Multi photon imaging of thick samples
Locked
 
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Hege Avsnes Dale |
Multi photon imaging of thick samples
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Hi
all. Thank you in advance :-) |
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Hege,
You don't
mention what detectors you are using. To get any sort of useful results
from
MP you have to use the non-descanned detectors - using
confocal detectors with the pinhole
open is completely useless whatever the manufacturers
say. With thick samples it's often
worthwhile to use both transmission and back-propagated
detectors - the trans detector will
start to pick up when the epi one dies.
It's not
possible to use epi-nd detectors at the same time as the confocal detectors, of
course, but you can easily use the trans detector for MP
and the confocal detectors for single
photon as two channels in a sequential imaging acquisition
- if that gives different depths for
the two channels of the same dataset something strange
is happening. But what I suspect is
that you have not set the beam-expander correctly - the
longer beam path of the Ti-S laser
means it needs less expansion. So if you take a
confocal set with the same beam-expander
setting it will not be using the full NA of the objective
in confocal mode and so will not be truly
confocal.
As to
cooking - any particulate matter will induce electric breakdown and void
formation,
often at moderate intensities when you could otherwise
expect to image for hours. I don't know
what in brain slices would cause this, but you may be able
to sort it out by carefully checking
which areas cause bubbling and which don't.
Guy Cox
Optical Imaging Techniques in Cell Biology From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Hege Avsnes Dale Sent: Thursday, 4 December 2008 10:23 PM To: [hidden email] Subject: Multi photon imaging of thick samples Hi all. Thank you in advance :-) No virus found in this incoming message. No virus found in this outgoing message. |
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Alison J. North |
Re: Multi photon imaging of thick samples
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In reply to this post by Hege Avsnes Dale
Dear Hege,
This is indeed a tricky question. There is certainly huge variability between the depth of imaging for different tissues and for fixed vs. unfixed tissues, and also depending on which microscope and laser you are using. I think this is why it's so difficult to glean from the literature what you can really expect. However, since you have asked for some examples of depth, we are happy to share a few numbers, though I'm sure other people will probably have contradictory ones. We have an old Zeiss 510 NLO system in the lab, and we are currently shopping for a new MP system, so we have been testing various of the systems that are around at the moment. The good news is that the new MP systems are definitely giving greatly increased depth penetration - certainly this applies to three of the systems we have tested so far on our own samples, namely the Olympus, the new Zeiss 710 and the LaVision TriM systems, all of which appear to be excellent instruments. However, I have not tested a Leica myself, so I am afraid that makes it difficult for you to compare with your own system. Our most extensive tests have been performed on a new Olympus MP system with a SpectraPhysics MaiTai DeepSee laser. We have had this system on loan to the lab for a few months now, so we have been able to really put it through its paces. On living mouse lymph nodes, we can image to around 450 microns depth with this system, in comparison to around 250 microns on an older BioRad system, and only 100 microns or so with the old Zeiss 510 NLO. When we fix these lymph nodes (but don't perform clearing), we can only image to around 200 depth now on the Olympus (haven't checked this on the others). With mouse brain tissue, we have seen variable results again, depending on e.g. the stain or fluorescent protein used. Our best depth so far on the Olympus was a massive 1.1 mm on mouse brain stained with Evans Blue. On skin, we haven't yet been able to go so deep - maybe only to around 300 microns. All of these figures, for all instruments, were using epi-NDDs. Please feel free to contact us offline if you have any further questions - I won't address your other questions because Guy Cox has already done so! Good luck and best wishes, Alison Hege Avsnes Dale wrote: > > Hi all. > I've recently started working with multi photon microscopy on a Leica > SP5 system with a Coherent Chameleon-Ultra laser using a 20X 1NA dip > in objective. We study both live and fixed thick samples, and we run > into different problems that maybe someone has experiences on that > they could share. > > 1) Cooking: Working on live brain slices in buffer the sample starts > "cooking" under conditions we believe is not too extreme. We don't use > more laser than we need to be able to visualize