Lightsheet test target
Locked
|
*****
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. ***** Does anyone know about any commercially available imaging target that can be used for testing a lightsheet set up? Thank you, Anjul |
|
|
*****
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 Anuj for posting this question. I was about to ask the same question. I wonder if people from Zeiss might answer this post and give us some idea what they use to test the performance of their Z1 lightsheet. Best wishes, Radek Radek MACHAN, Ph.D. | Senior Research Fellow NOBIC Imaging Facilities<https://www.nobic.sg/Facilities.html> Manager www.nobic.sg<http://www.nobic.sg> | [hidden email] SCELSE, Nanyang Technological University #B2, 60 Nanyang Drive, SBS-01N-27 Singapore 637551 ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Anjul Loiacono <[hidden email]> Sent: Tuesday, December 15, 2020 6:42 To: [hidden email] <[hidden email]> Subject: Lightsheet test target ***** 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. ***** Does anyone know about any commercially available imaging target that can be used for testing a lightsheet set up? Thank you, Anjul ________________________________ CONFIDENTIALITY: This email is intended solely for the person(s) named and may be confidential and/or privileged. If you are not the intended recipient, please delete it, notify us and do not copy, use, or disclose its contents. Towards a sustainable earth: Print only when necessary. Thank you. |
|
|
*****
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've never used a Z1 (or other Zeiss lightsheet) so I don't know if the right controls are exposed to the user for this but one beaty of a lightsheet is that almost all critical aspects of system performance and alignment can be trivially visualized using a sample spiked with a little bit of fluorescent dye, for example fluorescein. For any lightsheet system, your two main goals are to check to see whether the excitation beam (for scanned systems) or sheet (for stationary optics) is coming out straight and parallel to the image plane. Straightness is most easily assessed in a scanned system since the beam (when the scanning optic is stationary) should ideally be parallel to the X axis of the camera. To check whether the sheet is parallel, one merely needs to move the imaging objective in and out while observing the stationary beam or sheet. If parallel, it will go evenly in and out of focus across the entire FOV. If not parallel, the waist will translate laterally in the image when the imaging objective is moved. A final check (although one that shouldn't drift much) is to make sure that the scanning mirror that generates the sheet in a scanned system is parallel to the image plane by inspecting the top and bottom edges of the sheet and verifying that both are in focus. One parameter that needs a sample to measure is the thickness of the beam waist. For that we add sub-diffractive beads to the sample at a low concentration, focus on a single bead, and then acquire a stack without moving the imaging objective (just sweeping the sheet over the bead). The axial profile of the bead gives you the thickness of the sheet. Measuring system PSF is a good way to get a sense of whether the system as a whole is in decent optical shape, but isn't as directly informative as the lightsheet alignment checks. For this, we use the same beads as for measuring sheet thickness and pass the resulting stack through PSFj: https://github.com/cmongis/psfj We usually get 100 nm FluoSpheres or TetraSpeck beads for these checks. The diSPIM wiki has excellent illustrations of these alignment checks made by Jon Daniels from ASI. While some details are specific to the diSPIM (software and the dual view alignment) the general principles are common to all instruments. Of course this doesn't include alignment checks for the beam shaping optics for fancier beam profiles like Bessel, Airy or lattices, the overall principle of the alignment checks still hold, though. http://dispim.org/docs/manual#alignment On Mon, Dec 14, 2020, 7:03 PM Radek Machan (Dr) <[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 Anuj for posting this question. I was about to ask the same > question. I wonder if people from Zeiss might answer this post and give us > some idea what they use to test the performance of their Z1 lightsheet. > > Best wishes, > Radek > > Radek MACHAN, Ph.D. | Senior Research Fellow > NOBIC Imaging Facilities<https://www.nobic.sg/Facilities.html> Manager > www.nobic.sg<http://www.nobic.sg> | [hidden email] > > SCELSE, Nanyang Technological University > #B2, 60 Nanyang Drive, SBS-01N-27 > Singapore 637551 > > > ________________________________ > From: Confocal Microscopy List <[hidden email]> on > behalf of Anjul Loiacono <[hidden email]> > Sent: Tuesday, December 15, 2020 6:42 > To: [hidden email] <[hidden email]> > Subject: Lightsheet test target > > ***** > 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. > ***** > > Does anyone know about any commercially available imaging target that can > be used for testing a lightsheet set up? > > Thank you, > Anjul > ________________________________ > > CONFIDENTIALITY: This email is intended solely for the person(s) named and > may be confidential and/or privileged. If you are not the intended > recipient, please delete it, notify us and do not copy, use, or disclose > its contents. > Towards a sustainable earth: Print only when necessary. Thank you. > |
|
|
*****
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 Pavak, thanks for sharing your experiences. How do you prepare the beads for the lightsheet testing, embed them in an agarose block? I'm wondering if there's a way to prepare a more permanent testing sample analogous to bead slides commonly used for PSF measurement of "normal" microscopes. One possibility I know some people are using is a mirror at 45deg. to the lightsheet, which reflect the lightsheet to the imaging objective. Zeiss engineers out there, do you have any such contraption designed specifically for Z1? Best wishes, Radek Radek MACHAN, Ph.D. | Senior Research Fellow NOBIC Imaging Facilities<https://www.nobic.sg/Facilities.html> Manager www.nobic.sg<http://www.nobic.sg> | [hidden email] SCELSE, Nanyang Technological University #B2, 60 Nanyang Drive, SBS-01N-27 Singapore 637551 ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of PAVAK SHAH <[hidden email]> Sent: Wednesday, December 16, 2020 0:04 To: [hidden email] <[hidden email]> Subject: Re: Lightsheet test target ***** 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've never used a Z1 (or other Zeiss lightsheet) so I don't know if the right controls are exposed to the user for this but one beaty of a lightsheet is that almost all critical aspects of system performance and alignment can be trivially visualized