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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 Microscopists, Do you know if any other company, besides Leica, include white light laser in their confocals. Or if any other technology can replicate the free selection wavelength laser light? On the other hand, what are disadvantages of the WLL? Best regards, Dr. Konstantín Levitskiy Servicio de Microscopía InstitutodeBiomedicinadeSevilla - IBiS Email: <mailto:[hidden email]> [hidden email] Web: <http://www.ibis-sevilla.es/> www.ibis-sevilla.es |
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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 Konstantin, I am not aware of any other company than Leica offering a WLL on a confocal microscope (LaVision/Miltenyi offers it on their light sheet system too, for example), but I may well be wrong. The WLL is fantastic - very flexible, allows you to go down to pretty long wavelengths (670 nm, unless that has changed now), and also enables you to do time gating in conjunction with the HyDs. It does have the disadvantage of not being so powerful at a particular selected wavelength - for imaging we rarely found that to be an issue, as you can always activate two lines close together to increase the power, but it is a problem if you want to do FRAP. One other potential disadvantage is that if the WLL goes down, you have lost all of your lasers in effect, but our experience has been that Leica are pretty efficient at replacing it, though obviously service reliability varies hugely with geographical location. All the best, Alison On 10/21/2019 9:28 AM, Konstantín Levitskiy wrote: > ***** > 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=DwIFAw&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=lJCrUe1TWbbjJV_eY55c628vqUXom7PcfRow1JdtrwE&s=Ux1H_E9wq2esNcqrwX2zJNgTQfHAI1eVVODZz6oGop8&e= > Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAw&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=lJCrUe1TWbbjJV_eY55c628vqUXom7PcfRow1JdtrwE&s=iKNQUp7xCJMLGeEcdUdD1wfuQoA376CU5-gleTiyvic&e= and include the link in your posting. > ***** > > Dear Microscopists, > > Do you know if any other company, besides Leica, include white light laser > in their confocals. Or if any other technology can replicate the free > selection wavelength laser light? On the other hand, what are disadvantages > of the WLL? > > Best regards, > > Dr. Konstantín Levitskiy > > Servicio de Microscopía > > InstitutodeBiomedicinadeSevilla - IBiS > > Email: <mailto:[hidden email]> [hidden email] > > Web: <https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ibis-2Dsevilla.es_&d=DwIFAw&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=lJCrUe1TWbbjJV_eY55c628vqUXom7PcfRow1JdtrwE&s=SXPXE8wwcN15m7zKHNiptMvee6N4PddMVXM_SzkhdW0&e= > www.ibis-sevilla.es > > -- Alison J. North, Ph.D., Research Associate Professor and Senior Director of the Bio-Imaging Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065. Tel: office ++ 212 327 7488 Tel: lab ++ 212 327 7486 Fax: ++ 212 327 7489 |
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In reply to this post by Konstantín Levitskiy
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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. ***** The white light laser (WWL) is a continuum-based fiber laser. These mostly work, but if anything goes wrong are not easily repairable. The power of these sources shorter than ~480nm is also very low, and most are unusable shorter than 450nm, so you often need a separate 405 or 440nm laser if you really need those wavelengths. The tuning system relies on acoustic modulation or liquid crystal filtering, which works fairly well, but may need periodic checkups for tuning frequencies in the case of the AOM or 'twist' in the case of the LC for each wavelength band. I would recommend keeping your service contract up-to-date if you have one of these systems, just for peace of mind. The benefit of such a laser is you have any wavelength from cyan to deep red, and even into the NIR for some systems, on tap whenever you need it. You can also adjust the band of some of the filters to narrow or widen the spectral content. Wider filtering will allow for more power if you are struggling for signal. For core facilities the WLL provides a great deal of flexibility in terms of what fluorophores you can address, which is handy if you have a wide variety of users. It is also beneficial for users tagging a single sample with a large number of fluorophores as access to many spectral lines allows you to be very strategic with what colors you select. However, for a researcher purchasing a system for their own project, with known fixed fluorophores, the WLL is probably not necessary. The only alternative technology off hand I am aware of are some of the semi-tunable gas and solid-state lasers, but I know of no systems that are commonly available. In the past I have considered a frequency-doubled continuous-wave Ti:Saph which can access green through the NIR, or the very rare tunable He:Ne laser, which can access numerous discrete lines from green to NIR. Craig On Mon, Oct 21, 2019 at 7:40 AM Konstantín Levitskiy <[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. > ***** > > Dear Microscopists, > > Do you know if any other company, besides Leica, include white light laser > in their confocals. Or if any other technology can replicate the free > selection wavelength laser light? On the other hand, what are disadvantages > of the WLL? > > Best regards, > > Dr. Konstantín Levitskiy > > Servicio de Microscopía > > InstitutodeBiomedicinadeSevilla - IBiS > > Email: <mailto:[hidden email]> [hidden email] > > Web: <http://www.ibis-sevilla.es/> www.ibis-sevilla.es > > > |