the virus transdused > EGFP expressing cells, that are quite bright, but bobbles starts to > form on the turning points of the laser. Any tips on optimalization > steps either on the imaging side or the sample itself that prevent > burning the sample? > > 2) Depth penetration: This is what everybody wants to know, how deep > can you penetrate your sample? My experience so far is that it varies > with the density of the sample (and of course the staining), and that > I cannot really go very deep... In dense tumors (expressing EGFP) I'm > able to maybe reach a 100 um, which is less than most would expect > with a MP, I guess. > Does anyone have any experiences with different type of tissue and how > deep it might be possible to reach? I know that there are many > conditions that play a role, but any indications would be appreciated... > > 3) A puzzle: Our system is also equipped with lasers for confocal > imaging, and I wanted to compare a z-stack using the 488 laser line > and the MP 800 nm. This was not a very thick sample and had a defined > border of fluorescence so the two stacks were identically defined. > But; the confocal stack gave a volume of 34 um whereas the MP-stack > only a volume of 25 um. This is a difference of approx 40 % on this > small stack. Can this be explained in any way or do we need to contact > our manufactures? > > Thank you in advance :-) > > Hege -- Alison J. North, Ph.D., Research Assistant Professor and Director of the Bio-Imaging Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065. Tel: office ++ 212 327 7488 Tel: lab ++ 212 327 7486 Fax: ++ 212 327 7489 |
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Armstrong, Brian |
Re: Multi photon imaging of thick samples
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Hege, just to add another data point, I concur with Alison. Our LSM510
NLO NDD system ("old" as Alison put it :( )was quite poor at 2P imaging and the depths we measured were equivalent to Alison's. With our Prairie Ultima using a Coherent Chameleon Ultra II we image in-vivo lymph nodes to between ~300-500um, live mouse brain ~500-1000um, fixed pig brain embryo ~ 1000um, and in-vivo tumor ~200-400um. There are many variables that can affect depth that have been expounded on previously on the list (lambda [ex,emm] clearing, fluorophore, GVD/pre-chirp, etc.,). I also regret that I do not have experience with 2P NDD on the Lecia SP5. Yes, we see the bubbling as well. I assume that it is heat transfer that occurs from the huge amount of power we are able to generate with our laser. When doing in-vivo 2P imaging you do not have a lot of time to waste trying to perfect your acquisition. Once you start scanning the stop-watch is ticking! 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 Alison North Sent: Thursday, December 04, 2008 8:33 AM To: [hidden email] Subject: Re: Multi photon imaging of thick samples Dear Hege, This is indeed a tricky question. There is certainly huge variability between the depth of imaging for different tissues and for fixed vs. unfixed tissues, and also depending on which microscope and laser you are using. I think this is why it's so difficult to glean from the literature what you can really expect. However, since you have asked for some examples of depth, we are happy to share a few numbers, though I'm sure other people will probably have contradictory ones. We have an old Zeiss 510 NLO system in the lab, and we are currently shopping for a new MP system, so we have been testing various of the systems that are around at the moment. The good news is that the new MP systems are definitely giving greatly increased depth penetration - certainly this applies to three of the systems we have tested so far on our own samples, namely the Olympus, the new Zeiss 710 and the LaVision TriM systems, all of which appear to be excellent instruments. However, I have not tested a Leica myself, so I am afraid that makes it difficult for you to compare with your own system. Our most extensive tests have been performed on a new Olympus MP system with a SpectraPhysics MaiTai DeepSee laser. We have had this system on loan to the lab for a few months now, so we have been able to really put it through its paces. On living mouse lymph nodes, we can image to around 450 microns depth with this system, in comparison to around 250 microns on an older BioRad system, and only 100 microns or so with the old Zeiss 510 NLO. When we fix these lymph nodes (but don't perform clearing), we can only image to around 200 depth now on the Olympus (haven't checked this on the others). With mouse brain tissue, we have seen variable results again, depending on e.g. the stain or fluorescent protein used. Our best depth so far on the Olympus was a massive 1.1 mm on mouse brain stained with Evans Blue. On skin, we haven't yet been able to go so deep - maybe only to around 300 microns. All of these figures, for all instruments, were using epi-NDDs. Please feel free to contact us offline if you have