Acapella looping through files in a folder
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John Bradley |
Acapella looping through files in a folder
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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 have multiple pairs of tif images that I'd like to analyze using Acapella. I can hard code a particular pair of images I want to analyze and the analysis works. However, could someone please point me in the right direction for which approach to take for looping through many pairs of images in a folder. Many thanks, John |
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Emmanuel Gustin |
Re: Acapella looping through files in a folder
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Assuming that your pairs of images are numbered in a consistent pattern, the easiest way is probably to write a foreach() loop over the sequence of numbers, and then use the sprintf() function to create your filename. You can then use this filename for your further processing: foreach(1..numImages, "idx", 1) sprintf(filename1, "Blue%002d.tif", idx) sprintf(filename2, "Green%002d.tif", idx) ... end() Google for the syntax of the formatting expression in sprintf(), Acapella uses the same syntax as C++. A code in the form %002d generates a fields of three digits with leading zeroes, so 001, 002, 003, ... The alternative, but that is rather trickier, is to use glob() with a suitable pattern to get a list of the files for one channel, e.g. glob("Blue*.tif"), then use pathsplit() to isolate the filename from the full path, then regex() to break the filename into the relevant parts, and finally sprintf() to construct the filename for the matching file. I would use that only if the pattern of your filenames is really unpredictable. Best Regards, Emmanuel -- Emmanuel Gustin, Tel. (+32) 14 64 1586, e-mail: [hidden email] ! telephone number changed ! -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Bradley Sent: donderdag 18 november 2010 15:57 To: [hidden email] Subject: Acapella looping through files in a folder ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I have multiple pairs of tif images that I'd like to analyze using Acapella. I can hard code a particular pair of images I want to analyze and the analysis works. However, could someone please point me in the right direction for which approach to take for looping through many pairs of images in a folder. Many thanks, John |
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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 microscopists, I'm planning to go through a regimen of adjusting the MaiTai DeepSee on a variety of samples with different optics to see what changes there are in depth penetration (and, maybe, preservation of biological activity). Does anybody have experience with this? If so, have you found that the DeepSee provides a noticeable practical benefit over the MaiTai without it? Any comments welcome and please feel free to contact me offline at [hidden email] Thank you! Sincerely, Michael C. ------------------------------------------------------------ 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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Cameron Nowell |
Re: MaiTai DeepSee adjustments on multiphoton microscope
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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 Michael, We have the DeepSee on our MaiTai and have found that it doesn't do a lot on our system. At extreme depths, say greater than 500um you can get a small enhancement in signal strength and quality but it is only very minor. It may give you a few extra microns penetration but thats about it. It really depends on how much the optics of your system are dispersing/chirping the beam. If they are not altering it much then pre-chirping it will not do a lot. This is all based on qualitative assessment, i have not quantitated any of this. Also we have recently received a new Olympus 25x NA1.05 objective specifically designed for MP imaging. I have not seen how this performs yet with the DeepSee. Cheers Cam Cameron J. Nowell Microscopy Manager Centre for Advanced Microscopy Ludwig Institute for Cancer Research PO Box 2008 Royal Melbourne Hospital Victoria, 3050 AUSTRALIA Office: +61 3 9341 3155 Mobile: +61422882700 Fax: +61 3 9341 3104 Facility Website -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael Sent: Friday, 19 November 2010 4:08 AM To: [hidden email] Subject: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear microscopists, I'm planning to go through a regimen of adjusting the MaiTai DeepSee on a variety of samples with different optics to see what changes there are in depth penetration (and, maybe, preservation of biological activity). Does anybody have experience with this? If so, have you found that the DeepSee provides a noticeable practical benefit over the MaiTai without it? Any comments welcome and please feel free to contact me offline at [hidden email] Thank you! Sincerely, Michael C. ------------------------------------------------------------ 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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Andreas Bruckbauer |
Re: MaiTai DeepSee adjustments on multiphoton microscope