using a sample spiked with a little bit of fluorescent dye, for example fluorescein. For any lightsheet system, your two main goals are to check to see whether the excitation beam (for scanned systems) or sheet (for stationary optics) is coming out straight and parallel to the image plane. Straightness is most easily assessed in a scanned system since the beam (when the scanning optic is stationary) should ideally be parallel to the X axis of the camera. To check whether the sheet is parallel, one merely needs to move the imaging objective in and out while observing the stationary beam or sheet. If parallel, it will go evenly in and out of focus across the entire FOV. If not parallel, the waist will translate laterally in the image when the imaging objective is moved. A final check (although one that shouldn't drift much) is to make sure that the scanning mirror that generates the sheet in a scanned system is parallel to the image plane by inspecting the top and bottom edges of the sheet and verifying that both are in focus. One parameter that needs a sample to measure is the thickness of the beam waist. For that we add sub-diffractive beads to the sample at a low concentration, focus on a single bead, and then acquire a stack without moving the imaging objective (just sweeping the sheet over the bead). The axial profile of the bead gives you the thickness of the sheet. Measuring system PSF is a good way to get a sense of whether the system as a whole is in decent optical shape, but isn't as directly informative as the lightsheet alignment checks. For this, we use the same beads as for measuring sheet thickness and pass the resulting stack through PSFj: https://github.com/cmongis/psfj We usually get 100 nm FluoSpheres or TetraSpeck beads for these checks. The diSPIM wiki has excellent illustrations of these alignment checks made by Jon Daniels from ASI. While some details are specific to the diSPIM (software and the dual view alignment) the general principles are common to all instruments. Of course this doesn't include alignment checks for the beam shaping optics for fancier beam profiles like Bessel, Airy or lattices, the overall principle of the alignment checks still hold, though. http://dispim.org/docs/manual#alignment On Mon, Dec 14, 2020, 7:03 PM Radek Machan (Dr) <[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 Anuj for posting this question. I was about to ask the same > question. I wonder if people from Zeiss might answer this post and give us > some idea what they use to test the performance of their Z1 lightsheet. > > Best wishes, > Radek > > Radek MACHAN, Ph.D. | Senior Research Fellow > NOBIC Imaging Facilities<https://www.nobic.sg/Facilities.html> Manager > www.nobic.sg<http://www.nobic.sg> | [hidden email] > > SCELSE, Nanyang Technological University > #B2, 60 Nanyang Drive, SBS-01N-27 > Singapore 637551 > > > ________________________________ > From: Confocal Microscopy List <[hidden email]> on > behalf of Anjul Loiacono <[hidden email]> > Sent: Tuesday, December 15, 2020 6:42 > To: [hidden email] <[hidden email]> > Subject: Lightsheet test target > > ***** > 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. > ***** > > Does anyone know about any commercially available imaging target that can > be used for testing a lightsheet set up? > > Thank you, > Anjul > ________________________________ > > CONFIDENTIALITY: This email is intended solely for the person(s) named and > may be confidential and/or privileged. If you are not the intended > recipient, please delete it, notify us and do not copy, use, or disclose > its contents. > Towards a sustainable earth: Print only when necessary. Thank you. > |
|
|
In reply to this post by Anjul Loiacono
*****
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. ***** We have started working with quantum dots embedded in an epoxy resin. We are using a resin mix published a few years ago by Dodt's group that has an RI near 1.5. Ted |
 
|
Alessandro Ciccarelli |
Re: Lightsheet test target
|
|
In reply to this post by Anjul Loiacono
*****
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 Anjul, when we bought a light-sheet Lavision UMII we got an alignment tool; it is an intelligent glass cuvette filled with some fluo dye and with a thin opaque membrane in the middle; some holes in the membrane can help you to see if the L-sheets (6 in our case) are illuminating the same area. It is not perfect, but quite useful. Maybe you can ask LavisionBiotec /Miltenyi if they can send you one of this probe. I also use TetraSpek beads in agar for checking light-sheet alignments in PBS/water media. Very useful to see camera mis-alignment, chromatic aberrations and PFSs distortions. I ve tried to clear (ECi protcol ) some agar blocks containing beads, but I ve lost the fluo signal (or maybe the beads..), imaging after 1 week . I agree that it would be very useful to have some commercially available/standardized tools for light-sheet alignments. Hope it helps, cheers, Alessandro --- Alessandro Ciccarelli Light Sheet Specialist CALM - Crick Advanced Light Microscopy Facility The Francis Crick Institute 1 Midland Road - London -NW1 1AT United Kingdom T: +442037960913 E: [hidden email]<mailto:[hidden email]> W: www.crick.ac.uk<http://www.crick.ac.uk/> ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Anjul Loiacono <[hidden email]> Sent: 14 December 2020 22:42 To: [hidden email] <[hidden email]> Subject: Lightsheet test target ***** To join, leave or search the confocal microscopy listserv, go to: https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7C%7Ccfef0f6013b947c485a308d8a081984a%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637435825771727506%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=DPE0GBldFB2tMhI879ugALbvT0n5M1DIMAyFSWaHq1I%3D&reserved=0 Post images on https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=04%7C01%7C%7Ccfef0f6013b947c485a308d8a081984a%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637435825771737499%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=4sJJA7TpXEcc3Cd69Xpmi2zGnWapLIx1nrBDA2tSieU%3D&reserved=0 and include the link in your posting. ***** Does anyone know about any commercially available imaging target that can be used for testing a lightsheet set up? Thank you, Anjul The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 1 Midland Road London NW1 1AT |
|
|
