Posted by
Jerry (Gerald) Sedgewick on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Uniformity-of-2-photon-illumination-tp6719119p6720296.html
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To determine if the laser is not centered, you might want to do one of two
things that has worked for me. You can set the laser at a wavelength you
can see closer to 720nm. Place business card or the like under a 10X lens.
Activate the laser and then focus up and down. If the spot does not remain
centered as you move up and down, the laser is off axis with the optics. My
thought is that the laser would likely be badly misaligned in this scenario.
For another means of determining a misaligned laser, print a target with
larger and larger circles so that this can fit nicely over the field lens
(you will have to measure the diameter of the field lens and then make the
outermost circle at that diameter, cut out the target using the outermost
diameter as a guide, so that the target is centered). Use a 4x or 10x lens,
remove the condensor and activate laser. You should be able to see the
illumination pattern.
This target is a nice thing to use when aligning the laser yourself (if you
do not void your service agreement/contract). With 2 people in the room,
one aligning and the other looking at the target, the task of alignment is
made easier. While it's true that an attachment for the microscope can be
had for centering, the fact that head movement in relation to that device
always made me less certain about its efficacy.
Otherwise, like Guy said, the Chroma slide is a good test.
Jerry Sedgewick
On Wed, Aug 24, 2011 at 2:31 AM, Guy Cox <
[hidden email]> wrote:
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> The Chroma slide should be a pretty good test. You should do 2 or 3
> focal positions in case the effect is actually a tilt in the stage or
> the optics. Do you see the same effect in the confocal and
> non-descanned detectors? If so it's clearly not something interfering
> with the detection optics, and you'd have to blame the illumination.
>
> Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox CRC Press / Taylor & Francis
>
http://www.guycox.com/optical.htm> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
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> Phone +61 2 9351 3176 Fax +61 2 9351 7682
> Mobile 0413 281 861
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> -----Original Message-----
> From: Confocal Microscopy List [mailto:
[hidden email]]
> On Behalf Of stu_the_flat
> Sent: Wednesday, 24 August 2011 5:14 PM
> To:
[hidden email]
> Subject: Uniformity of 2-photon illumination?
>
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> Dear list users,
>
> We have a Zeiss 510 two-photon system, it seems to have a an non-uniform
> field of illumination,
>
> I have tried to test this by imaging a chroma fluorescent test slide
> there
> is a two fold drop in fluorescent signal across the X axis, the there
> appears to be a slight variation on the Y axis as well,
>
> What is most likely to be causing this? Is imaging a test slide a fair
> test?
>
> Any advice would be most welcome.
>
> Thank you
>
> Stuart McIntyre
>
>
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