any further questions - I won't address your other questions because Guy Cox has already done so! Good luck and best wishes, Alison Hege Avsnes Dale wrote: > > Hi all. > I've recently started working with multi photon microscopy on a Leica > SP5 system with a Coherent Chameleon-Ultra laser using a 20X 1NA dip > in objective. We study both live and fixed thick samples, and we run > into different problems that maybe someone has experiences on that > they could share. > > 1) Cooking: Working on live brain slices in buffer the sample starts > "cooking" under conditions we believe is not too extreme. We don't use > more laser than we need to be able to visualize the virus transdused > EGFP expressing cells, that are quite bright, but bobbles starts to > form on the turning points of the laser. Any tips on optimalization > steps either on the imaging side or the sample itself that prevent > burning the sample? > > 2) Depth penetration: This is what everybody wants to know, how deep > can you penetrate your sample? My experience so far is that it varies > with the density of the sample (and of course the staining), and that > I cannot really go very deep... In dense tumors (expressing EGFP) I'm > able to maybe reach a 100 um, which is less than most would expect > with a MP, I guess. > Does anyone have any experiences with different type of tissue and how > deep it might be possible to reach? I know that there are many > conditions that play a role, but any indications would be appreciated... > > 3) A puzzle: Our system is also equipped with lasers for confocal > imaging, and I wanted to compare a z-stack using the 488 laser line > and the MP 800 nm. This was not a very thick sample and had a defined > border of fluorescence so the two stacks were identically defined. > But; the confocal stack gave a volume of 34 um whereas the MP-stack > only a volume of 25 um. This is a difference of approx 40 % on this > small stack. Can this be explained in any way or do we need to contact > our manufactures? > > Thank you in advance :-) > > Hege -- Alison J. North, Ph.D., Research Assistant Professor and Director of the Bio-Imaging Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065. Tel: office ++ 212 327 7488 Tel: lab ++ 212 327 7486 Fax: ++ 212 327 7489 --------------------------------------------------------------------- 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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Qi, Hai (NIH/NIAID) [F] |
Re: Multi photon imaging of thick samples
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As implied in what Brian wrote, there is always a trade-off for how deep
one can see with how long one can continuously image. "When doing in-vivo 2P imaging you do not have a lot of time to waste trying to perfect your acquisition. Once you start scanning the stop-watch is ticking!", is probably not always desirable or necessarily right, if it is really due to heat damage. As far as live imaging is concerned, it is not so useful if the power required for seeing much of anything rapidly cooks the cells. We routinely do continuous intravital imaging on lymph nodes for hours (30~60s per cycle), and cells surely do not get cooked. Of course we are mostly within the 1st 100 um. On the other hand, it is no question that newer MP systems such as Zeiss 710 NLO are more sensitive than old 510 NLO. Hai Hai Qi, M.D., Ph.D. Postdoctoral Fellow Lymphocyte Biology Section, Laboratory of Immunology National Institute of Allergy and Infectious Diseases, NIH Bldg. 10 Rm. 11N250 10 Center Dr. MSC-1892 Bethesda, MD 20892-1892 Tel: 301-4961868 Fax: 301-4807352 Email: [hidden email] ====== Disclaimer ====== The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. National Institute of Allergy and Infectious Diseases shall not accept liability for any statements made that are sender's own and not expressly made on behalf of the NIAID by one of its representatives. -----Original Message----- From: Armstrong, Brian [mailto:[hidden email]] Sent: Thursday, December 04, 2008 12:34 PM To: [hidden email] Subject: Re: Multi photon imaging of thick samples Hege, just to add another data point, I concur with Alison. Our LSM510 NLO NDD system ("old" as Alison put it :( )was quite poor at 2P imaging and the depths we measured were equivalent to Alison's. With our Prairie Ultima using a Coherent Chameleon Ultra II we image in-vivo lymph nodes to between ~300-500um, live mouse brain ~500-1000um, fixed pig brain embryo ~ 1000um, and in-vivo tumor ~200-400um. There are many variables that can affect depth that have been expounded on previously on the list (lambda [ex,emm] clearing, fluorophore, GVD/pre-chirp, etc.,). I also regret that I do not have experience with 2P NDD on the Lecia SP5. Yes, we see the bubbling as well. I assume that it is heat transfer that occurs from the huge amount of power we are able to generate with our laser. When doing in-vivo 2P imaging you do not have a lot of time to waste trying to perfect your acquisition. Once you start scanning the stop-watch is ticking! 