Re: Bad fluorescence uniformity across the field of v iew with a Yokogawa W1 unit - Commercial reply

[DENNIS Andrew]

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Dear Emmanuel,

Andor has now developed Borealis for the W1 system, as expected it provides considerable uniformity improvement over the larger area of an sCMOS, or megapixel EMCCD sensor.

If you are interested in getting more info then please drop me a line.

Andrew
[hidden email]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Emmanuel Levy
Sent: 13 December 2014 19:23
To: [hidden email]
Subject: Re: Bad fluorescence uniformity across the field of v iew with a Yokogawa W1 unit

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Dear Zdenek,

For these measurements we used a 60X UplanApo, 1.35NA so I do not believe that the objective is the problem. The Borealis option does not exist for the W1 but it does suggest that things could be improved.

If there are other users of the W1, I would be keen to know what values you get.

The sample observed is a specific yeast cell tagged with YFP, we avoided the Chroma fluorescent slides as we read that it was not optimal to check uniformity. I should mention that under the conditions tested bleaching was negligible (the "center" positioning was measured at the beginning and at the end of all measurements, and gave the same value).

A colleague suggested that the laser was not properly focused at the back focal plane on the objective. But I cannot check this as I cannot open the
W1 box myself (or I'd loose the warranty).

Thanks for your feedback,

Emmanuel


On 13 December 2014 at 20:36, Zdenek Svindrych <[hidden email]> wrote:

>
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> 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
>
>
>
>
>
>
> ---------- Původní zpráva ----------
> Od: Emmanuel Levy <[hidden email]>
> Komu: [hidden email]
> Datum: 13. 12. 2014 12:29:45
> Předmět: 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"
>