http://confocal-microscopy-list.275.s1.nabble.com/Two-photon-microscope-questions-tp7583010p7583022.html
P.S. Tsai, and D. Kleinfeld. In vivo two-photon laser scanning microscopy with concurrent plasma-mediated ablation: Principles and hardware realization in Methods for In Vivo Optical Imaging, Second Edition, R. D. Frostig, editor, CRC Press, 3:59-115 (2009)
> On 30 Nov 2014, at 00:41, Zdenek Svindrych <
[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.
> *****
>
> Yes,
> the BFP of the objective should be the only aperture that limits your
> optical system. By properly imaging this onto the detector you don't loose
> any useful light. It is convenient to use black tubes, as any light leaking
> into the system will degrade your image considerably.
>
> With a high magnification objective (say 60x 1.4NA) the rays between the
> objective and the tube lens are usually well collimated, so you don't need
> to be concerned with 'long' relay lenses between the objective and the non-
> descanned detector (this is not entirely true for scattered two-photon
> fluorescence, you need to perform some sort of simulation or calculation to
> get an idea about the diameter/focal length of your relay lenses necessary
> to collect out-of-focus light) .
>
> Best, Zdenek, www.kcci.virginia.edu
>
>
>
> ---------- Původní zpráva ----------
> Od: Michael Giacomelli <
[hidden email]>
> Komu:
[hidden email]
> Datum: 29. 11. 2014 18:27:33
> Předmět: Re: Two-photon microscope questions
>
> "*****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> Post images on
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> *****
>
> Hi Peter,
>
> With good optical design you can collect nearly the full radiance of the
> objective back aperture (that is, the only place where light will hit the
> sides is inside the objective). There should be negligible signal hitting
> the lens tubes, but perhaps a bit of light leakage from outside the
> microscope if any part is not fully light tight. I think this is the main
> concern, although in practice I'm not sure how much it really matters.
>
> Mike
>
> On Sat, Nov 29, 2014 at 6:07 PM, Peter Rupprecht <
[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.
>> *****
>>
>> Dear Heping,
>>
>> To your 2nd question:
>>>> 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?
>>
>> This sounds difficult to me. The detector size is usually so big that it
>> would be difficult to find the optimal alignment by this method.
>>
>> For a quick alignment, I would, if the excitation light path is perfectly
>> aligned, put a homogeneous plastic fluoslide under the objective and align
>> the detection pathway such that the picture you get looks as good as
>> possible. Do this with the lowest zoom setting (highest scanning angle),
>> otherwise it will always look perfect. With "good", I mean a FOV with the
>> highest brightness in its center. Simply play around with mirror angles
>> while imaging and check the picture live. Usually, due to aperture
> clipping
>> effects while scanning, the FOV is not totally homogeneous. This also
>> depends on the highest zoom factor allowed by your scanners or your
>> software. -- If someone has a better idea, I'd be happy to learn them!
>>
>> As already mentioned, I would also vote for putting the dichroic as close
>> to the objective as possible.
>>
>>
>> As a sidenote, I have always been wondering why some 2P-microscopes I've
>> seen use such long detection pathways (>15 cm) for 2P microscopy, with all
>> the tubes and inner parts being completely black. Wouldn't it be much
>> better to coat the full detection pathway surfaces with an aluminium or
>> silver coating in order to trap every scattered photon until it impinges
> on
>> the detector? Has anyone tried this out and compared it to non-coated
>> surfaces for highly scattering conditions?
>> Best,
>> Peter
>> "