*****
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 Anjul, We did almost the same as Alessandro, to make a test sample for our Miltenyi / Lavision Biotec Ultramicroscope II microscope. We have a fixed 12x objective for this microscope and the standard alignment tool (with a kind of a crosshair slit) was not good enough. We also made an ECI cleared agar block with fluorescent beads, though we used another brand which might be more stable (1 µm fluorobrite multicolor beads 1:100 in 1% agarose). At least they are very bright and they are sold at large quantity (ml range). Kind regards, Gert-Jan G.J. Bakker, PhD Researcher / Multiphoton microscopy specialist Dept. of Cell Biology (283) and Microscopic Imaging Centre [hidden email]<mailto:[hidden email]> T +31 (0)24 36 142 96 / T(lab) +31 (0)24 36 515 81 Radboud university medical center P.O.Box 9101, 6500 HB Nijmegen (283), The Netherlands Visiting address: Geert Grooteplein 28 (route 283) https://www.radboudumc.nl/Research/Organisationofresearch/Departments/cellbiology/Pages/default.aspx ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Alessandro Ciccarelli <[hidden email]> Sent: Thursday, December 17, 2020 12:39 PM To: [hidden email] <[hidden email]> Subject: Re: Lightsheet test target ***** 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 Anjul, when we bought a light-sheet Lavision UMII we got an alignment tool; it is an intelligent glass cuvette filled with some fluo dye and with a thin opaque membrane in the middle; some holes in the membrane can help you to see if the L-sheets (6 in our case) are illuminating the same area. It is not perfect, but quite useful. Maybe you can ask LavisionBiotec /Miltenyi if they can send you one of this probe. I also use TetraSpek beads in agar for checking light-sheet alignments in PBS/water media. Very useful to see camera mis-alignment, chromatic aberrations and PFSs distortions. I ve tried to clear (ECi protcol ) some agar blocks containing beads, but I ve lost the fluo signal (or maybe the beads..), imaging after 1 week . I agree that it would be very useful to have some commercially available/standardized tools for light-sheet alignments. Hope it helps, cheers, Alessandro --- Alessandro Ciccarelli Light Sheet Specialist CALM - Crick Advanced Light Microscopy Facility The Francis Crick Institute 1 Midland Road - London -NW1 1AT United Kingdom T: +442037960913 E: [hidden email]<mailto:[hidden email]> W: www.crick.ac.uk<http://www.crick.ac.uk/> ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Anjul Loiacono <[hidden email]> Sent: 14 December 2020 22:42 To: [hidden email] <[hidden email]> Subject: Lightsheet test target ***** To join, leave or search the confocal microscopy listserv, go to: https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7C%7Ccfef0f6013b947c485a308d8a081984a%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637435825771727506%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=DPE0GBldFB2tMhI879ugALbvT0n5M1DIMAyFSWaHq1I%3D&reserved=0 Post images on https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=04%7C01%7C%7Ccfef0f6013b947c485a308d8a081984a%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637435825771737499%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=4sJJA7TpXEcc3Cd69Xpmi2zGnWapLIx1nrBDA2tSieU%3D&reserved=0 and include the link in your posting. ***** Does anyone know about any commercially available imaging target that can be used for testing a lightsheet set up? Thank you, Anjul The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 1 Midland Road London NW1 1AT De informatie in dit bericht is uitsluitend bestemd voor de geadresseerde. Aan dit bericht en de bijlagen kunnen geen rechten worden ontleend. Heeft u deze e-mail onbedoeld ontvangen? Dan verzoeken wij u het te vernietigen en de afzender te informeren. Openbaar maken, kopiëren en verspreiden van deze e-mail of informatie uit deze e-mail is alleen toegestaan met voorafgaande schriftelijke toestemming van de afzender. Het Radboudumc staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The content of this message is intended solely for the addressee. No rights can be derived from this message or its attachments. If you are not the intended recipient, we kindly request you to delete the message and inform the sender. It is strictly prohibited to disclose, copy or distribute this email or the information inside it, without a written consent from the sender. Radboud university medical center is registered with the Dutch Chamber of Commerce trade register with number 41055629. |
|
|
*****
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 Anjul, dear all, Correction, we did not use the fluorobrite beads, but melamine resin based fluorescent beads, see https://www.sigmaaldrich.com/technical-documents/articles/biofiles/fluorescent-microparticles.html . It is mentioned that these are very stable in organic solvents. It worked well to characterize PSF and to perform light sheet alignment, especially with the 12x objective. Sorry for the confusion. I hope this helps. Best regards, Gert-Jan G.J. Bakker, PhD Researcher / Multiphoton microscopy specialist Dept. of Cell Biology (283) and Microscopic Imaging Centre [hidden email]<mailto:[hidden email]> T +31 (0)24 36 142 96 / T(lab) +31 (0)24 36 515 81 Radboud university medical center P.O.Box 9101, 6500 HB Nijmegen (283), The Netherlands Visiting address: Geert Grooteplein 28 (route 283) https://www.radboudumc.nl/Research/Organisationofresearch/Departments/cellbiology/Pages/default.aspx ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Bakker, Gert-Jan <[hidden email]> Sent: Thursday, December 17, 2020 5:30 PM To: [hidden email] <[hidden email]> Subject: Re: Lightsheet test target ***** 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 Anjul, We did almost the same as Alessandro, to make a test sample for our Miltenyi / Lavision Biotec Ultramicroscope II microscope. We have a fixed 12x objective for this microscope and the standard alignment tool (with a kind of a crosshair slit) was not good enough. We also made an ECI cleared agar block with fluorescent beads, though we used another brand which might be more stable (1 µm fluorobrite multicolor beads 1:100 in 1% agarose). At least they are very bright and they are sold at large quantity (ml range). Kind regards, Gert-Jan G.J. Bakker, PhD Researcher / Multiphoton microscopy specialist Dept. of Cell Biology (283) and Microscopic Imaging Centre [hidden email]<mailto:[hidden email]> T +31 (0)24 36 142 96 / T(lab) +31 (0)24 36 515 81 Radboud university medical center P.O.Box 9101, 6500 HB Nijmegen (283), The Netherlands Visiting address: Geert Grooteplein 28 (route 283) https://www.radboudumc.nl/Research/Organisationofresearch/Departments/cellbiology/Pages/default.aspx ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Alessandro Ciccarelli <[hidden email]> Sent: Thursday, December 17, 2020 12:39 PM To: [hidden email] <[hidden email]> Subject: Re: Lightsheet test target ***** 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 Anjul, when we bought a light-sheet Lavision UMII we got an alignment tool; it is an intelligent glass cuvette filled with some fluo dye and with a thin opaque membrane in the middle; some holes in the membrane can help you to see if the L-sheets (6 in our case) are