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In reply to this post by Konstantín Levitskiy
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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 Konstantin, I am also very interested in the advantages and disadvantages of the WLL. Our systems all have 4-5 lasers that cover the visible spectrum well (405 - 635nm). Given that most fluorophores don't have a very narrow absorption spectrum, how important is the flexibility of the WLL? I think it would be very useful perhaps for spectral unmixing but how often do users change the excitation when using the WLL? The only laser line that I miss having now and which would be great to have on any new system is a UV. We recently decommissioned our last confocal which had the water-cooled argon with 351/364nm. I was really sad to see it go. Cheers, Jacqui ________________________________________ From: Confocal Microscopy List [[hidden email]] on behalf of Konstantín Levitskiy [[hidden email]] Sent: 22 October 2019 02:28 To: [hidden email] Subject: white light laser ***** 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 Microscopists, Do you know if any other company, besides Leica, include white light laser in their confocals. Or if any other technology can replicate the free selection wavelength laser light? On the other hand, what are disadvantages of the WLL? Best regards, Dr. Konstantín Levitskiy Servicio de Microscopía InstitutodeBiomedicinadeSevilla - IBiS Email: <mailto:[hidden email]> [hidden email] Web: <http://www.ibis-sevilla.es/> www.ibis-sevilla.es |
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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 have also noticed fewer options for NUV sources for microscopy. The last time we tried it I had to take the extreme step of frequency-doubling a Ti:Saph connected to our multiphoton/confocal system and re-routing the resulting 380nm beam through the confocal optics. It was a very crude solution, but the Ti:Saph could be tuned and doubled from 350-540nm with a crystal, then fiber-coupled for delivery to the confocal portion of the scan head. I was mainly limited by the coatings on our optics or glass absorption, as there was a significant drop-off of transmission at wavelengths shorter than 375nm with the lens we were using. https://spie.org/Publications/Proceedings/Paper/10.1117/12.2079625?SSO=1 Craig On Mon, Oct 21, 2019 at 2:11 PM Jacqueline Ross <[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. > ***** > > Hi Konstantin, > > I am also very interested in the advantages and disadvantages of the WLL. > Our systems all have 4-5 lasers that cover the visible spectrum well (405 - > 635nm). Given that most fluorophores don't have a very narrow absorption > spectrum, how important is the flexibility of the WLL? I think it would be > very useful perhaps for spectral unmixing but how often do users change the > excitation when using the WLL? > > The only laser line that I miss having now and which would be great to > have on any new system is a UV. We recently decommissioned our last > confocal which had the water-cooled argon with 351/364nm. I was really sad > to see it go. > > Cheers, > > Jacqui > ________________________________________ > From: Confocal Microscopy List [[hidden email]] on > behalf of Konstantín Levitskiy [[hidden email]] > Sent: 22 October 2019 02:28 > To: [hidden email] > Subject: white light laser > > ***** > 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 Microscopists, > > Do you know if any other company, besides Leica, include white light laser > in their confocals. Or if any other technology can replicate the free > selection wavelength laser light? On the other hand, what are disadvantages > of the WLL? > > Best regards, > > Dr. Konstantín Levitskiy > > Servicio de Microscopía > > InstitutodeBiomedicinadeSevilla - IBiS > > Email: <mailto:[hidden email]> [hidden email] > > Web: <http://www.ibis-sevilla.es/> www.ibis-sevilla.es > |
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In reply to this post by Konstantín Levitskiy
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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 Konstantin, Zeiss can mount a white laser in their confocal but for me it has no sense to mount a white laser in a dichoric system. At the end, you are limited by the spliters. Best regards Juan Luis El 21/10/2019 a las 15:28, Konstantín Levitskiy escribió: > ***** > 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 Microscopists, > > Do you know if any other company, besides Leica, include white light laser > in their confocals. Or if any other technology can replicate the free > selection wavelength laser light? On the other hand, what are disadvantages > of the WLL? > > Best regards, > > Dr. Konstantín Levitskiy > > Servicio de Microscopía > > InstitutodeBiomedicinadeSevilla - IBiS > > Email: <mailto:[hidden email]> [hidden email] > > Web: <http://www.ibis-sevilla.es/> www.ibis-sevilla.es > > > . > /Juan Luis Ribas/ Servicio de Microscopía Centro de Investigación, Tecnología e Innovación Universidad de Sevilla Av. Reina Mercedes 4b 41012 Sevilla Spain |
