photoactivation Dendra2 with LSM710
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Claudia Florindo |
photoactivation Dendra2 with LSM710
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear all confocal list members I need some help on how to activate pDendra2 on LSM710. In our facility we have an lsm710 with 405, Argon, 564 and 633 lasers. I am trying to establish condition to photo activate pDendra2. I checked in some papers that pDendra can be activated with mercury bulbs, metal halide bulbs. I have tested that and with dendra metal halide works pretty well. I have also saw that 405 laser line could be use to activate dendra. It would be very useful for me to use the laser from the confocal, in that way I could direct to activation to a very small region, compared to all cell with metal halide bulbs. The problem is that: I either blast out the fluorescence and I get no activation (to strong condition) or, on the other hand if I do not blast I still do not activate dendra. I should say that after some attempt with the lasers if I use the mercury bulb I am able to photo activate dendra. Can anybody help me? Does anyone have a protocol that I could use? Feel free to write me outside the list, Thank you all, Best regards Claudia. ____________________________ Claudia Florindo PhD, Microscopy Unit Manager Email : [hidden email] phone: 00351 289 244 489 address: Departamento de Ciências Biomédicas e Medicina Ed 8 Lab 1.18 Campus de Gamelas Universidade do Algarve 8005-139 Faro Portugal |
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Scott, Mark |
Re: photoactivation Dendra2 with LSM710
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Claudia, A colleague of mine has tried this (amongst others) and while we ended up switching/activating with a multi-photon laser, we did it to coincide with the 405nm laser wavelength since that is what is suggested and we did not have a 405 laser hooked up to the microscope we were using. I did notice that the switching itself usually went quite quickly but the 2-photon laser we were using was much more powerful than our 405 would have been. Other proteins we have tried took longer so I think while you are correct in using 405nm to activate this, it may just be a case of trialling different power and exposure times to get it to activate without bleaching. Good luck Mark Scott FILM - Facility for Imaging by Light Microscopy Senior Research Technician Sir Alexander Fleming Building, desk 408 Imperial College London / South Kensington Exhibition Road London SW7 2AZ UK Tel: ++44(0)20-759-49793 E-mail: [hidden email] Website: http://imperial.ac.uk/imagingfacility -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Claudia Florindo Sent: 27 May 2011 15:26 To: [hidden email] Subject: photoactivation Dendra2 with LSM710 ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear all confocal list members I need some help on how to activate pDendra2 on LSM710. In our facility we have an lsm710 with 405, Argon, 564 and 633 lasers. I am trying to establish condition to photo activate pDendra2. I checked in some papers that pDendra can be activated with mercury bulbs, metal halide bulbs. I have tested that and with dendra metal halide works pretty well. I have also saw that 405 laser line could be use to activate dendra. It would be very useful for me to use the laser from the confocal, in that way I could direct to activation to a very small region, compared to all cell with metal halide bulbs. The problem is that: I either blast out the fluorescence and I get no activation (to strong condition) or, on the other hand if I do not blast I still do not activate dendra. I should say that after some attempt with the lasers if I use the mercury bulb I am able to photo activate dendra. Can anybody help me? Does anyone have a protocol that I could use? Feel free to write me outside the list, Thank you all, Best regards Claudia. ____________________________ Claudia Florindo PhD, Microscopy Unit Manager Email : [hidden email] phone: 00351 289 244 489 address: Departamento de Ciências Biomédicas e Medicina Ed 8 Lab 1.18 Campus de Gamelas Universidade do Algarve 8005-139 Faro Portugal |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I want to second this comment. A few years ago I worked out photoconverting dendra using a 405 laser on a Leica SP5. The trick was to keep the laser and exposure times high enough to convert without bleaching. This was a very tricky balance, but we were able to get it to work in cultured cells. Subsequently, people in the lab did it in live animals though a surgically implanted window. -Michael C. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Scott, Mark Sent: Friday, May 27, 2011 11:16 AM To: [hidden email] Subject: Re: photoactivation Dendra2 with LSM710 ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Claudia, A colleague of mine has tried this (amongst others) and while we ended up switching/activating with a multi-photon laser, we did it to coincide with the 405nm laser wavelength since that is what is suggested and we did not have a 405 laser hooked up to the microscope we were using. I did notice that the switching itself usually went quite quickly but the 2-photon laser we were using was much more powerful than our 405 would have been. Other proteins we have tried took longer so I think while you are correct in using 405nm to activate this, it may just be a case of trialling different power and exposure times to get it to activate without bleaching. Good luck Mark Scott FILM - Facility for Imaging by Light Microscopy Senior Research Technician Sir Alexander Fleming Building, desk 408 Imperial College