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:[log in to unmask]]
On Behalf Of Alison North
Sent: Thursday, December 04, 2008 8:33 AM
To: [log in to unmask]
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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