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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 Michael, i tried a quantification at 800 nm excitation 450 - 500 micrometer into fixed lymph nodes using the 25x Olympus objective. Over the range of the deepsee compensation i found a change of a factor two in fluorescence. One thing is that i could not set the compensation to zero with our deepsee model so i don't know what the fluorescence would be without deepsee. Anyhow with a good estimate of the GDD of the system from the deepsee motor position one can calculate how much this would broaden the 100 fs pulse out of the MaiTai and for our system this is about 220 fs. so you would have to use 1.5x more laser power to get the same signal without pre-chirp. In my experience you don't need to optimise for different samples as the less than one mm depth into the sample is insignificant compared to the thickness of the optics and GVD of an AOM. There is a slight difference for different objectives though (more or less glass). My conclusion was that for live samples the pre-chirp is definetly a benefit because you can use lower laser power. In strongly scattering samples like lymph nodes the signal decreases so rapidly with depth that you hardly notice a difference in penetration depth when adjusting the pre-chirp. best wishes Andreas -----Original Message----- From: Cameron Nowell <[hidden email]> To: [hidden email] Sent: Thu, 18 Nov 2010 21:58 Subject: Re: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Michael, We have the DeepSee on our MaiTai and have found that it doesn't do a lot on our system. At extreme depths, say greater than 500um you can get a small enhancement in signal strength and quality but it is only very minor. It may give you a few extra microns penetration but thats about it. It really depends on how much the optics of your system are dispersing/chirping the beam. If they are not altering it much then pre-chirping it will not do a lot. This is all based on qualitative assessment, i have not quantitated any of this. Also we have recently received a new Olympus 25x NA1.05 objective specifically designed for MP imaging. I have not seen how this performs yet with the DeepSee. Cheers Cam Cameron J. Nowell Microscopy Manager Centre for Advanced Microscopy Ludwig Institute for Cancer Research PO Box 2008 Royal Melbourne Hospital Victoria, 3050 AUSTRALIA Office: +61 3 9341 3155 Mobile: +61422882700 Fax: +61 3 9341 3104 Facility Website -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael Sent: Friday, 19 November 2010 4:08 AM To: [hidden email] Subject: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear microscopists, I'm planning to go through a regimen of adjusting the MaiTai DeepSee on a variety of samples with different optics to see what changes there are in depth penetration (and, maybe, preservation of biological activity). Does anybody have experience with this? If so, have you found that the DeepSee provides a noticeable practical benefit over the MaiTai without it? Any comments welcome and please feel free to contact me offline at [hidden email] Thank you! Sincerely, Michael C. ------------------------------------------------------------ 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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Armstrong, Brian |
Re: MaiTai DeepSee adjustments on multiphoton microscope
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Andreas, can expound on the sentence below? I'm not sure what you mean exactly. Thank you! "In strongly scattering samples like lymph nodes the signal decreases so rapidly with depth that you hardly notice a difference in penetration depth when adjusting the pre-chirp". Brian D Armstrong PhD Light Microscopy Core Manager Beckman Research Institute City of Hope Dept of Neuroscience 1450 E Duarte Rd Duarte, CA 91010 626-256-4673 x62872 http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag ing/Pages/default.aspx -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andreas Bruckbauer Sent: Thursday, November 18, 2010 11:09 PM To: [hidden email] Subject: Re: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Michael, i tried a quantification at 800 nm excitation 450 - 500 micrometer into fixed lymph nodes using the 25x Olympus objective. Over the range of the deepsee compensation i found a change of a factor two in fluorescence. One thing is that i could not set the compensation to zero with our deepsee model so i don't know what the fluorescence would be without deepsee. Anyhow with a good estimate of the GDD of the system from the deepsee motor position one can calculate how much this would broaden the 100 fs pulse out of the MaiTai and for our system this is about 220 fs. so you would have to use 1.5x more laser power to get the same signal without pre-chirp. In my experience you don't need to optimise for different samples as the less than one mm depth into the sample is insignificant compared to the thickness of the optics and GVD of an AOM. There is a slight difference for different objectives though (more or less glass). My conclusion was that for live samples the pre-chirp is definetly a benefit because you can use lower laser power. In strongly scattering samples like lymph nodes the signal decreases so rapidly with depth that you hardly notice a difference in penetration depth when adjusting the pre-chirp. best wishes Andreas -----Original Message----- From: Cameron Nowell <[hidden email]> To: [hidden email] Sent: Thu, 18 Nov 2010 21:58 Subject: Re: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Michael, We have the DeepSee on our MaiTai and have found that it doesn't do a lot on our system. At extreme depths, say greater than 500um you can get a small enhancement in signal strength and quality but it is only very minor. It may give you a few extra microns penetration but thats about it. It really depends on how much the optics of your system are dispersing/chirping the beam. If they are not altering it much then pre-chirping it will not do a lot. This is all based on qualitative assessment, i have not quantitated any of this. Also we have recently received a new Olympus 25x NA1.05 objective specifically designed for MP imaging. I have not seen how this performs yet with the DeepSee. Cheers Cam Cameron J. Nowell Microscopy Manager Centre for Advanced Microscopy Ludwig Institute for Cancer Research PO Box 2008 Royal Melbourne Hospital Victoria, 3050 AUSTRALIA Office: +61 3 9341 3155 Mobile: +61422882700 Fax: +61 3 9341 3104 Facility Website -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael Sent: Friday, 19 November 2010 4:08 AM To: [hidden email] Subject: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear microscopists, I'm planning to go through a regimen of adjusting the MaiTai DeepSee on a variety of samples with different optics to see what changes there are in depth penetration (and, maybe, preservation of biological activity). Does anybody have experience with this? If so, have you found that the DeepSee provides a noticeable practical benefit over the MaiTai without it? Any comments welcome and please feel free to contact me offline at [hidden email] Thank you! Sincerely, Michael C. ------------------------------------------------------------ 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. ================================= --------------------------------------------------------------------- SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. --------------------------------------------------------------------- |