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 Alison North Sent: Thursday, December 04, 2008 8:33 AM To: [hidden email] Subject: Re: Multi photon imaging of thick samples Dear Hege, This is indeed a tricky question. There is certainly huge variability between the depth of imaging for different tissues and for fixed vs. unfixed tissues, and also depending on which microscope and laser you are using. I think this is why it's so difficult to glean from the literature what you can really expect. However, since you have asked for some examples of depth, we are happy to share a few numbers, though I'm sure other people will probably have contradictory ones. We have an old Zeiss 510 NLO system in the lab, and we are currently shopping for a new MP system, so we have been testing various of the systems that are around at the moment. The good news is that the new MP systems are definitely giving greatly increased depth penetration - certainly this applies to three of the systems we have tested so far on our own samples, namely the Olympus, the new Zeiss 710 and the LaVision TriM systems, all of which appear to be excellent instruments. However, I have not tested a Leica myself, so I am afraid that makes it difficult for you to compare with your own system. Our most extensive tests have been performed on a new Olympus MP system with a SpectraPhysics MaiTai DeepSee laser. We have had this system on loan to the lab for a few months now, so we have been able to really put it through its paces. On living mouse lymph nodes, we can image to around 450 microns depth with this system, in comparison to around 250 microns on an older BioRad system, and only 100 microns or so with the old Zeiss 510 NLO. When we fix these lymph nodes (but don't perform clearing), we can only image to around 200 depth now on the Olympus (haven't checked this on the others). With mouse brain tissue, we have seen variable results again, depending on e.g. the stain or fluorescent protein used. Our best depth so far on the Olympus was a massive 1.1 mm on mouse brain stained with Evans Blue. On skin, we haven't yet been able to go so deep - maybe only to around 300 microns. All of these figures, for all instruments, were using epi-NDDs. Please feel free to contact us offline if you have any further questions - I won't address your other questions because Guy Cox has already done so! Good luck and best wishes, Alison Hege Avsnes Dale wrote: > > Hi all. > I've recently started working with multi photon microscopy on a Leica > SP5 system with a Coherent Chameleon-Ultra laser using a 20X 1NA dip > in objective. We study both live and fixed thick samples, and we run > into different problems that maybe someone has experiences on that > they could share. > > 1) Cooking: Working on live brain slices in buffer the sample starts > "cooking" under conditions we believe is not too extreme. We don't use > more laser than we need to be able to visualize the virus transdused > EGFP expressing cells, that are quite bright, but bobbles starts to > form on the turning points of the laser. Any tips on optimalization > steps either on the imaging side or the sample itself that prevent > burning the sample? > > 2) Depth penetration: This is what everybody wants to know, how deep > can you penetrate your sample? My experience so far is that it varies > with the density of the sample (and of course the staining), and that > I cannot really go very deep... In dense tumors (expressing EGFP) I'm > able to maybe reach a 100 um, which is less than most would expect > with a MP, I guess. > Does anyone have any experiences with different type of tissue and how > deep it might be possible to reach? I know that there are many > conditions that play a role, but any indications would be appreciated... > > 3) A puzzle: Our system is also equipped with lasers for confocal > imaging, and I wanted to compare a z-stack using the 488 laser line > and the MP 800 nm. This was not a very thick sample and had a defined > border of fluorescence so the two stacks were identically defined. > But; the confocal stack gave a volume of 34 um whereas the MP-stack > only a volume of 25 um. This is a difference of approx 40 % on this > small stack. Can this be explained in any way or do we need to contact > our manufactures? > > Thank you in advance :-) > > Hege -- Alison J. North, Ph.D., Research Assistant Professor and Director of the Bio-Imaging Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065. Tel: office ++ 212 327 7488 Tel: lab ++ 212 327 7486 Fax: ++ 212 327 7489 --------------------------------------------------------------------- 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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Hege Avsnes Dale |
Re: Multi photon imaging of thick samples
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In reply to this post by Hege Avsnes Dale
First of all; thanks lot to all repliers! It's most
appreciated.
