Two-photon microscope questions

Posted by Heping Yuan on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Two-photon-microscope-questions-tp7583010.html

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi everyone, I was looking to modify an old confocal system into a two-photon system. I
had two questions and would appreciate any help.

1) A section of the schematic of typical two-photon is as follows
(http://www.thorlabs.com/tutorials.cfm?tabID=32729):

Galvos -> Scan Lens -> Tube Lens -> Dichroic (690 LP) -> Objective

where fluorescence emission is reflected off Dichroic towards a collection lens and PMT's

I'm wondering if there are some unforeseen problems in changing the order as follows:

Galvos -> Scan Lens -> Dichroic (690 LP)  -> Tube Lens -> Objective

where the tube lens can focus the fluorescence emission back to the Dichroic and directly
into the PMT's (without a collection lens).

2) What is a common procedure to image the back aperture of the objective to the input
window of the PMT? My first thought is to create a collimated source with size > than
back aperture and shine directly into the back aperture with objective off (to see spot size
at PMT input). Would this be correct? If so, practically speaking what type of source is
typically used?

Thanks,
Heping