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Craig Brideau |
Re: MaiTai DeepSee adjustments on multiphoton microscope
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** For deep imaging I think you would benefit more from adaptive optics than pulse compression. I have a home-built compressor that we have used to run some experiments. We found that shortening the pulse increases your fluorescence as advertised. If you are doing live imaging though, it kills your tissue pretty quickly, even if you back off on average power. The shorter pulses generate more higher-order effects which typically includes free radical production. We found that a happy medium of 150 to 200 fs at the sample seems to be good for viability yet still gives reasonable fluorescence. We have not examined the effect for tissue penetration yet. Craig On Fri, Nov 19, 2010 at 9:30 AM, Armstrong, Brian <[hidden email]>wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Andreas, can expound on the sentence below? I'm not sure what you mean > exactly. > Thank you! > > "In strongly scattering samples like lymph nodes the signal decreases so > rapidly with depth that you hardly notice a difference in penetration > depth when adjusting the pre-chirp". > > Brian D Armstrong PhD > Light Microscopy Core Manager > Beckman Research Institute > City of Hope > Dept of Neuroscience > 1450 E Duarte Rd > Duarte, CA 91010 > 626-256-4673 x62872 > http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag > ing/Pages/default.aspx<http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag%0Aing/Pages/default.aspx> > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Andreas Bruckbauer > Sent: Thursday, November 18, 2010 11:09 PM > To: [hidden email] > Subject: Re: MaiTai DeepSee adjustments on multiphoton microscope > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > > Hi Michael, > > i tried a quantification at 800 nm excitation 450 - 500 micrometer into > fixed lymph nodes using the 25x Olympus objective. Over the range of the > deepsee compensation i found a change of a factor two in fluorescence. > One thing is that i could not set the compensation to zero with our > deepsee model so i don't know what the fluorescence would be without > deepsee. Anyhow with a good estimate of the GDD of the system from the > deepsee motor position one can calculate how much this would broaden the > 100 fs pulse out of the MaiTai and for our system this is about 220 fs. > so you would have to use 1.5x more laser power to get the same signal > without pre-chirp. > > In my experience you don't need to optimise for different samples as the > less than one mm depth into the sample is insignificant compared to the > thickness of the optics and GVD of an AOM. There is a slight difference > for different objectives though (more or less glass). My conclusion was > that for live samples the pre-chirp is definetly a benefit because you > can use lower laser power. In strongly scattering samples like lymph > nodes the signal decreases so rapidly with depth that you hardly notice > a difference in penetration depth when adjusting the pre-chirp. > > best wishes > > Andreas > > > > > > > -----Original Message----- > From: Cameron Nowell <[hidden email]> > To: [hidden email] > Sent: Thu, 18 Nov 2010 21:58 > Subject: Re: MaiTai DeepSee adjustments on multiphoton microscope > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > ***** > > > > Hi Michael, > > > > We have the DeepSee on our MaiTai and have found that it doesn't do a > > lot on our system. At extreme depths, say greater than 500um you can get > > a small enhancement in signal strength and quality but it is only very > > minor. It may give you a few extra microns penetration but thats about > > it. > > > > It really depends on how much the optics of your system are > > dispersing/chirping the beam. If they are not altering it much then > > pre-chirping it will not do a lot. > > > > This is all based on qualitative assessment, i have not quantitated any > > of this. Also we have recently received a new Olympus 25x NA1.05 > > objective specifically designed for MP imaging. I have not seen how this > > performs yet with the DeepSee. > > > > > > Cheers > > > > Cam > > > > > > > > Cameron J. Nowell > > Microscopy Manager > > Centre for Advanced Microscopy > > Ludwig Institute for Cancer Research > > PO Box 2008 > > Royal Melbourne Hospital > > Victoria, 3050 > > AUSTRALIA > > Office: +61 3 9341 3155 > > Mobile: +61422882700 > > Fax: +61 3 9341 3104 > > Facility Website > > > > > > > > -----Original Message----- > > From: Confocal Microscopy List [mailto:[hidden email]] > > On Behalf Of Cammer, Michael > > Sent: Friday, 19 November 2010 4:08 AM > > To: [hidden email] > > Subject: MaiTai DeepSee adjustments on multiphoton microscope > > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > ***** > > > > Dear microscopists, > > > > I'm planning to go through a regimen of adjusting the MaiTai DeepSee > > on a variety of samples with different optics to see what changes there > > are in depth penetration (and, maybe, preservation of biological > > activity). > > > > Does anybody have experience with this? If so, have you found that the > > DeepSee provides a noticeable practical benefit over the MaiTai without > > it? > > > > Any comments welcome and please feel free to contact me offline at > > [hidden email] > > > > Thank you! > > > > Sincerely, > > > > Michael C. > > > > ------------------------------------------------------------ > > 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. > > ================================= > > > > > > --------------------------------------------------------------------- > SECURITY/CONFIDENTIALITY WARNING: > This message and any attachments are intended solely for the individual or > entity to which they are addressed. This communication may contain > information that is privileged, confidential, or exempt from disclosure > under applicable law (e.g., personal health information, research data, > financial information). Because this e-mail has been sent without > encryption, individuals other than the intended recipient may be able to > view the information, forward it to others or tamper with the information > without the knowledge or consent of the sender. If you are not the intended > recipient, or the employee or person responsible for delivering the message > to the intended recipient, any dissemination, distribution or copying of the > communication is strictly prohibited. If you received the communication in > error, please notify the sender immediately by replying to this message and > deleting the message and any accompanying files from your system. If, due to > the security risks, you do not wish to receive further communications via > e-mail, please reply to this message and inform the sender that you do not > wish to receive further e-mail from the sender. > > --------------------------------------------------------------------- > |
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Andreas Bruckbauer |
Re: MaiTai DeepSee adjustments on multiphoton microscope
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In reply to this post by Armstrong, Brian
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Brian, the reasoning behind this sentence is that the laser power decreases exponentionally with depth and I estimate the decay length (ld) in our lymph nodes to be about 170 micrometers. The 2 photon signal decreases then with exp(-2z/ld) (see e.g. Helmchen, Denk Nature Methods 2005). If you have double the signal you would penetrate "only" ln(2)/2 * ld = 0.35 ld or 60 microns more in the example above. I must admid that this is more than "hardly noticable", however experimentally we often find a "brick wall" which we cannot penetrate, the laser seems not to be focused any more. From tissue sections we know that there are still labelled cells in this depth. best wishes Andreas -----Original Message----- From: Armstrong, Brian <[hidden email]> To: [hidden email] Sent: Fri, 19 Nov 2010 16:30 Subject: Re: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Andreas, can expound on the sentence below? I'm not sure what you mean exactly. Thank you! "In strongly scattering samples like lymph nodes the signal decreases so rapidly with depth that you hardly notice a difference in penetration depth when adjusting the pre-chirp". Brian D Armstrong PhD Light Microscopy Core Manager Beckman Research Institute City of Hope Dept of Neuroscience 1450 E Duarte Rd Duarte, CA 91010 626-256-4673 x62872 http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag ing/Pages/default.aspx -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andreas Bruckbauer Sent: Thursday, November 18, 2010 11:09 PM To: [hidden email] Subject: Re: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Michael, i tried a quantification at 800 nm excitation 450 - 500 micrometer into fixed lymph nodes using the 25x Olympus objective. Over the range of the deepsee compensation i found a change of a factor two in fluorescence. One thing is that i could not set the compensation to zero with our deepsee model so i don't know what the fluorescence would be without deepsee. Anyhow with a good estimate of the GDD of the system from the deepsee motor position one can calculate how much this would broaden the 100 fs pulse out of the MaiTai and for our system this is about 220 fs. so you would have to use 1.5x more laser power to get the same signal without pre-chirp. In my experience you don't need to optimise for different samples as the less than one mm depth into the sample is insignificant compared to the thickness of the optics and GVD of an AOM. There is a slight difference for different objectives though (more or less glass). My conclusion was that for live samples the pre-chirp is definetly a benefit because you can use lower laser power. In strongly scattering samples like lymph nodes the signal decreases so rapidly with depth that you hardly notice a difference in penetration depth when adjusting the pre-chirp. best wishes Andreas -----Original Message----- From: Cameron Nowell <[hidden email]> To: [hidden email] Sent: Thu, 18 Nov 2010 21:58 Subject: Re: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Michael, We have the DeepSee on our MaiTai and have found that it doesn't do a lot on our system. At extreme depths, say greater than 500um you can get a small enhancement in signal strength and quality but it is only very minor. It may give you a few extra microns penetration but thats about it. It really depends on how much the optics of your system are dispersing/chirping the beam. If they are not altering it much then pre-chirping it will not do a lot. This is all based on qualitative assessment, i have not quantitated any of this. Also we have recently received a new Olympus 25x NA1.05 objective specifically designed for MP imaging. I have not seen how this performs yet with the DeepSee. Cheers Cam Cameron J. Nowell Microscopy Manager Centre for Advanced Microscopy Ludwig Institute for Cancer Research PO Box 2008 Royal