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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. ***** Your point is very valid, Juan, which is why Leica typically offers this with their adjustable band 5-channel detector system. It is essentially a 5-band monochrometer system that can be adjusted to match the lines you have selected from the WLL. Zeiss also has a spectral offering that can do much the same thing, although I am not sure if it is as good at blocking the excitation as I have never personally used or observed it. Craig On Tue, Oct 22, 2019 at 3:21 AM Juan Luis Ribas <[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. > ***** > > Dear Konstantin, > > Zeiss can mount a white laser in their confocal but for me it has no > sense to mount a white laser in a dichoric system. At the end, you are > limited by the spliters. > > Best regards > > Juan Luis > > > El 21/10/2019 a las 15:28, Konstantín Levitskiy escribió: > > ***** > > 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 Microscopists, > > > > Do you know if any other company, besides Leica, include white light > laser > > in their confocals. Or if any other technology can replicate the free > > selection wavelength laser light? On the other hand, what are > disadvantages > > of the WLL? > > > > Best regards, > > > > Dr. Konstantín Levitskiy > > > > Servicio de Microscopía > > > > InstitutodeBiomedicinadeSevilla - IBiS > > > > Email: <mailto:[hidden email]> [hidden email] > > > > Web: <http://www.ibis-sevilla.es/> www.ibis-sevilla.es > > > > > > . > > > -- > /Juan Luis Ribas/ > Servicio de Microscopía > Centro de Investigación, Tecnología e Innovación > Universidad de Sevilla > Av. Reina Mercedes 4b > 41012 Sevilla > Spain > |
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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 Craig The differentiator here is on the illumination side as opposed to the detection side of the instrument. The Leica AOBS beamsplitter allows free selection of illumination lines. Multispectral parallel detection would potentially be as effective as the broadband spectrophotometer arrangement Leica uses as a detection system if all else was roughly equal, but Zeiss and the Nikon multichannel PMT as well as the spectrophotometer arrangement in the Fluoview are not supported with the AOBS, their filter beamsplitters mean that the flexible illumination options inherent in a supercontinuum white light laser can't be realised to the extent the AOBS makes possible. Each system has its own strengths and weaknesses, but the AOBS was a great innovation, and is an excellent fit for the white light laser. Darran Clements Imaging Facility Assistant Wellcome-MRC Stem Cell Institute Jeffrey Cheah Biomedical Centre Cambridge Biomedical Campus University of Cambridge Puddicombe Way Cambridge CB2 0AW [hidden email] 01223 760 201 -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Craig Brideau Sent: 22 October 2019 16:44 To: [hidden email] Subject: Re: white light laser ***** 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. ***** Your point is very valid, Juan, which is why Leica typically offers this with their adjustable band 5-channel detector system. It is essentially a 5-band monochrometer system that can be adjusted to match the lines you have selected from the WLL. Zeiss also has a spectral offering that can do much the same thing, although I am not sure if it is as good at blocking the excitation as I have never personally used or observed it. Craig On Tue, Oct 22, 2019 at 3:21 AM Juan Luis Ribas <[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. > ***** > > Dear Konstantin, > > Zeiss can mount a white laser in their confocal but for me it has no > sense to mount a white laser in a dichoric system. At the end, you are > limited by the spliters. > > Best regards > > Juan Luis > > > El 21/10/2019 a las 15:28, Konstantín Levitskiy escribió: > > ***** > > 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 Microscopists, > > > > Do you know if any other company, besides Leica, include white light > laser > > in their confocals. Or if any other technology can replicate the > > free selection wavelength laser light? On the other hand, what are > disadvantages > > of the WLL? > > > > Best regards, > > > > Dr. Konstantín Levitskiy > > > > Servicio de Microscopía > > > > InstitutodeBiomedicinadeSevilla - IBiS > > > > Email: <mailto:[hidden email]> [hidden email] > > > > Web: <http://www.ibis-sevilla.es/> www.ibis-sevilla.es > > > > > > . > > > -- > /Juan Luis Ribas/ > Servicio de Microscopía > Centro de Investigación, Tecnología e Innovación Universidad de > Sevilla Av. Reina Mercedes 4b > 41012 Sevilla > Spain > |
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In reply to this post by Konstantín Levitskiy