To clarify a bit; we have 3 conf. detectors, 2 NDDs and 1 transmission detector, I've been using both confocal detectors with open pinhole and NDDs, but did not realize I could use the trans detector. This will be tested! I'm aware that we need to use the NDDs to go deep, but feel we must have more depth potential than what we are currently achieving... Comparing confocal and MP I used confocal detectors. When it comes to the beam expander I'm awaiting an answer from Leica, because I thought the beam expander was "out of reach" on the SP5 system (on the SP2 it can be adjusted). Is it possible that this somehow can be related to a misaligned laser? These new lasers are aligned by the manufacturer and we're not supposed to adjust anything ourselves... I don't even know how to evaluate if our laser is misaligned when it already gives a nice XY image, since I do not know exactly what to expect in Z. All the best, Hege Alison: All the best, Hege Hege Avsnes Dale wrote:
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In reply to this post by Qi, Hai (NIH/NIAID) [F]
Hai, Brian,
I would think that the heat transfer is due to relatively high average power. To get the photons deep into the sample the only thing you can do is play with (shorten) the pulse width to temporally focus the photons. Ashley <quote author="Qi, Hai (NIH/NIAID) [F]"> As implied in what Brian wrote, there is always a trade-off for how deep one can see with how long one can continuously image. "When doing in-vivo 2P imaging you do not have a lot of time to waste trying to perfect your acquisition. Once you start scanning the stop-watch is ticking!", is probably not always desirable or necessarily right, if it is really due to heat damage. As far as live imaging is concerned, it is not so useful if the power required for seeing much of anything rapidly cooks the cells. We routinely do continuous intravital imaging on lymph nodes for hours (30~60s per cycle), and cells surely do not get cooked. Of course we are mostly within the 1st 100 um. On the other hand, it is no question that newer MP systems such as Zeiss 710 NLO are more sensitive than old 510 NLO. Hai Hai Qi, M.D., Ph.D. Postdoctoral Fellow Lymphocyte Biology Section, Laboratory of Immunology National Institute of Allergy and Infectious Diseases, NIH Bldg. 10 Rm. 11N250 10 Center Dr. MSC-1892 Bethesda, MD 20892-1892 Tel: 301-4961868 Fax: 301-4807352 Email: hqi@niaid.nih.gov |
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Armstrong, Brian |
Re: Multi photon imaging of thick samples
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Hi Ashley, sounds like an ad for the Mai Tai Deep See! There was a
thread recently about GVD vs pulse width, and therefore Deep See vs Chameleon II, and it seems as though when the pulse is shorter it is much more susceptible to GVD. Many experts agree that that pre-chirp does gain you very much overall and is instead only valuable in certain experimental paradigms. Coherent is now selling the Chameleon Pre-Comp even though the longer pulse (~140fsec) is less susceptible to GVD. I assume this is an answer to the Deep See and is more about marketing than functionality. I have no plans to purchase one! Cheers, 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 A Crane Sent: Friday, December 05, 2008 5:42 AM To: [hidden email] Subject: Re: Multi photon imaging of thick samples Hai, Brian, I would think that the heat transfer is due to relatively high average power. To get the photons deep into the sample the only thing you can do is play with (shorten) the pulse width to temporally focus the photons. Ashley As implied in what Brian wrote, there is always a trade-off for how deep one can see with how long one can continuously image. "When doing in-vivo 2P imaging you do not have a lot of time to waste trying to perfect your acquisition. Once you start scanning the stop-watch is ticking!", is probably not always desirable or necessarily right, if it is really due to heat damage. As far as live imaging is concerned, it is not so useful if the power required for seeing much of anything rapidly cooks the cells. We routinely do continuous intravital imaging on lymph nodes for hours (30~60s per cycle), and cells surely do not get cooked. Of course we are mostly within the 1st 100 um. On the other hand, it is no question that newer MP systems such as Zeiss 710 NLO are more sensitive than old 510 NLO. Hai Hai Qi, M.D., Ph.D. Postdoctoral Fellow Lymphocyte Biology Section, Laboratory of Immunology National Institute of Allergy and Infectious Diseases, NIH Bldg. 10 Rm. 11N250 10 Center Dr. MSC-1892 Bethesda, MD 20892-1892 Tel: 301-4961868 Fax: 301-4807352 Email: [hidden email] -- View this message in context: http://n2.nabble.com/Multi-photon-imaging-of-thick-samples-tp1613145p161 8387.html Sent from the Confocal Microscopy List mailing list archive at Nabble.com. --------------------------------------------------------------------- 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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Stephen Cody-2 |
Re: Multi photon imaging of thick samples
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This is the reason why we were keen to buy a system with pre-chirp. The old argument that GVD doesn't matter because you just increase IR laser power is flawed in that you will eventually get direct IR damage. It is much more gentle on the specimen to pre-chirp the laser pulse so that it is very narrow within your specimen, and then you can use minimal laser power.
We are very happy with the Deep-See system, it is a simple matter of tweaking the pre-chirp to maximise the signal from the specimen.