illuminating the same area. It is not perfect, but quite useful. Maybe you can ask LavisionBiotec /Miltenyi if they can send you one of this probe. I also use TetraSpek beads in agar for checking light-sheet alignments in PBS/water media. Very useful to see camera mis-alignment, chromatic aberrations and PFSs distortions. I ve tried to clear (ECi protcol ) some agar blocks containing beads, but I ve lost the fluo signal (or maybe the beads..), imaging after 1 week . I agree that it would be very useful to have some commercially available/standardized tools for light-sheet alignments. Hope it helps, cheers, Alessandro --- Alessandro Ciccarelli Light Sheet Specialist CALM - Crick Advanced Light Microscopy Facility The Francis Crick Institute 1 Midland Road - London -NW1 1AT United Kingdom T: +442037960913 E: [hidden email]<mailto:[hidden email]> W: www.crick.ac.uk<http://www.crick.ac.uk/> ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Anjul Loiacono <[hidden email]> Sent: 14 December 2020 22:42 To: [hidden email] <[hidden email]> Subject: Lightsheet test target ***** To join, leave or search the confocal microscopy listserv, go to: https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7C%7Ccfef0f6013b947c485a308d8a081984a%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637435825771727506%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=DPE0GBldFB2tMhI879ugALbvT0n5M1DIMAyFSWaHq1I%3D&reserved=0 Post images on https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=04%7C01%7C%7Ccfef0f6013b947c485a308d8a081984a%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637435825771737499%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=4sJJA7TpXEcc3Cd69Xpmi2zGnWapLIx1nrBDA2tSieU%3D&reserved=0 and include the link in your posting. ***** Does anyone know about any commercially available imaging target that can be used for testing a lightsheet set up? Thank you, Anjul The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 1 Midland Road London NW1 1AT De informatie in dit bericht is uitsluitend bestemd voor de geadresseerde. Aan dit bericht en de bijlagen kunnen geen rechten worden ontleend. Heeft u deze e-mail onbedoeld ontvangen? Dan verzoeken wij u het te vernietigen en de afzender te informeren. Openbaar maken, kopiëren en verspreiden van deze e-mail of informatie uit deze e-mail is alleen toegestaan met voorafgaande schriftelijke toestemming van de afzender. Het Radboudumc staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The content of this message is intended solely for the addressee. No rights can be derived from this message or its attachments. If you are not the intended recipient, we kindly request you to delete the message and inform the sender. It is strictly prohibited to disclose, copy or distribute this email or the information inside it, without a written consent from the sender. Radboud university medical center is registered with the Dutch Chamber of Commerce trade register with number 41055629. De informatie in dit bericht is uitsluitend bestemd voor de geadresseerde. Aan dit bericht en de bijlagen kunnen geen rechten worden ontleend. Heeft u deze e-mail onbedoeld ontvangen? Dan verzoeken wij u het te vernietigen en de afzender te informeren. Openbaar maken, kopiëren en verspreiden van deze e-mail of informatie uit deze e-mail is alleen toegestaan met voorafgaande schriftelijke toestemming van de afzender. Het Radboudumc staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The content of this message is intended solely for the addressee. No rights can be derived from this message or its attachments. If you are not the intended recipient, we kindly request you to delete the message and inform the sender. It is strictly prohibited to disclose, copy or distribute this email or the information inside it, without a written consent from the sender. Radboud university medical center is registered with the Dutch Chamber of Commerce trade register with number 41055629. |
|
|
*****
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. ***** We also have a LaVision light-sheet system and mostly use the calibration tool that comes with it. However, when we've tried beads we've used iDISCO+ cleared agarose blocks with the following beads: https://www.micromod.de/de/produkte-3-fluoreszent.html (example: Prod.-Nr.: 40-02-103, 1um diameter red) These do not dissolve in DBE, and were recommended by someone from LaVision. Unfortunately, there are no multi-fluorophore versions to evaluate chromatic aberrations. If anyone knows of solvent-stable multi-fluorophore beads (Tetra-speck like), I would be very interested. Best, Pablo Pablo Ariel, Ph.D. Assistant Professor Director of the Microscopy Services Laboratory Brinkhous-Bullitt Bldg B04 Department of Pathology and Laboratory Medicine University of North Carolina at Chapel Hill http://www.med.unc.edu/microscopy Tel: 919-966-2413 -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Bakker, Gert-Jan Sent: Friday, December 18, 2020 3:13 AM To: [hidden email] Subject: Re: Lightsheet test target ***** 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 Anjul, dear all, Correction, we did not use the fluorobrite beads, but melamine resin based fluorescent beads, see https://www.sigmaaldrich.com/technical-documents/articles/biofiles/fluorescent-microparticles.html . It is mentioned that these are very stable in organic solvents. It worked well to characterize PSF and to perform light sheet alignment, especially with the 12x objective. Sorry for the confusion. I hope this helps. Best regards, Gert-Jan G.J. Bakker, PhD Researcher / Multiphoton microscopy specialist Dept. of Cell Biology (283) and Microscopic Imaging Centre [hidden email]<mailto:[hidden email]> T +31 (0)24 36 142 96 / T(lab) +31 (0)24 36 515 81 Radboud university medical center P.O.Box 9101, 6500 HB Nijmegen (283), The Netherlands Visiting address: Geert Grooteplein 28 (route 283) https://www.radboudumc.nl/Research/Organisationofresearch/Departments/cellbiology/Pages/default.aspx ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Bakker, Gert-Jan <[hidden email]> Sent: Thursday, December 17, 2020 5:30 PM To: [hidden email] <[hidden email]> Subject: Re: Lightsheet test target ***** 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 Anjul, We did almost the same as Alessandro, to make a test sample for our Miltenyi / Lavision Biotec Ultramicroscope II microscope. We have a fixed 12x objective for this microscope and the standard alignment tool (with a kind of a crosshair slit) was not good enough. We also made an ECI cleared agar block with fluorescent beads, though we used another brand which might be more stable (1 µm fluorobrite multicolor beads 1:100 in 1% agarose). At least they are very bright and they are sold at large quantity (ml range). Kind regards, Gert-Jan G.J. Bakker, PhD Researcher / Multiphoton