Re: Two-photon microscope questions

Posted by Michael Giacomelli on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Two-photon-microscope-questions-tp7583010p7583015.html

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The main problem with changing the order will be a loss of collection NA,
particularly for larger fields of view. If you take apart the thorlabs
optics you will see that they have placed the dichroic nearly touching the
back of the objective. This maximizes light collection.

This may or may not matter depending on how deep you intend to image into
samples, the field of view you want to scan, and the effective focal length
of your objective. I would measure the displacement you intend to add
between the dichroic and objective and compute the approximate collection
NA and FOV that will give you.

Mike

On Sat, Nov 29, 2014, 12:33 PM Andrew York <
[hidden email]> wrote:

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>
>  Short answer to (1) is, astigmatism.
>
>  Transmission through a flat, tilted piece of glass gives only a lateral
> deflection to a collimated beam, but it gives astigmatism to a focusing
> beam. You could imagine using a dichroic that reflects the excitation
> rather than transmitting it, but be careful about flatness (most dichroics
> are quite curved, sadly) which can also give astigmatism.
>
>  Because of some bad decisions, I ended up using a dichroic in the same
> manner you describe. You can cancel a lot of the resulting astigmatism with
> a second piece of glass tilted the same amount but rotated 90 degrees about
> the optic axis. On the other hand, why bother solving a problem you don't
> have to have?
>
> On Sat, Nov 29, 2014 at 11:23 AM, Heping Yuan <[hidden email]>
> wrote:
>
> > *****
> > 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
> >
>