Melbourne Hospital Victoria, 3050 AUSTRALIA Office: +61 3 9341 3155 Mobile: +61422882700 Fax: +61 3 9341 3104 Facility Website -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael Sent: Friday, 19 November 2010 4:08 AM To: [hidden email] Subject: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear microscopists, I'm planning to go through a regimen of adjusting the MaiTai DeepSee on a variety of samples with different optics to see what changes there are in depth penetration (and, maybe, preservation of biological activity). Does anybody have experience with this? If so, have you found that the DeepSee provides a noticeable practical benefit over the MaiTai without it? Any comments welcome and please feel free to contact me offline at [hidden email] Thank you! Sincerely, Michael C. ------------------------------------------------------------ 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. ================================= --------------------------------------------------------------------- SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. --------------------------------------------------------------------- |
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Armstrong, Brian |
Re: MaiTai DeepSee adjustments on multiphoton microscope
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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 Andreas, we are imaging lymph nodes and notice the same thing. What was not clear to me in your sentence is whether or not the pre-chirp helps at these greater depths. Are you saying that because of this "Denk effect" (2005) the pre-chirp does not help much? Or, are you saying that the pre-chirp helps SO much that you hardly notice this "Denk effect" any more. Thanks, Brian Armstrong PhD Light Microscopy and Digital Imaging Beckman Research Institute of the City of Hope x62872 office x62874 lab http://www.coh.org/shared-resources/light-microscopy-imaging/Pages/defau lt.aspx -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andreas Bruckbauer Sent: Friday, November 19, 2010 1:48 PM To: [hidden email] Subject: Re: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Brian, the reasoning behind this sentence is that the laser power decreases exponentionally with depth and I estimate the decay length (ld) in our lymph nodes to be about 170 micrometers. The 2 photon signal decreases then with exp(-2z/ld) (see e.g. Helmchen, Denk Nature Methods 2005). If you have double the signal you would penetrate "only" ln(2)/2 * ld = 0.35 ld or 60 microns more in the example above. I must admid that this is more than "hardly noticable", however experimentally we often find a "brick wall" which we cannot penetrate, the laser seems not to be focused any more. From tissue sections we know that there are still labelled cells in this depth. best wishes Andreas -----Original Message----- From: Armstrong, Brian <[hidden email]> To: [hidden email] Sent: Fri, 19 Nov 2010 16:30 Subject: Re: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Andreas, can expound on the sentence below? I'm not sure what you mean exactly. Thank you! "In strongly scattering samples like lymph nodes the signal decreases so rapidly with depth that you hardly notice a difference in penetration depth when adjusting the pre-chirp". Brian D Armstrong PhD Light Microscopy Core Manager Beckman Research Institute City of Hope Dept of Neuroscience 1450 E Duarte Rd Duarte, CA 91010 626-256-4673 x62872 http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag ing/Pages/default.aspx -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andreas Bruckbauer Sent: Thursday, November 18, 2010 11:09 PM To: [hidden email] Subject: Re: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Michael, i tried a quantification at 800 nm excitation 450 - 500 micrometer into fixed lymph nodes using the 25x Olympus objective. Over the range of the deepsee compensation i found a change of a factor two in fluorescence. One thing is that i could not set the compensation to zero with our deepsee model so i don't know what the fluorescence would be without deepsee. Anyhow with a good estimate of the GDD of the system from the deepsee motor position one can calculate how much this would broaden the 100 fs pulse out of the MaiTai and for our system this is about 220 fs. so you would have to use 1.5x more laser power to get the same signal without pre-chirp. In my experience you don't need to optimise for different samples as the less than one mm depth into the sample is insignificant compared to the thickness of the optics and GVD of an AOM. There is a slight difference for different objectives though (more or less glass). My conclusion was that for live samples the pre-chirp is definetly a benefit because you can use lower laser power. In strongly scattering samples like lymph nodes the signal decreases so rapidly with depth that you hardly notice a difference in penetration depth when adjusting the pre-chirp. best wishes Andreas -----Original Message----- From: Cameron Nowell <[hidden email]> To: [hidden email] Sent: Thu, 18 Nov 2010 21:58 Subject: Re: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Michael, We have the DeepSee on our MaiTai and have found that it doesn't do a lot on our system. At extreme depths, say greater than 500um you can get a small enhancement in signal strength and quality but it is only very minor. It may give you a few extra microns penetration but thats about it. It really depends on how much the optics of your system are dispersing/chirping the beam. If they are not altering it much then pre-chirping it will not do a lot. This is all based on qualitative assessment, i have not quantitated any of this. Also we have recently received a new Olympus 25x NA1.05 objective specifically designed for MP imaging. I have not seen how this performs yet with the DeepSee. Cheers Cam Cameron J. Nowell Microscopy Manager Centre for Advanced Microscopy Ludwig Institute for Cancer Research PO Box 2008 