Re: Acquiring a multiphoton system-some questions

Posted by George McNamara on
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Hi Jan,

If you get a Coherent Chameleon II with 3.3 Watts (average, my recollection is that a 100 fs pulse repeated at 100 MHz is 1e6 higher peak, so 200 fs pulses would be 5e5 peak) power - or its Spectra-Physics equivalent - the depth limit is the burning smell of the surface of the tissue (see also Kleinfeld and Tsien's laser ablation article, pubmed 12848930 - I'm curious whether they breathed the ablated molecules).

Overall performance: NDD location (and PMT quality) and objective lenses.

You may want to talk with Martin Kohler in Per-Olaf Berggren's group at the Karolinska about intravital MP imaging. See http://ki.se/ki/jsp/polopoly.jsp?l=sv&d=2061 (full disclosure: PO also works at my location http://www.diabetesresearch.org/AbouttheDRI/DRIFaculty/PerOlofBerggren.htm from his lack of tan, the photo was probably taken at the beginning of one of his visits).


Most people I've asked about pre-chirp have told me it is not worth the bother. I've gone a about 1 mm into a freshly removed mouse brain (Hoechst staining for a few minutes, did perfuse to get rid of the blood) - I think the limit was either how far the (Zeiss 10x lens focused or how much time we were willing to spend acquiring one field of view). A group in the UK - see

Girkin JM, McConnell G.
 
Advances in laser sources for confocal and multiphoton microscopy.
Microsc Res Tech. 2005 May;67(1):8-14. Review.
PMID: 16025485


for example, is one of the few who are into short pulses (how many diseases have they cured with it?). The other use of pre-chirp is with LaVision Biotec's TriMScope, but it is for high speed calcium imaging not deep tissue imaging.



At 04:28 PM 2/24/2008, you wrote:

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Dear all,

we are in the process  of acquiring a multiphoton system intended mainly for intravital imaging, and I have a couple of questions regarding performance:

1. If I understand it correctly (correct me otherwise!),  intensity modulation using an AOM is a major source of chirp. At least one manufacturer offers an alternative without AOM, which is said to improve imaging depth. With the advent of pre-chirping, is this a relevant issue? We would of course like the utility and bleaching capabilities afforded by an AOM, but perhaps not if it significantly reduces the imaging depth.

2. Connected to the above, to those of you that have experience with pre-chirping. How much could we expect this to improve our imaging depth? Is the improvement greater if we choose a laser with shorter pulses ~80fs as compared to 140fs?

2. Since no manufacturer will give any hard numbers with regard to the depth reach of their systems, we would like to be able to test this for ourselves. Do you know of a test sample that can be made several hundred um thick, that is or mimics some type of tissue, that has specific fluorescent structures at various depths, and that preferably is stable at least over some weeks. Are there commercially available MP test samples?
A tall order maybe, but I´m hoping..

4. What components of the system do you feel are the most critical for the overall performance. Is it the laser pulse shape, NDD performance, obcectives, other??


All responses are appreciated!

Best regards,

Jan Grawé




Jan Grawé
Cell Analysis Core Facility
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George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
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http://www.sylvester.org/research/SR_lab_analytical.asp?ana=desc (Analytical Imaging Core Facility)