microscopy specialist Dept. of Cell Biology (283) and Microscopic Imaging Centre [hidden email]<mailto:[hidden email]> T +31 (0)24 36 142 96 / T(lab) +31 (0)24 36 515 81 Radboud university medical center P.O.Box 9101, 6500 HB Nijmegen (283), The Netherlands Visiting address: Geert Grooteplein 28 (route 283) https://www.radboudumc.nl/Research/Organisationofresearch/Departments/cellbiology/Pages/default.aspx ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Alessandro Ciccarelli <[hidden email]> Sent: Thursday, December 17, 2020 12:39 PM To: [hidden email] <[hidden email]> Subject: Re: Lightsheet test target ***** 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 Anjul, when we bought a light-sheet Lavision UMII we got an alignment tool; it is an intelligent glass cuvette filled with some fluo dye and with a thin opaque membrane in the middle; some holes in the membrane can help you to see if the L-sheets (6 in our case) are illuminating the same area. It is not perfect, but quite useful. Maybe you can ask LavisionBiotec /Miltenyi if they can send you one of this probe. I also use TetraSpek beads in agar for checking light-sheet alignments in PBS/water media. Very useful to see camera mis-alignment, chromatic aberrations and PFSs distortions. I ve tried to clear (ECi protcol ) some agar blocks containing beads, but I ve lost the fluo signal (or maybe the beads..), imaging after 1 week . I agree that it would be very useful to have some commercially available/standardized tools for light-sheet alignments. Hope it helps, cheers, Alessandro --- Alessandro Ciccarelli Light Sheet Specialist CALM - Crick Advanced Light Microscopy Facility The Francis Crick Institute 1 Midland Road - London -NW1 1AT United Kingdom T: +442037960913 E: [hidden email]<mailto:[hidden email]> W: www.crick.ac.uk<http://www.crick.ac.uk/> ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Anjul Loiacono <[hidden email]> Sent: 14 December 2020 22:42 To: [hidden email] <[hidden email]> Subject: Lightsheet test target ***** To join, leave or search the confocal microscopy listserv, go to: https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7C%7Ccfef0f6013b947c485a308d8a081984a%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637435825771727506%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=DPE0GBldFB2tMhI879ugALbvT0n5M1DIMAyFSWaHq1I%3D&reserved=0 Post images on https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=04%7C01%7C%7Ccfef0f6013b947c485a308d8a081984a%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637435825771737499%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=4sJJA7TpXEcc3Cd69Xpmi2zGnWapLIx1nrBDA2tSieU%3D&reserved=0 and include the link in your posting. ***** Does anyone know about any commercially available imaging target that can be used for testing a lightsheet set up? Thank you, Anjul The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 1 Midland Road London NW1 1AT De informatie in dit bericht is uitsluitend bestemd voor de geadresseerde. Aan dit bericht en de bijlagen kunnen geen rechten worden ontleend. Heeft u deze e-mail onbedoeld ontvangen? Dan verzoeken wij u het te vernietigen en de afzender te informeren. Openbaar maken, kopiëren en verspreiden van deze e-mail of informatie uit deze e-mail is alleen toegestaan met voorafgaande schriftelijke toestemming van de afzender. Het Radboudumc staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The content of this message is intended solely for the addressee. No rights can be derived from this message or its attachments. If you are not the intended recipient, we kindly request you to delete the message and inform the sender. It is strictly prohibited to disclose, copy or distribute this email or the information inside it, without a written consent from the sender. Radboud university medical center is registered with the Dutch Chamber of Commerce trade register with number 41055629. De informatie in dit bericht is uitsluitend bestemd voor de geadresseerde. Aan dit bericht en de bijlagen kunnen geen rechten worden ontleend. Heeft u deze e-mail onbedoeld ontvangen? Dan verzoeken wij u het te vernietigen en de afzender te informeren. Openbaar maken, kopiëren en verspreiden van deze e-mail of informatie uit deze e-mail is alleen toegestaan met voorafgaande schriftelijke toestemming van de afzender. Het Radboudumc staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The content of this message is intended solely for the addressee. No rights can be derived from this message or its attachments. If you are not the intended recipient, we kindly request you to delete the message and inform the sender. It is strictly prohibited to disclose, copy or distribute this email or the information inside it, without a written consent from the sender. Radboud university medical center is registered with the Dutch Chamber of Commerce trade register with number 41055629. |
|
|
*****
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. ***** We have always used mirror & grid test targets inserted under 45 degree into the chamber. You can homebuilt them by cutting a microscope test slide with a diamond pen, and then gluing it (eg with UV curable optics glue). If you translate the mirror through the chamber along the light sheet, you sample the beam profile at different positions and can measure light sheet waist. Here’s an image different versions I used during my PhD in Jan Huisken's lab. I don’t think you could ever buy anything ready made though. https://seafile.ist.ac.at/f/90fec884a8f24cfaa721/ Regarding bead samples, if you store the agarose embedded beads in a falcon with some distilled water to prevent the agarose from drying out, I found them to be good for several months. I’ve also used dye solution mixed with agarose to visualise the light sheets. These bleach after 1-2 weeks when stored at room temperature. For something more durable: I’ve been wondering whether the UV curable polymer from this pre-print could be used to embed beads for something more durable. The company has polymers with a range of refractive indices as well. I have not tried it though. https://www.biorxiv.org/content/10.1101/2020.10.04.324996v1.article-metrics https://www.mypolymers.com/bio-133-enables-diverse-applications-in-fluorescence-microscopy Best, Wiebke -- ------------------------------------------------------------------ Wiebke Jahr HFSP postdoctoral fellow Danzl Group, Institute of Science and Technology Austria Am Campus 1, 3400 Klosterneuburg phone: +43 2243 9000-2063 (office)/ -2061 (lab) ------------------------------------------------------------------ On 18.12.2020, at 13:12, Ariel, Pablo <[hidden email]<mailto:[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. ***** We also have a LaVision light-sheet system and mostly use the calibration tool that comes with it. However, when we've tried beads we've used iDISCO+ cleared agarose blocks with the following beads: https://www.micromod.de/de/produkte-3-fluoreszent.html (example: Prod.