Royal Melbourne Hospital Victoria, 3050 AUSTRALIA Office: +61 3 9341 3155 Mobile: +61422882700 Fax: +61 3 9341 3104 Facility Website -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael Sent: Friday, 19 November 2010 4:08 AM To: [hidden email] Subject: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear microscopists, I'm planning to go through a regimen of adjusting the MaiTai DeepSee on a variety of samples with different optics to see what changes there are in depth penetration (and, maybe, preservation of biological activity). Does anybody have experience with this? If so, have you found that the DeepSee provides a noticeable practical benefit over the MaiTai without it? Any comments welcome and please feel free to contact me offline at [hidden email] Thank you! Sincerely, Michael C. ------------------------------------------------------------ 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. ================================= --------------------------------------------------------------------- SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. --------------------------------------------------------------------- |
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Johannes Helm |
Re: MaiTai DeepSee adjustments on multiphoton microscope
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In reply to this post by mcammer
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Good evening, I do neither know whether this has been mentioned before nor do I want to start a Coherent vs. Spectra Physics battle (I have actually both, DeepSees and Vision IIs here and I have been working with both, Spectra Physics and Coherent lasers since 1985 and I like the products of both companies, and the engineers of both companies, as far as I can judge, do a great job). While the DeepSees can be bought with pulse lengths specified to be shorter than 70fsec - the Visions are not specified to attain pulse lengths that short - the DeepSee GDC has a disadvantage compared to the Vision II GDC: While the Visions can compensate on all wavelengths from 0 to the max. possible compensation value at the resp. wavelength, the DeepSee has a minimum compensation value, which is, according to http://www.newport.com/images/webDocuments-EN/images/15374.pdf , Standard Dispersion Compensation Range 1 690 nm: -22,500 fs2 to -41,700 fs2 800 nm: -8,900 fs2 to -24,500 fs2 1040 nm: 0 fs2 to -9,600 fs2. (wavelengths between these values can be coarsly approximated by linear interpolation, there also is a complete curve somewhere but I do not find it right now). Optionally, a "low dispersion version" can be purchased, but this does then only cover the values from 0 up to the LOWER limits of the standard compensation bands. As a consequence, in certain configurations GVD cannot be DeepSee compensated completely in certain wavelength ranges. This is the case, e.g., when using EOMs with KD*P crystals (which have a somewhat "strange" GVD vs. lambda behaviour since the refractive index of KD*P changes from normal to anomalous dispersion somewhere around 1000nm). EOMs good for MPLSM setups do in many cases have KD*P crystals. If you calculate the GDC values, which you need, using appropriate Sellmeier formulas, you'll find out that you can compensate, in the described case, quite well in the central wavelength range of the DeepSee, while you always over resp. undercompensate in the short- resp. long wavelength regions of the emission spectrum of the laser. Since the DeepSee emits comparatively short pulses and since pulse stretching is a non-linear phenomenon, you might end up in the preparation with longer pulses when using a DeepSee compared to when using a Vision not in spite of but BECAUSE the DeepSee's original pulse length is shorter than the Vision's. The easiest way to find out how good your pulses are is, of course, to measure them with an autocorrelator; then you do not have to bother about programming the Sellmeier formulas for all the material the laser beam has to pass in your setup before arriving in the objective focus. Best wishes and have a good sunday! Johannes > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Dear microscopists, > > I'm planning to go through a regimen of adjusting the MaiTai DeepSee on > a variety of samples with different optics to see what changes there are > in depth penetration (and, maybe, preservation of biological activity). > > Does anybody have experience with this? If so, have you found that the > DeepSee provides a noticeable practical benefit over the MaiTai without > it? > > Any comments welcome and please feel free to contact me offline at > [hidden email] > > Thank you! > > Sincerely, > > Michael C. > > ------------------------------------------------------------ > 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. > ================================= > -- P. Johannes Helm, M.Sc. PhD Seniorengineer CMBN University of Oslo Institute of Basic Medical Science Department of Anatomy Postboks 1105 - Blindern NO-0317 Oslo Voice: +47 228 51159 Fax: +47 228 51499 WWW: folk.uio.no/jhelm |
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Andreas Bruckbauer |
Re: MaiTai DeepSee adjustments on multiphoton microscope
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In reply to this post by Armstrong, Brian