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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 Konstantin, apart from things that were already mentioned by others, the fact that they are pulsed is an advantage and disadvantage. Gated detection with the HyDs is an extremely cool feature to get rid of reflected background noise. Plant people also seem to love this since you can gate out the very short lifetime chlorophyll fluorescence. On the downside, you can excite any fluorochrome only every 12.5 seconds, so you may run into saturation faster than with a cw laser. Another downside is that the excitation line is not as narrow as with single lasers. In other words, it seems that the AOTF is not 100% tight against neighboring lines. In spectral detection we advise our users to keep 10 nm between excitation and detection. This is a very cautious approach, and if you are careful and know what you are doing you can get closer. But as a general rule we'd prefer to be on the safe side for the HyDs. Now you could argue that the gated detection reflection suppression is a problem that you wouldn't have without the WLL, but I don't think so. Some samples somehow create a strong reflection near the coverslip that I don't think is coming from the WLL, I know this pattern also from single line systems. Also, the option of exciting with 670 opens up another channel above 630 excitation that you don't reach with a 488/552/594/633 battery of lasers. Maybe in some experiments it is helpful to know that there will be zero misalignment between the laser lines (except for the 405 and the WLL) Just my two euro-cents. Steffen Am 21.10.2019 um 15:28 schrieb Konstantín Levitskiy: > ***** > 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 Microscopists, > > Do you know if any other company, besides Leica, include white light laser > in their confocals. Or if any other technology can replicate the free > selection wavelength laser light? On the other hand, what are disadvantages > of the WLL? > > Best regards, > > Dr. Konstantín Levitskiy > > Servicio de Microscopía > > InstitutodeBiomedicinadeSevilla - IBiS > > Email: <mailto:[hidden email]> [hidden email] > > Web: <http://www.ibis-sevilla.es/> www.ibis-sevilla.es > > > ------------------------------------------------------------ Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Biomedical Center (BMC) Head of the Core Facility Bioimaging Großhaderner Straße 9 D-82152 Planegg-Martinsried Germany http://www.bioimaging.bmc.med.uni-muenchen.de |
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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. ***** Let me add - it was kind of mentioned already - that Leica WLL+AOBS does not have the excitation-suppression capability of filter-based systems. The're trying to improve it with the time gating, but that only works with HyDs. As an example, with LSM780 and their low-angle dichroics (and the meta detector, 488nm Ar-ion) you can see the Raman scattering of water and you don't even have to try hard. You can't achieve this with AOBS. Also, the fluorophore saturation (and other nonlinearities) will manifest at lower power (at least 10x lower, whatever that means practically, but it depends on temporal pulse width, which is not specified in Fianium or NKT datasheets, maybe because it varies with wavelength - ask your Leica rep!). My $0.02 (and no further commercial interest). zdenek On Thu, Oct 24, 2019 at 10:53 AM Steffen Dietzel <[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. > ***** > > Dear Konstantin, > > apart from things that were already mentioned by others, the fact that > they are pulsed is an advantage and disadvantage. Gated detection with > the HyDs is an extremely cool feature to get rid of reflected background > noise. Plant people also seem to love this since you can gate out the > very short lifetime chlorophyll fluorescence. On the downside, you can > excite any fluorochrome only every 12.5 seconds, so you may run into > saturation faster than with a cw laser. Another downside is that the > excitation line is not as narrow as with single lasers. In other words, > it seems that the AOTF is not 100% tight against neighboring lines. In > spectral detection we advise our users to keep 10 nm between excitation > and detection. This is a very cautious approach, and if you are careful > and know what you are doing you can get closer. But as a general rule > we'd prefer to be on the safe side for the HyDs. Now you could argue > that the gated detection reflection suppression is a problem that you > wouldn't have without the WLL, but I don't think so. Some samples > somehow create a strong reflection near the coverslip that I don't think > is coming from the WLL, I know this pattern also from single line systems. > > Also, the option of exciting with 670 opens up another channel above 630 > excitation that you don't reach with a 488/552/594/633 battery of lasers. > > Maybe in some experiments it is helpful to know that there will be zero > misalignment between the laser lines (except for the 405 and the WLL) > > Just my two euro-cents. > > Steffen > > > > Am 21.10.2019 um 15:28 schrieb Konstantín Levitskiy: > > ***** > > 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 Microscopists, > > > > Do you know if any other company, besides Leica, include