*****
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,

I'm not familiar with that specific scan unit, but I have aligned
scanning system before, and excessive vignetting at large scan angles often
means that the scanners are axially displaced from the back aperture of the
scan lens (or tube lens if the scan lens is integrated into the scanner).
This results in reduced back-coupling into the pinhole off axis, but
relatively normal coupling on-axis.

The other possibility is just that your objective or scanner do not perform
well, but if you are seeing significant loss of signal at reasonable field
(say half the field number or less), I would try to check the alignment.

Mike

On Sat, Dec 13, 2014 at 2:22 PM, Emmanuel Levy <[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 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 <[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.
> > *****
> >
> > 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 <[log in to unmask]>
> > Komu: [log in to unmask]
> > 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"
> >
>