-Nr.: 40-02-103, 1um diameter red) These do not dissolve in DBE, and were recommended by someone from LaVision. Unfortunately, there are no multi-fluorophore versions to evaluate chromatic aberrations. If anyone knows of solvent-stable multi-fluorophore beads (Tetra-speck like), I would be very interested. Best, Pablo Pablo Ariel, Ph.D. Assistant Professor Director of the Microscopy Services Laboratory Brinkhous-Bullitt Bldg B04 Department of Pathology and Laboratory Medicine University of North Carolina at Chapel Hill http://www.med.unc.edu/microscopy Tel: 919-966-2413 -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Bakker, Gert-Jan Sent: Friday, December 18, 2020 3:13 AM To: [hidden email] Subject: Re: Lightsheet test target ***** 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 Anjul, dear all, Correction, we did not use the fluorobrite beads, but melamine resin based fluorescent beads, see https://www.sigmaaldrich.com/technical-documents/articles/biofiles/fluorescent-microparticles.html . It is mentioned that these are very stable in organic solvents. It worked well to characterize PSF and to perform light sheet alignment, especially with the 12x objective. Sorry for the confusion. I hope this helps. Best regards, Gert-Jan G.J. Bakker, PhD Researcher / Multiphoton microscopy specialist Dept. of Cell Biology (283) and Microscopic Imaging Centre [hidden email]<mailto:[hidden email]> T +31 (0)24 36 142 96 / T(lab) +31 (0)24 36 515 81 Radboud university medical center P.O.Box 9101, 6500 HB Nijmegen (283), The Netherlands Visiting address: Geert Grooteplein 28 (route 283) https://www.radboudumc.nl/Research/Organisationofresearch/Departments/cellbiology/Pages/default.aspx ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Bakker, Gert-Jan <[hidden email]> Sent: Thursday, December 17, 2020 5:30 PM To: [hidden email] <[hidden email]> Subject: Re: Lightsheet test target ***** 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 Anjul, We did almost the same as Alessandro, to make a test sample for our Miltenyi / Lavision Biotec Ultramicroscope II microscope. We have a fixed 12x objective for this microscope and the standard alignment tool (with a kind of a crosshair slit) was not good enough. We also made an ECI cleared agar block with fluorescent beads, though we used another brand which might be more stable (1 µm fluorobrite multicolor beads 1:100 in 1% agarose). At least they are very bright and they are sold at large quantity (ml range). Kind regards, Gert-Jan G.J. Bakker, PhD Researcher / Multiphoton microscopy specialist Dept. of Cell Biology (283) and Microscopic Imaging Centre [hidden email]<mailto:[hidden email]> T +31 (0)24 36 142 96 / T(lab) +31 (0)24 36 515 81 Radboud university medical center P.O.Box 9101, 6500 HB Nijmegen (283), The Netherlands Visiting address: Geert Grooteplein 28 (route 283) https://www.radboudumc.nl/Research/Organisationofresearch/Departments/cellbiology/Pages/default.aspx ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Alessandro Ciccarelli <[hidden email]> Sent: Thursday, December 17, 2020 12:39 PM To: [hidden email] <[hidden email]> Subject: Re: Lightsheet test target ***** 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 Anjul, when we bought a light-sheet Lavision UMII we got an alignment tool; it is an intelligent glass cuvette filled with some fluo dye and with a thin opaque membrane in the middle; some holes in the membrane can help you to see if the L-sheets (6 in our case) are illuminating the same area. It is not perfect, but quite useful. Maybe you can ask LavisionBiotec /Miltenyi if they can send you one of this probe. I also use TetraSpek beads in agar for checking light-sheet alignments in PBS/water media. Very useful to see camera mis-alignment, chromatic aberrations and PFSs distortions. I ve tried to clear (ECi protcol ) some agar blocks containing beads, but I ve lost the fluo signal (or maybe the beads..), imaging after 1 week . I agree that it would be very useful to have some commercially available/standardized tools for light-sheet alignments. Hope it helps, cheers, Alessandro --- Alessandro Ciccarelli Light Sheet Specialist CALM - Crick Advanced Light Microscopy Facility The Francis Crick Institute 1 Midland Road - London -NW1 1AT United Kingdom T: +442037960913 E: [hidden email]<mailto:[hidden email]> W: www.crick.ac.uk<http://www.crick.ac.uk/> ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Anjul Loiacono <[hidden email]> Sent: 14 December 2020 22:42 To: [hidden email] <[hidden email]> Subject: Lightsheet test target ***** To join, leave or search the confocal microscopy listserv, go to: https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7C%7Ccfef0f6013b947c485a308d8a081984a%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637435825771727506%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=DPE0GBldFB2tMhI879ugALbvT0n5M1DIMAyFSWaHq1I%3D&reserved=0 Post images on https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=04%7C01%7C%7Ccfef0f6013b947c485a308d8a081984a%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637435825771737499%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=4sJJA7TpXEcc3Cd69Xpmi2zGnWapLIx1nrBDA2tSieU%3D&reserved=0 and include the link in your posting. ***** Does anyone know about any commercially available imaging target that can be used for testing a lightsheet set up? Thank you, Anjul The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 1 Midland Road London NW1 1AT De informatie in dit bericht is uitsluitend bestemd voor de geadresseerde. Aan dit bericht en de bijlagen kunnen geen rechten worden ontleend. Heeft u deze e-mail onbedoeld ontvangen? Dan verzoeken wij u het te vernietigen en de afzender te informeren. Openbaar maken, kopiëren en verspreiden van deze e-mail of informatie uit deze e-mail is alleen toegestaan met voorafgaande schriftelijke toestemming van de afzender. Het Radboudumc staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The content of this message is intended solely for the addressee. No rights can be derived from this message or its attachments. If you are not the intended recipient, we kindly request you to delete the message and inform the sender. It is strictly prohibited to disclose, copy or distribute this email or the information inside it, without a written consent from the sender. Radboud university medical center is registered with the Dutch Chamber of Commerce trade register with number 41055629. De informatie in dit bericht is uitsluitend bestemd voor de geadresseerde. Aan dit bericht en de bijlagen kunnen geen rechten worden ontleend. Heeft u deze e-mail onbedoeld ontvangen? Dan verzoeken wij u het te vernietigen en de afzender te informeren. Openbaar maken, kopiëren en verspreiden van deze e-mail of informatie uit deze e-mail is alleen toegestaan met voorafgaande schriftelijke toestemming van de afzender. Het Radboudumc staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The content of this message is intended solely for the addressee. No rights can be derived from this message or its attachments. If you are not the intended recipient, we kindly request you to delete the message and inform the sender. It is strictly prohibited to disclose, copy or distribute this email or the information inside it, without a written consent from the sender. Radboud university medical center is registered with the Dutch Chamber of Commerce trade register with number 41055629. |
