Multi photon imaging of thick samples

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Hege Avsnes Dale Hege Avsnes Dale
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Multi photon imaging of thick samples

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
Guy Cox Guy Cox
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Re: Multi photon imaging of thick samples

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
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

 


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.
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

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Alison J. North Alison J. North
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Re: Multi photon imaging of thick samples

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
Armstrong, Brian Armstrong, Brian
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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


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Qi, Hai (NIH/NIAID) [F] Qi, Hai (NIH/NIAID) [F]
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Re: Multi photon imaging of thick samples

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
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-----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


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Hege Avsnes Dale Hege Avsnes Dale
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Re: Multi photon imaging of thick samples

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:

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

--


 

A Crane A Crane
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Re: Multi photon imaging of thick samples

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
Armstrong, Brian Armstrong, Brian
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Re: Multi photon imaging of thick samples

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.


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Stephen Cody-2 Stephen Cody-2
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Re: Multi photon imaging of thick samples

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
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.


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Stephen Cody
Armstrong, Brian Armstrong, Brian
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Re: Multi photon imaging of thick samples

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 Hope

Dept of Neuroscience

1450 E Duarte Rd

Duarte, CA 91010

626-256-4673 x62872

http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx


From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stephen Cody
Sent: Friday, December 05, 2008 3:58 PM
To: [hidden email]
Subject: Re: Multi photon imaging of thick samples

 

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
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.

---------------------------------------------------------------------

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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

Craig Brideau Craig Brideau
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Re: Multi photon imaging of thick samples

In reply to this post by Stephen Cody-2

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.

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.

 
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

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

 
 
2008/12/6 Armstrong, Brian <[hidden email]>

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.


---------------------------------------------------------------------

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Stephen Cody

Stephen Cody-2 Stephen Cody-2
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Re: Multi photon imaging of thick samples

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]>

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 Hope

Dept of Neuroscience

1450 E Duarte Rd

Duarte, CA 91010

626-256-4673 x62872

http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx


From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stephen Cody
Sent: Friday, December 05, 2008 3:58 PM


To: [hidden email]
Subject: Re: Multi photon imaging of thick samples

 

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
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




--
Stephen Cody
Andreas Bruckbauer Andreas Bruckbauer
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Re: Multi photon imaging of thick samples

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?
Guy Cox Guy Cox
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Re: Multi photon imaging of thick samples

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?

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Re: Multi photon imaging of thick samples

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?

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Csúcs  Gábor Csúcs Gábor
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Question about Hamamatsu cameras

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]