London / South Kensington Exhibition Road London SW7 2AZ UK Tel: ++44(0)20-759-49793 E-mail: [hidden email] Website: http://imperial.ac.uk/imagingfacility -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Claudia Florindo Sent: 27 May 2011 15:26 To: [hidden email] Subject: photoactivation Dendra2 with LSM710 ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear all confocal list members I need some help on how to activate pDendra2 on LSM710. In our facility we have an lsm710 with 405, Argon, 564 and 633 lasers. I am trying to establish condition to photo activate pDendra2. I checked in some papers that pDendra can be activated with mercury bulbs, metal halide bulbs. I have tested that and with dendra metal halide works pretty well. I have also saw that 405 laser line could be use to activate dendra. It would be very useful for me to use the laser from the confocal, in that way I could direct to activation to a very small region, compared to all cell with metal halide bulbs. The problem is that: I either blast out the fluorescence and I get no activation (to strong condition) or, on the other hand if I do not blast I still do not activate dendra. I should say that after some attempt with the lasers if I use the mercury bulb I am able to photo activate dendra. Can anybody help me? Does anyone have a protocol that I could use? Feel free to write me outside the list, Thank you all, Best regards Claudia. ____________________________ Claudia Florindo PhD, Microscopy Unit Manager Email : [hidden email] phone: 00351 289 244 489 address: Departamento de Ciências Biomédicas e Medicina Ed 8 Lab 1.18 Campus de Gamelas Universidade do Algarve 8005-139 Faro Portugal ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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Claudia Florindo |
Re: photoactivation Dendra2 with LSM710
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear confocal list members Thank you all for your help, I will try again using the informations you gave me Very best regards Claudia From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael Sent: sexta-feira, 27 de Maio de 2011 16:47 To: [hidden email] Subject: Re: photoactivation Dendra2 with LSM710 ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I want to second this comment. A few years ago I worked out photoconverting dendra using a 405 laser on a Leica SP5. The trick was to keep the laser and exposure times high enough to convert without bleaching. This was a very tricky balance, but we were able to get it to work in cultured cells. Subsequently, people in the lab did it in live animals though a surgically implanted window. -Michael C. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Scott, Mark Sent: Friday, May 27, 2011 11:16 AM To: [hidden email] Subject: Re: photoactivation Dendra2 with LSM710 ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Claudia, A colleague of mine has tried this (amongst others) and while we ended up switching/activating with a multi-photon laser, we did it to coincide with the 405nm laser wavelength since that is what is suggested and we did not have a 405 laser hooked up to the microscope we were using. I did notice that the switching itself usually went quite quickly but the 2-photon laser we were using was much more powerful than our 405 would have been. Other proteins we have tried took longer so I think while you are correct in using 405nm to activate this, it may just be a case of trialling different power and exposure times to get it to activate without bleaching. Good luck Mark Scott FILM - Facility for Imaging by Light Microscopy Senior Research Technician Sir Alexander Fleming Building, desk 408 Imperial College London / South Kensington Exhibition Road London SW7 2AZ UK Tel: ++44(0)20-759-49793 E-mail: [hidden email] Website: http://imperial.ac.uk/imagingfacility -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Claudia Florindo Sent: 27 May 2011 15:26 To: [hidden email] Subject: photoactivation Dendra2 with LSM710 ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear all confocal list members I need some help on how to activate pDendra2 on LSM710. In our facility we have an lsm710 with 405, Argon, 564 and 633 lasers. I am trying to establish condition to photo activate pDendra2. I checked in some papers that pDendra can be activated with mercury bulbs, metal halide bulbs. I have tested that and with dendra metal halide works pretty well. I have also saw that 405 laser line could be use to activate dendra. It would be very useful for me to use the laser from the confocal, in that way I could direct to activation to a very small region, compared to all cell with metal halide bulbs. The problem is that: I either blast out the fluorescence and I get no activation (to strong condition) or, on the other hand if I do not blast I still do not activate dendra. I should say that after some attempt with the lasers if I use the mercury bulb I am able to photo activate dendra. Can anybody help me? Does anyone have a protocol that I could use? Feel free to write me outside the list, Thank you all, Best regards Claudia. ____________________________ Claudia Florindo PhD, Microscopy Unit Manager Email : [hidden email] phone: 00351 289 244 489 address: Departamento de Ciências Biomédicas e Medicina Ed 8 Lab 1.18 Campus de Gamelas Universidade do Algarve 8005-139 Faro Portugal ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= _____ Nenhum vírus encontrado nessa mensagem. Verificado por AVG - www.avgbrasil.com.br Versão: 10.0.1325 / Banco de dados de vírus: 1509/3663 - Data de Lançamento: 05/27/11 |
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