, however. Best, Pavak On Fri, Apr 24, 2020, 9:56 AM SANJUAN SAMARRA, XAVIER < [hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Thanks for all the feedback! I did not want this for a photobleaching > experiment, we have been using a multiphoton laser at 830 nm to heat > nanoparticles within cells, but I guess this does not affect the > calculations! > > Best, > > Xavi. > > ___________________________________ > > *Xavier Sanjuan* > Advanced Light Microscopy Unit > > Parc de Recerca Biomèdica de Barcelona > Doctor Aiguader, 88 > 08003 Barcelona - Spain > Tel: + 34 93 316 0195 (ext 1195 dins el PRBB) > Fax: + 34 93 316 09 01 > E-mail: [hidden email] > Web: > http://www.crg.eu/en/core/programmes-groups/advanced-light-microscopy-unit > |
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