white light > laser > > in their confocals. Or if any other technology can replicate the free > > selection wavelength laser light? On the other hand, what are > disadvantages > > of the WLL? > > > > Best regards, > > > > Dr. Konstantín Levitskiy > > > > Servicio de Microscopía > > > > InstitutodeBiomedicinadeSevilla - IBiS > > > > Email: <mailto:[hidden email]> [hidden email] > > > > Web: <http://www.ibis-sevilla.es/> www.ibis-sevilla.es > > > > > > > -- > ------------------------------------------------------------ > Steffen Dietzel, PD Dr. rer. nat > Ludwig-Maximilians-Universität München > Biomedical Center (BMC) > Head of the Core Facility Bioimaging > > Großhaderner Straße 9 > D-82152 Planegg-Martinsried > Germany > > http://www.bioimaging.bmc.med.uni-muenchen.de > -- -- Zdenek Svindrych, Ph.D. Research Associate - Imaging Specialist Department of Biochemistry and Cell Biology Geisel School of Medicine at Dartmouth |
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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 Zdenk, this Raman thing sounds very interesting, but I have not much knowledge about this field. Can you explain at which wavelength you expect this signal when exciting with 488 nm? Best Steffen Am 24.10.2019 um 18:17 schrieb Zdenek Svindrych: > ***** > 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. > ***** > > Let me add - it was kind of mentioned already - that Leica WLL+AOBS does > not have the excitation-suppression capability of filter-based systems. > The're trying to improve it with the time gating, but that only works with > HyDs. > As an example, with LSM780 and their low-angle dichroics (and the meta > detector, 488nm Ar-ion) you can see the Raman scattering of water and you > don't even have to try hard. You can't achieve this with AOBS. > Also, the fluorophore saturation (and other nonlinearities) will manifest > at lower power (at least 10x lower, whatever that means practically, but it > depends on temporal pulse width, which is not specified in Fianium or NKT > datasheets, maybe because it varies with wavelength - ask your Leica rep!). > My $0.02 (and no further commercial interest). > zdenek > > On Thu, Oct 24, 2019 at 10:53 AM Steffen Dietzel <[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. >> ***** >> >> Dear Konstantin, >> >> apart from things that were already mentioned by others, the fact that >> they are pulsed is an advantage and disadvantage. Gated detection with >> the HyDs is an extremely cool feature to get rid of reflected background >> noise. Plant people also seem to love this since you can gate out the >> very short lifetime chlorophyll fluorescence. On the downside, you can >> excite any fluorochrome only every 12.5 seconds, so you may run into >> saturation faster than with a cw laser. Another downside is that the >> excitation line is not as narrow as with single lasers. In other words, >> it seems that the AOTF is not 100% tight against neighboring lines. In >> spectral detection we advise our users to keep 10 nm between excitation >> and detection. This is a very cautious approach, and if you are careful >> and know what you are doing you can get closer. But as a general rule >> we'd prefer to be on the safe side for the HyDs. Now you could argue >> that the gated detection reflection suppression is a problem that you >> wouldn't have without the WLL, but I don't think so. Some samples >> somehow create a strong reflection near the coverslip that I don't think >> is coming from the WLL, I know this pattern also from single line systems. >> >> Also, the option of exciting with 670 opens up another channel above 630 >> excitation that you don't reach with a 488/552/594/633 battery of lasers. >> >> Maybe in some experiments it is helpful to know that there will be zero >> misalignment between the laser lines (except for the 405 and the WLL) >> >> Just my two euro-cents. >> >> Steffen >> >> >> >> Am 21.10.2019 um 15:28 schrieb Konstantín Levitskiy: >>> ***** >>> 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 Microscopists, >>> >>> Do you know if any other company, besides Leica, include white light >> laser >>> in their confocals. Or if any other technology can replicate the free >>> selection wavelength laser light? On the other hand, what are >> disadvantages >>> of the WLL? >>> >>> Best regards, >>> >>> Dr. Konstantín Levitskiy >>> >>> Servicio de Microscopía >>> >>> InstitutodeBiomedicinadeSevilla - IBiS >>> >>> Email: <mailto:[hidden email]> [hidden email] >>> >>> Web: <http://www.ibis-sevilla.es/> www.ibis-sevilla.es >>> >>> >>> >> -- >> ------------------------------------------------------------ >> Steffen Dietzel, PD Dr. rer. nat >> Ludwig-Maximilians-Universität München >> Biomedical Center (BMC) >> Head of the Core Facility Bioimaging >> >> Großhaderner Straße 9 >> D-82152 Planegg-Martinsried >> Germany >> >> http://www.bioimaging.bmc.med.uni-muenchen.de >> > ------------------------------------------------------------ Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Biomedical Center (BMC) Head of the Core Facility Bioimaging Großhaderner Straße 9 D-82152 Planegg-Martinsried Germany http://www.bioimaging.bmc.med.uni-muenchen.de |
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