It would be a nice idea if the confocal software allowed you to set a few different pre-chirp settings at different depths in the specimen, and then the correct pre-chirp could be set for various depths throughout the specimen.
Stephen Cody
2008/12/6 Armstrong, Brian <[hidden email]> Hi Ashley, sounds like an ad for the Mai Tai Deep See! There was a -- Stephen Cody |
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Armstrong, Brian |
Re: Multi photon imaging of thick samples
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Hi Stephen, do you automatically change
the pre-chirp (inter-prism distance) when you change wavelengths? How fast is
this tuning? Does it gain you more in shorter wavelengths? How deep can you go,
and in what tissue? Any idea how your performance with the Deep See would
compare to a Chameleon Ultra II that has a longer pulse width (140fsec)? Can you calculate the theoretical difference
in GVD in: A) 80fsec pulse that is pre-chirped vs B) 140fsec pulse that is not
pre-chirp compensated? Best wishes, Brian D Armstrong PhD Light Microscopy Core Manager Beckman Research Institute City of Dept of Neuroscience 626-256-4673 x62872 http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx From: This is the reason why we were keen to buy a system with pre-chirp. The
old argument that GVD doesn't matter because you just increase IR laser power
is flawed in that you will eventually get direct IR damage. It is much more
gentle on the specimen to pre-chirp the laser pulse so that it is very narrow
within your specimen, and then you can use minimal laser power. We are very happy with the Deep-See system, it is a simple matter of
tweaking the pre-chirp to maximise the signal from the specimen. It would be a nice idea if the confocal software allowed you
to set a few different pre-chirp settings at different depths in the specimen,
and then the correct pre-chirp could be set for various depths throughout the
specimen. Stephen Cody 2008/12/6 Hi Ashley, sounds like an ad for the Mai Tai Deep See! There was a
On Behalf Of A Crane Subject: Re: Multi photon
imaging of thick samples ---------------------------------------------------------------------
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Craig Brideau |
Re: Multi photon imaging of thick samples
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In reply to this post by Stephen Cody-2
Do you notice any phototoxic effects from the shorter pulses? There seems to be some ongoing debate about whether free radical production from higher-energy pulses is actually worse for your sample than the heating from lower peak energy but higher average power pulses.
Do you find the GVD varies much with sample depth? We're talking 100's of microns here which is pretty insignificant compared to the centimeters of glass in the rest of your system. As you go really deep do you find the pre-chirp needs to be adjusted with increasing depth? Craig
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Stephen Cody-2 |
Re: Multi photon imaging of thick samples
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In reply to this post by Armstrong, Brian
Dear List,
I'm sorry if my previous posting sounded like an add for Deep-See, the truth is Coherent had not released their Pre-Chirp system at the time we purchased our system. I'm not able to comment on comparisons as unfortunately I have had no experience on the Coherent system.
The Deep-See system can be set with several saved settings for different wavelengths and objectives currently there is no control from the Olympus software, although I think the Zeiss software may control the pre-chirp (can some one please confirm this). We generally only use the one objective (high NA 20X dipping) for everything. At the moment we generally just set the pre-chirp manually with the control panel, you just set it back and forth until the brightest signal is achieved. It is almost as easy as focusing the microscope. Unfortunately we can't compare a result with and without pre-chirp as it is not a trivial matter to bypass the pre-chirp system, and this would take the system offline in a core facility for some time. At the moment we are easily getting about 300um into zebrafish, we should do a lot better as we have yet to make modifications to keep the room dark so that we can use the NDDs to their full advantage. By the way the system is built on an Olympus FV1000.