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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 Brian, good (or bad) that you find the same, adjusting the pre-chirp definetly helps to increase the signal deep in the tissue, the cells in the last 50 micrometers are 2x brighter with the right deepsee settings but for some reason cells deeper in the tissues remain invisible. best wishes Andreas -----Original Message----- From: Armstrong, Brian <[hidden email]> To: [hidden email] Sent: Fri, 19 Nov 2010 22:08 Subject: Re: MaiTai DeepSee adjustments on multiphoton microscope ***** o join, leave or search the confocal microscopy listserv, go to: ttp://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy **** Hi Andreas, we are imaging lymph nodes and notice the same thing. What was not clear to me in your sentence is whether or not the re-chirp helps at these greater depths. Are you saying that because of his "Denk effect" (2005) the pre-chirp does not help much? r, are you saying that the pre-chirp helps SO much that you hardly otice this "Denk effect" any more. Thanks, Brian Armstrong PhD ight Microscopy and Digital Imaging eckman Research Institute of the City of Hope 62872 office 62874 lab ttp://www.coh.org/shared-resources/light-microscopy-imaging/Pages/defau t.aspx ----Original Message----- rom: Confocal Microscopy List [mailto:[hidden email]] n Behalf Of Andreas Bruckbauer ent: Friday, November 19, 2010 1:48 PM o: [hidden email] ubject: Re: MaiTai DeepSee adjustments on multiphoton microscope ***** o join, leave or search the confocal microscopy listserv, go to: ttp://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy **** Brian, the reasoning behind this sentence is that the laser power decreases xponentionally with depth and I estimate the decay length (ld) in our ymph nodes to be about 170 micrometers. The 2 photon signal decreases hen with exp(-2z/ld) (see e.g. Helmchen, Denk Nature Methods 2005). If ou have double the signal you would penetrate "only" ln(2)/2 * ld = .35 ld or 60 microns more in the example above. I must admid that this s more than "hardly noticable", however experimentally we often find a brick wall" which we cannot penetrate, the laser seems not to be ocused any more. From tissue sections we know that there are still abelled cells in this depth. best wishes Andreas -----Original Message----- rom: Armstrong, Brian <[hidden email]> o: [hidden email] ent: Fri, 19 Nov 2010 16:30 ubject: Re: MaiTai DeepSee adjustments on multiphoton microscope **** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Andreas, can expound on the sentence below? I'm not sure what you mean exactly. Thank you! "In strongly scattering samples like lymph nodes the signal decreases so rapidly with depth that you hardly notice a difference in penetration depth when adjusting the pre-chirp". Brian D Armstrong PhD Light Microscopy Core Manager Beckman Research Institute City of Hope Dept of Neuroscience 1450 E Duarte Rd Duarte, CA 91010 626-256-4673 x62872 http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag ing/Pages/default.aspx -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andreas Bruckbauer Sent: Thursday, November 18, 2010 11:09 PM To: [hidden email] Subject: Re: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Michael, i tried a quantification at 800 nm excitation 450 - 500 micrometer into fixed lymph nodes using the 25x Olympus objective. Over the range of the deepsee compensation i found a change of a factor two in fluorescence. One thing is that i could not set the compensation to zero with our deepsee model so i don't know what the fluorescence would be without deepsee. Anyhow with a good estimate of the GDD of the system from the deepsee motor position one can calculate how much this would broaden the 100 fs pulse out of the MaiTai and for our system this is about 220 fs. so you would have to use 1.5x more laser power to get the same signal without pre-chirp. In my experience you don't need to optimise for different samples as the less than one mm depth into the sample is insignificant compared to the thickness of the optics and GVD of an AOM. There is a slight difference for different objectives though (more or less glass). My conclusion was that for live samples the pre-chirp is definetly a benefit because you can use lower laser power. In strongly scattering samples like lymph nodes the signal decreases so rapidly with depth that you hardly notice a difference in penetration depth when adjusting the pre-chirp. best wishes Andreas -----Original Message----- From: Cameron Nowell <[hidden email]> To: [hidden email] Sent: Thu, 18 Nov 2010 21:58 Subject: Re: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Michael, We have the DeepSee on our MaiTai and have found that it doesn't do a lot on our system. At extreme depths, say greater than 500um you can get a small enhancement in signal strength and quality but it is only very minor. It may give you a few extra microns penetration but thats about it. It really depends on how much the optics of your system are dispersing/chirping the beam. If they are not altering it much then pre-chirping it will not do a lot. This is all based on qualitative assessment, i have not quantitated any of this. Also we have recently received a new Olympus 25x NA1.05 objective specifically designed for MP imaging. I have not seen how this performs yet with the DeepSee. Cheers Cam Cameron J. Nowell Microscopy Manager Centre for Advanced Microscopy Ludwig Institute for Cancer Research PO Box 2008 Royal Melbourne Hospital Victoria, 3050 AUSTRALIA Office: +61 3 9341 3155 Mobile: +61422882700 Fax: +61 3 9341 3104 Facility Website -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael Sent: Friday, 19 November 2010 4:08 AM To: [hidden email] Subject: MaiTai DeepSee adjustments on multiphoton microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear microscopists, I'm planning to go through a regimen of adjusting the MaiTai DeepSee on a variety of samples with different optics to see what changes there are in depth penetration (and, maybe, preservation of biological activity). Does anybody have experience with this? If so, have you found that the DeepSee provides a noticeable practical benefit over the MaiTai without it? Any comments welcome and please feel free to contact me offline at [hidden email] Thank you! Sincerely, Michael C. ------------------------------------------------------------ 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. ================================= --------------------------------------------------------------------- SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are