Re: Acquiring a multiphoton system-some questions

Posted by Craig Brideau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Acquiring-a-multiphoton-system-some-questions-tp592485p592490.html

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal If you are trying to approach 1mm in depth, pre-chirp will matter.  If you are only going 300-500 um approx then you might be able to just crank up your power instead.  If you have an AOM I'd recommend pre-chirping.  The shorter your pulse is out of the laser the more it will benefit from pre-chirping.  Right now there are a few options that I've personally investigated (this is not a complete list by any means!):

Coherent makes the Chameleon, which is about 140fs out of the laser.  This laser is the least broadened by AOMs, glass in prisms/lenses in the microscope, etc. because it starts out with a fairly broad transform-limited pulse.  If you don't have much technical confidence, then this is the simplest way to go, but may not give optimal results depending on how deep you really need to go.

Spectra Physics makes the Mai Tai which typically has 70-80fs out of the laser.  It will be vulnerable to chirp, but at the same time if it is used with pre-chirping (prism compressor or the like) it is capable of good depth performance.  Systems using lasers like this have gone to 700um or so (check papers).

For a more 'far out' solution, I saw a nifty gadget from Coherent at this past Photonics West show.  It's a 10 or 20fs laser with a pre-chirping module add on/built-in.  You could probably go pretty deep with this thing, but it would require some adjustment of the chirp parameters.  The pulse is so short that it will be very sensitive to chirping.  The one drawback is that it is not tunable, although at 10fs the bandwidth (spectral content) of the laser is already quite broad so it will most likely stimulate most dyes.  If you want to be able to 'tune off' a dye though to check something else it wouldn't be possible with this laser.

Craig

On Sun, Feb 24, 2008 at 2:28 PM, Jan Grawé <[hidden email]> wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

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
Rudbecklaboratoriet/C5
Dag hammarskjölds väg 20
SE-75185 Uppsala
SWEDEN

Phone: +(0)18-4714657
Cell:   +(0)70-2577874
[hidden email]
www.rudbeck.uu.se/cellanalys