Cheers
Steve
2008/12/6 Armstrong, Brian <[hidden email]>
-- Stephen Cody |
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Andreas Bruckbauer |
Re: Multi photon imaging of thick samples
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In reply to this post by Hege Avsnes Dale
Does anyone know how the scan speed effects IR heating of the sample? I
imagine that fast scanning with lower average laser power and frame averaging to get sufficient SNR could be beneficial. Instead of hitting the sample at the same point for a longer time and therefore cooking it, you could hit it several times below the damage threshold and give it enough time to cool in-between. We usually use faster scan times when imaging live lymph nodes to get sufficient time resolution in the time series. This works fine (no damage) with penetration up to 350 micrometer into the tissue on the Olympus Fluoview 1000 multiphoton system with Olympus prechirp. The software allows to adjust laser power and PMT settings for different depths using as many points as you like to define the curve, not only two as in the Zeiss software. I could imagine that when using picosecond pulses and therefore higher average power on the standard Leica setup, you could avoid cooking the sample by faster scan rates using the resonant scanner. Has anyone tried this? |
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In my experience, damage is generally not caused by heating. It's
the electric field causing molecular breakdown. So the key thing is to keep below that threshhold for your particular specimen. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andreas Bruckbauer Sent: Sunday, 7 December 2008 8:31 AM To: [hidden email] Subject: Re: Multi photon imaging of thick samples Does anyone know how the scan speed effects IR heating of the sample? I imagine that fast scanning with lower average laser power and frame averaging to get sufficient SNR could be beneficial. Instead of hitting the sample at the same point for a longer time and therefore cooking it, you could hit it several times below the damage threshold and give it enough time to cool in-between. We usually use faster scan times when imaging live lymph nodes to get sufficient time resolution in the time series. This works fine (no damage) with penetration up to 350 micrometer into the tissue on the Olympus Fluoview 1000 multiphoton system with Olympus prechirp. The software allows to adjust laser power and PMT settings for different depths using as many points as you like to define the curve, not only two as in the Zeiss software. I could imagine that when using picosecond pulses and therefore higher average power on the standard Leica setup, you could avoid cooking the sample by faster scan rates using the resonant scanner. Has anyone tried this? No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.14/1832 - Release Date: 5/12/2008 9:57 AM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.14/1832 - Release Date: 5/12/2008 9:57 AM |
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In reply to this post by Hege Avsnes Dale
Dear Guy,
this may be an ignorant question, but how would one assess the heating versus electric field effects in a sample. To a relatively inexperienced person this doesn't sound trivial. Darran -original message- Subject: Re: Multi photon imaging of thick samples From: Guy Cox <[hidden email]> Date: 07/12/2008 11:25 In my experience, damage is generally not caused by heating. It's the electric field causing molecular breakdown. So the key thing is to keep below that threshhold for your particular specimen. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andreas Bruckbauer Sent: Sunday, 7 December 2008 8:31 AM To: [hidden email] Subject: Re: Multi photon imaging of thick samples Does anyone know how the scan speed effects IR heating of the sample? I imagine that fast scanning with lower average laser power and frame averaging to get sufficient SNR could be beneficial. Instead of hitting the sample at the same point for a longer time and therefore cooking it, you could hit it several times below the damage threshold and give it enough time to cool in-between. We usually use faster scan times when imaging live lymph nodes to get sufficient time resolution in the time series. This works fine (no damage) with penetration up to 350 micrometer into the tissue on the Olympus Fluoview 1000 multiphoton system with Olympus prechirp. The software allows to adjust laser power and PMT settings for different depths using as many points as you like to define the curve, not only two as in the Zeiss software. I could imagine that when using picosecond pulses and therefore higher average power on the standard Leica setup, you could avoid cooking the sample by faster scan rates using the resonant scanner. Has anyone tried this? No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.14/1832 - Release Date: 5/12/2008 9:57 AM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.14/1832 - Release Date: 5/12/2008 9:57 AM |
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In reply to this post by Armstrong, Brian
Dear All,
Again this is not directly a confocal question, but perhaps someone knows the answer. We have a number of Hamamatsu Orca ER cameras in our facility. The older models use a Snapper frame grabber board for grabbing the images, which (on some systems) were exchanged (upgraded) to a Phoenix card. We have experienced that the various (commercial) software packages support these cards in a very heterogeneous manner. Sometimes it works, sometimes not etc. On the other hand the newer Orca ERs, which already have a FireWire connection, always work well. Therefore we though, why not to upgrade (= exchange/modify the controller) the old cameras also to FireWire. Unfortunately, the official Hamamatsu answer was that they can't do this, because they don't have any components left. I's wondering if anyone of you have succeeded to make such an upgrade, and if yes, would you be ready to share your experiences with us? Thanks Gabor -- Gabor Csucs Light Microscopy Centre, ETH Zurich Schafmattstrasse 18, HPM F16 CH-8093, Zurich, Switzerland Web: www.lmc.ethz.ch Phone: +41 44 633 6221 Fax: +41 44 632 1298 e-mail: [hidden email] |
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