intended solely for the individual r entity to which they are addressed. This communication may contain nformation that is privileged, confidential, or exempt from disclosure under pplicable law (e.g., personal health information, research data, financial nformation). Because this e-mail has been sent without encryption, individuals other han the intended recipient may be able to view the information, forward it to thers or tamper with the information without the knowledge or consent of the ender. If you are not the intended recipient, or the employee or person esponsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If ou received the communication in error, please notify the sender mmediately by replying to this message and deleting the message and any accompanying iles from your system. If, due to the security risks, you do not wish to eceive further communications via e-mail, please reply to this message and nform the sender that you do not wish to receive further e-mail from the sender. --------------------------------------------------------------------- |
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*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Whoops, my last message wasn't supposed to go to everyone. Haven't done that in years... Thanks to everyone responding to my questions and extending this into a discussion. The responses are extremely helpful. Sincerely, Michael _________________________________________ Michael Cammer, Assistant Research Scientist Skirball Institute of Biomolecular Medicine Lab: (212) 263-3208 Cell: (914) 309-3270 __ ------------------------------------------------------------ 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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S. Pagakis (IIBEAA) |
Re: MaiTai DeepSee adjustments on multiphoton microscope
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In reply to this post by Armstrong, Brian
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello list I have been following the above thread and I think I do now understand the issues with pre-chirping. MaiTai sells 2 DeepSee models the eHP and the HP. The first with a 70fs pulse whereas the second with a 100fs pulse. Their difference in price is extremely small (I think is about 5K Euros). Does anyone want to comment whether the first has a significant advantage over the other? Will I even be able to use the extra power of the eHP? In terms of tissue penetration, with such a small difference in pulse width, will I see better performance with the 70fs? Thank you all in advance ********************************* Stamatis Pagakis Ph.D. Biological Imaging Unit Biomedical Research Foundation, Academy of Athens Soranou Efessiou 4, Athens 115 27 - Greece M: +306946644955 W: +302106597481 FAX: +302106597545 http://www.bioacademy.gr/Faculty/core.php?cr=17 |
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Craig Brideau |
Re: MaiTai DeepSee adjustments on multiphoton microscope
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** We have done some research on this in our lab. We have an old Tsunami with minimum pulse time of 60 fs @ 850nm. The beauty of the Tsunami is you can adjust the width of the pulse spectrum from anywhere within the range of 18nm to 10nm. This means (with the appropriate pulse compressor) I can create transform-limited pulses of anywhere from 60 to 100fs approximately. If I am willing to allow the pulse to chirp, the compressor I have allows up to ~1ps pulses as well, although these are not transform-limited. We image live tissue sections which are being perfused, under an upright microscope. There is a lot of oxygen present because of the perfusion. We found that if we put ~60fs pulses at the sample the sample dies/breaks down very quickly. We suspect this is due to singlet oxygen production. Fixed samples do not break down as quickly if they have been treated with an oxygen scrubber (prolong gold, etc) which is what leads us to believe it is the oxygen. If we use 100 fs xfrm limited pulses we get a bit less damage while still getting reasonable signal, but we finally found that the sweet-spot for us was about 200fs slightly chirped. We got decent signal without harming the tissue over the time scales we ran our experiments. For samples with more or less oxygen present I suspect you could alter the pulse-width accordingly. Keep in mind that as long as you have a compressor you can modify your pulse width to whatever you want, down to the minimum the bandwidth of the laser allows. Finally, the larger the bandwidth of the laser, the more sensitive it is to dispersion effects. So in this case, the 70fs laser mentioned will be more responsive to small compressor adjustments than the 100fs laser. I have a paper detailing all this that made the rounds amongst the forum goers a few months ago. Email me privately if you are interested. Craig On Wed, Dec 1, 2010 at 4:45 AM, Stamatis Pagakis <[hidden email]>wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > > Hello list > > I have been following the above thread and I think I do now understand the > issues with pre-chirping. > > MaiTai sells 2 DeepSee models the eHP and the HP. The first with a 70fs > pulse whereas the second with a 100fs pulse. > > Their difference in price is extremely small (I think is about 5K Euros). > Does anyone want to comment whether the first has a significant advantage > over the other? > Will I even be able to use the extra power of the eHP? > In terms of tissue penetration, with such a small difference in pulse > width, will I see better performance with the 70fs? > > Thank you all in advance > > > > > ********************************* > Stamatis Pagakis Ph.D. > Biological Imaging Unit > Biomedical Research Foundation, Academy of Athens > Soranou Efessiou 4, Athens 115 27 - Greece > M: +306946644955 > W: +302106597481 > FAX: +302106597545 > http://www.bioacademy.gr/Faculty/core.php?cr=17 > |
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