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Dear All,

Thank you for your feedback, I would like to address a few points that were
raised:

- The objective is 60X oil, plan Apo
- The sample is thin and did not bleach
- The laser is indeed not perfectly aligned but I do not think it could
explain the >80% variation seen across the FOV.

One important addition: this problem is absent in brightfield (even with
the disk engaged of course). Therefore, the field number should be OK along
the light path. To me, this confirms that the problem is in the
illumination and not the collection.

If there are W1 users, I'd be really grateful if you could share your
experience.

Thank you,

Emmanuel



On 14 December 2014 at 03:46, James Pawley <[log in to unmask]> 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 Emmanuel,
>
> The fact that your intensity pattern is asymmetric says to me that the
> system may have an alignment problem (emitting end of the laser fibre not
> exactly on axis or the coupling optics not lined up with the optical axis
> of the objective etc. ...) . I suggest that you turn on the system with no
> specimen,  and hold a white card say 6-10 cm from the objective. If the BFP
> is properly illuminated, you should see a FULL, UNIFORM circle of
> excitation light and the circle should get larger (and dimmer) as you move
> the card away from the objective.
>
> Another problem might be the coupling optics between the disk and the
> CMOS. It needs to pass any light that makes it through the pinholes on to
> the chip. Vignetting here will make the corners darker. Talk to Yokogawa to
> make sure you have the proper lens.
>
> What is the specimen? If it is planar (and thin) you will need a "Plan"
> objective to keep it in focus. You mention moving the same object to 9
> positions in the FOV. Are you sure it didn't bleach during this process? if
> your specimen is thick and your lens is not a "Plan" lens, then the CMOS
> will be focused on a curved surface in the specimen. If the RI of
> everything (embedment, coverslip, immersion oil or other immersion liquid)
> isn't exactly correct, you will have different amounts of spherical
> aberration throughout the field of view and this is a great way to lose
> signal.
>
> Finally, as shown in Chapter 11, the performance of even very expensive
> optics (Zeiss 1.2 40x cPlanAPO, or 1.4 plan APO oil) drops off as one moves
> to the edge of the FOV. At the very least this is caused by vignetting.
> (Some of the high-NA rays emerging from the near the edge of the FOV just
> "hit the wall". (The glass just isn't large enough in diameter. Often this
> is limited to the available diameter of the threaded hole in the objective
> nosepiece.)
>
> Cheers,
>
> Jim P.
>
>
>  *****
>> 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 Emmanuel, what objective are you using? Maybe the objective has
>> smaller FOV, or the aberrations at the edge of FOV are so severe that the
>> fluorescence can't make it back through the pinholes... What sample are you
>> using? Is it thin fluorescent layer or 'sea of fluorescence'? I have only
>> worked with CSU X1 (with 8mm CCD), the uniformity was far from perfect, but
>> much better than yours. Also some systems seem to be tuned better than
>> others. With 'UPlanSApo' objectives you should be able to get better
>> results. Ask Andor! They may offer you a (not quite cheap) upgrade called
>> Borealis, a simple setup with multimode fibers (instead if singlemode),
>> vibrating homogenizer and critical illumination setup (instead of
>> Kohler-like). I liked this illumination style very much in Vutara
>> superresolution scopes, though I haven't tried it with Andor's confocals.
>> ---- absolutely no commercial interest ------ Best, zdenek,
>> kcci.virginia.edu ---------- PÛvodní zpráva ---------- Od: Emmanuel Levy
>> <[log in to unmask]> Komu: [log in to unmask] Datum:
>> 13. 12. 2014 12:29:45 PÞedmût: Bad fluorescence uniformity across the field
>> of view with a Yokogawa W1 unit "***** 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 All, We just acquired a Yokogawa W1
>> system coupled to a X83 Olympus microscope. It is working fine. However, we
>> noticed significant non-uniformity across the field of view. We are using
>> sCMOS cameras (flash4) that have a large chip, but the W1 was designed
>> specifically for such a large field of view so I doubt that the non
>> uniformity we observe is normal. Hence I'd like to ask if anyone
>> encountered a similar problem, and most importantly, of the same magnitude.
>> To give specific numbers, you can see below a table reflecting the
>> intensity that we measured for a specific object (cell), which we moved
>> from the center to all corners of the image. The first thing one can notice
>> is that the laser is not centered but considering the differences observed
>> (over 80%!) this would not improve things much even if it was. Fluorescence
>> intensity of an object moved in different parts of the field of view:
>> --------------------------------- | Left Center Right|
>> --------------------------------- | Top 386 1110 760 | | Center 540 1120
>> 1086 | | Bottom 200 450 275 | --------------------------------- We see
>> the same effect with both 488 and 561 lasers. If you have any experience
>> with such an issue I'd be grateful to hear about it, and hopefully how it
>> may be fixed. Thank you, Emmanuel"
>>
>
>
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