|
|
*****
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 typically use 200nm fluorescent beads in agarose to acquire PSFs. We have Zeiss Lightsheet Z1 with dual side illumination. Although it is a very fine microscope, the ours appears to have some problem: the waists of left and right lightsheets do not exactly coincide in X dimension (along the lightsheet). I think they should (correct me if I am wrong). I know the serviceman uses the mirror prep to evaluate the system. I imagine that, ideally, the mirror prep after rotation (i.e. changing between 45-degree-position for left and right illumination) should reflect the laser light towards the detection lens at exactly the same position in X dimension. However, this might not always be the case, especially with home-made mirror preps. This might be the reason why everything looks fine after service with the mirror prep but the waists fail to coincide in dual-side illumination systems. best, Tomasz Tomasz Wegierski, PhD International Institute of Molecular and Cell Biology Trojdena 4, 02-109 Warsaw, POLAND tel: +48-22 597 0763 fax: +48 22 597 0715 http://www.iimcb.gov.pl/ ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Wiebke JAHR <[hidden email]> Sent: Monday, December 21, 2020 10:57 AM To: [hidden email] <[hidden email]> Subject: Re: Lightsheet test target ***** 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. ***** We have always used mirror & grid test targets inserted under 45 degree into the chamber. You can homebuilt them by cutting a microscope test slide with a diamond pen, and then gluing it (eg with UV curable optics glue). If you translate the mirror through the chamber along the light sheet, you sample the beam profile at different positions and can measure light sheet waist. Here’s an image different versions I used during my PhD in Jan Huisken's lab. I don’t think you could ever buy anything ready made though. https://seafile.ist.ac.at/f/90fec884a8f24cfaa721/ Regarding bead samples, if you store the agarose embedded beads in a falcon with some distilled water to prevent the agarose from drying out, I found them to be good for several months. I’ve also used dye solution mixed with agarose to visualise the light sheets. These bleach after 1-2 weeks when stored at room temperature. For something more durable: I’ve been wondering whether the UV curable polymer from this pre-print could be used to embed beads for something more durable. The company has polymers with a range of refractive indices as well. I have not tried it though. https://www.biorxiv.org/content/10.1101/2020.10.04.324996v1.article-metrics https://www.mypolymers.com/bio-133-enables-diverse-applications-in-fluorescence-microscopy Best, Wiebke -- ------------------------------------------------------------------ Wiebke Jahr HFSP postdoctoral fellow Danzl Group, Institute of Science and Technology Austria Am Campus 1, 3400 Klosterneuburg phone: +43 2243 9000-2063 (office)/ -2061 (lab) ------------------------------------------------------------------ On 18.12.2020, at 13:12, Ariel, Pablo <[hidden email]<mailto:[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. ***** We also have a LaVision light-sheet system and mostly use the calibration tool that comes with it. However, when we've tried beads we've used iDISCO+ cleared agarose blocks with the following beads: https://www.micromod.de/de/produkte-3-fluoreszent.html (example: Prod.-Nr.: 40-02-103, 1um diameter red) These do not dissolve in DBE, and were recommended by someone from LaVision. Unfortunately, there are no multi-fluorophore versions to evaluate chromatic aberrations. If anyone knows of solvent-stable multi-fluorophore beads (Tetra-speck like), I would be very interested. Best, Pablo Pablo Ariel, Ph.D. Assistant Professor Director of the Microscopy Services Laboratory Brinkhous-Bullitt Bldg B04 Department of Pathology and Laboratory Medicine University of North Carolina at Chapel Hill http://www.med.unc.edu/microscopy Tel: 919-966-2413 -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Bakker, Gert-Jan Sent: Friday, December 18, 2020 3:13 AM To: [hidden email] Subject: Re: Lightsheet test target ***** 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 Anjul, dear all, Correction, we did not use the fluorobrite beads, but melamine resin based fluorescent beads, see https://www.sigmaaldrich.com/technical-documents/articles/biofiles/fluorescent-microparticles.html . It is mentioned that these are very stable in organic solvents. It worked well to characterize PSF and to perform light sheet alignment, especially with the 12x objective. Sorry for the confusion. I hope this helps. Best regards, Gert-Jan G.J. Bakker, PhD Researcher / Multiphoton microscopy specialist Dept. of Cell Biology (283) and Microscopic Imaging Centre [hidden email]<mailto:[hidden email]> T +31 (0)24 36 142 96 / T(lab) +31 (0)24 36 515 81 Radboud university medical center P.O.Box 9101, 6500 HB Nijmegen (283), The Netherlands Visiting address: Geert Grooteplein 28 (route 283) https://www.radboudumc.nl/Research/Organisationofresearch/Departments/cellbiology/Pages/default.aspx ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Bakker, Gert-Jan <[hidden email]> Sent: Thursday, December 17, 2020 5:30 PM To: [hidden email] <[hidden email]> Subject: Re: Lightsheet test target ***** 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 Anjul, We did almost the same as Alessandro, to make a test sample for our Miltenyi / Lavision Biotec Ultramicroscope II microscope. We have a fixed 12x objective for this microscope and the standard alignment tool (with a kind of a crosshair slit) was not good enough. We also made an ECI cleared agar block with fluorescent beads, though we used another brand which might be more stable (1 µm fluorobrite multicolor beads 1:100 in 1% agarose). At least they are very bright and they are sold at large quantity (ml range). Kind regards, Gert-Jan G.J. Bakker, PhD Researcher / Multiphoton microscopy specialist Dept. of Cell Biology (283) and Microscopic Imaging Centre [hidden email]<mailto:[hidden email]> T +31 (0)24 36 142 96 / T(lab) +31 (0)24 36 515 81 Radboud university medical center P.O.Box 9101, 6500 HB Nijmegen (283), The Netherlands Visiting address: Geert Grooteplein 28 (route 283) https://www.radboudumc.nl/Research/Organisationofresearch/Departments/cellbiology/Pages/default.aspx ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Alessandro Ciccarelli <[hidden email]> Sent: Thursday, December 17, 2020 12:39 PM To: [hidden email] <[hidden email]> Subject: Re: Lightsheet test target ***** 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 Anjul, when we bought a light-sheet Lavision UMII we got an alignment tool; it is an intelligent glass cuvette filled with some fluo dye and with a thin opaque membrane in the middle; some holes in the membrane can help you to see if the L-sheets (6 in our case) are illuminating the same area. It is not perfect, but quite useful. Maybe you can ask LavisionBiotec /Miltenyi if they can send you one of this probe. I also use TetraSpek beads in agar for checking light-sheet alignments in PBS/water media. Very useful to see camera mis-alignment, chromatic aberrations and PFSs distortions. I ve tried to clear (ECi protcol ) some agar blocks containing beads, but I ve lost the fluo signal (or maybe the beads..), imaging after 1 week . I agree that it would be very useful to have some commercially available/standardized tools for light-sheet alignments. Hope it helps, cheers, Alessandro --- Alessandro Ciccarelli Light Sheet Specialist CALM - Crick Advanced Light Microscopy Facility The Francis Crick Institute 1 Midland Road - London -NW1 1AT United Kingdom T: +442037960913 E: [hidden email]<mailto:[hidden email]> W: www.crick.ac.uk<http://www.crick.ac.uk/> ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Anjul Loiacono <[hidden email]> Sent: 14 December 2020 22:42 To: [hidden email] <[hidden email]> Subject: Lightsheet test target ***** To join, leave or search the confocal microscopy listserv, go to: https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7C%7Ccfef0f6013b947c485a308d8a081984a%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637435825771727506%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=DPE0GBldFB2tMhI879ugALbvT0n5M1DIMAyFSWaHq1I%3D&reserved=0 Post images on https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=04%7C01%7C%7Ccfef0f6013b947c485a308d8a081984a%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637435825771737499%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=4sJJA7TpXEcc3Cd69Xpmi2zGnWapLIx1nrBDA2tSieU%3D&reserved=0 and include the link in your posting. ***** Does anyone know about any commercially available imaging target that can be used for testing a lightsheet set up? Thank you, Anjul The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 1 Midland Road London NW1 1AT De informatie in dit bericht is uitsluitend bestemd voor de geadresseerde. Aan dit bericht en de bijlagen kunnen geen rechten worden ontleend. Heeft u deze e-mail onbedoeld ontvangen? Dan verzoeken wij u het te vernietigen en de afzender te informeren. Openbaar maken, kopiëren en verspreiden van deze e-mail of informatie uit deze e-mail is alleen toegestaan met voorafgaande schriftelijke toestemming van de afzender. Het Radboudumc staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The content of this message is intended solely for the addressee. No rights can be derived from this message or its attachments. If you are not the intended recipient, we kindly request you to delete the message and inform the sender. It is strictly prohibited to disclose, copy or distribute this email or the information inside it, without a written consent from the sender. Radboud university medical center is registered with the Dutch Chamber of Commerce trade register with number 41055629. De informatie in dit bericht is uitsluitend bestemd voor de geadresseerde. Aan dit bericht en de bijlagen kunnen geen rechten worden ontleend. Heeft u deze e-mail onbedoeld ontvangen? Dan verzoeken wij u het te vernietigen en de afzender te informeren. Openbaar maken, kopiëren en verspreiden van deze e-mail of informatie uit deze e-mail is alleen toegestaan met voorafgaande schriftelijke toestemming van de afzender. Het Radboudumc staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The content of this message is intended solely for the addressee. No rights can be derived from this message or its attachments. If you are not the intended recipient, we kindly request you to delete the message and inform the sender. It is strictly prohibited to disclose, copy or distribute this email or the information inside it, without a written consent from the sender. Radboud university medical center is registered with the Dutch Chamber of Commerce trade register with number 41055629. |
|
|
In reply to this post by Anjul Loiacono
*****
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 Tomasz et al. on a light sheet microscope with double-sided illumination, it is not uncommon to have the waists of the two LS separated a bit. If you assume that you will use the left LS primarily to illuminate the left part of the sample, you may want to have the waist more on that side. You could even put the waist of the left LS all the way in the middle of the left half of your FOV and the waist of the right LS in the middle of the right half. As a result, both LS can be made thinner by a factor of 1 / sqrt(2) = 0.7, because each LS only needs to cover half of the FOV (Huisken & Stainier, Opt. Lett. 2007). However, since the sample never fills the entire FOV exactly and you want some overlap of the two datasets in the middle, you would want to move the two waists a bit closer... Regarding the alignment mirror, we started making our own little mirrors at some point. We are happy to share them with anyone who is interested. They have a fully reflective part, a grid, parallel lines, and a target similar to the USAF target. A grid is fantastic as an alignment tool as it nicely shows you all the critical parameters of the light sheet(s). You can see precisely where the waist is and if the sheet is in focus, at an angle or turned, etc. It actually does not matter if the mirror's angle is accurately 45deg. You are basically looking at the bright stripe that the LS produces on a tilted surface. The stripes or grid lines on the target simply help you see where the focal plane of your detection system is. This figure may give you an idea of the alignment process: https://imgur.com/tUrwtuM Please let me know if you need any more help! Best Jan — Jan Huisken Director of Medical Engineering Lead Investigator of Multiscale Imaging Morgridge Institute for Research 330 N Orchard St Madison, WI 53715, USA www.morgridge.org/huisken www.involv3d.org @huiskenlab |
«
Return to Confocal Microscopy List
|
1 view|%1 views
| Free forum by Nabble | Edit this page |