Hi Mike and list,
Putting the beads on the coverglass is a good
start, but #1.5 is way too high a tolerance range
for best performing fluorescence microscopes and
nanoscopes. How about switching to the Zeiss 170
um coverglasses or equivalent. $40 for pack of 1,000 pieces, 18x18 mm.
I also encourage mattek
(www.glassbottomdishes.com) and everyone else to switch to 170 um coverglasses.
I also have a negative recommendation with
respect to Lab-Tek chamber slides. I had a
confocal user Friday who had their cells on a
Lab-Tek chamber slides with (unknown thickness)
coverglass applied by the user. While I like the
convenience of the blue ink ridges for finding
the focus, the Lab-Tek slides ridge thickness is
a significant gap between the objective lens and
cells. I also have no idea what kind of
coverglass thickness the user had or their
mounting medium. I also suspect they had left the
slides in a refrigerator, and managed to get
water condensation mixed with immersion oil on
imaging. What I can say is that their image
quality was mediocre, even after cleaned the oil
off the lens and coverglass and reapplied fresh
oil. The Lab-Tek slides also have the wonderful
property of warping during focusing - making it a
waste of time to try to do Z-series.
Sincerely,
George
At 05:31 PM 3/26/2010, you wrote:
>Dear Listservors,
>
>Thank you all very much for your contributions to this discussion.
>
>We have always used #1.5 coverslips(which range
>from 160 to 190 microns). All biological
>objectives (except those with correction
>collars) are designed for these. Going to #1
>coverslips would only narrow things by 30
>microns at best and defeat the optical correction factor.
>
>We continue to encourage do-it-yourselfers (DYI)
>slides as others have mentioned, and include
>protocols to do this. We are very generous in
>the quantities of beads shipped - you can make
>dozens to hundreds of slides from one vial.
>However we developed the multiple bead slides as
>a cost savings to the customer needing more than
>one bead type. Buying 10 bead preps is 2-5x
>what one of the prepared slides will cost.
>
>Unfortunately, the prepared slides are a quite a
>challenges to make in quantity. We have strict
>QC standards - our scrap rate and thus costs are
>high. We haven't implemented a QC step with oil
>objectives because of the damage to the slide
>that would entail. As mentioned in an earlier
>post we are addressing this going forward (first up mounting on coverslips).
>
>We will refund anyone that is unable to see
>their beads on our slides of course. Just call
>our Tech Support for a credit or replacement, at
>800-955-6288, then selection 2, then selection 4 to request this.
>
>Regards,
>
>Mike Ignatius
>
>Molecular Probes/Lifetechnologies
>
>
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[log in to unmask]] On Behalf Of Peter Pitrone
>Sent: Friday, March 26, 2010 6:03 AM
>To: [log in to unmask]
>Subject: Re: FocalCheck test slide
>
>They sell these "test slides" for hundreds of
>dollars, yet they don't take the time to make
>them right?!?! They should be made with correct
>coverslips chosen for their thickness, deposited
>on them and then mounted on the glass slide... I
>see that there is a disconnect some where, how
>much does it cost them to produce these slides? 10 bucks maybe...
>
>Pete
>
>On Mar 26, 2010, at 12:25 PM, Guy Cox wrote:
>
> > No, no, it IS the problem! As Mike Ignatius
> explained, MP put the beads on the slide, not
> the coverslip, then add mountant and then the
> coverslip. If you are using a #1.5 coverslip
> you need to put the beads directly on the
> coverslip. With the beads on the slide the
> extra thickness of the mountant needs to be
> corrected for by using a thinner coverslip - #1
> or #0 - which must be found by trial and
> error. (But since I guess they are using very
> reproducible conditions they only need to do
> the test once). At least it seems they have realized they have a problem.
> >
> > Guy
> >
> > -----Original Message-----
> > From: Confocal Microscopy List
> [mailto:[log in to unmask]] On
> Behalf Of MICROSCOPIA CONFOCAL y CCD
> > Sent: Thursday, 25 March 2010 10:15 PM
> > To: [log in to unmask]
> > Subject: Re: FocalCheck test slide
> >
> >> Thanks a lot for your answers.
> >
> >> Of course I put the slide upside down when using
> >> inverted microscope and I asked for Molecular
> >> probes about coverslip and they answered me:
> >> "The coverslip thickness for the FocalCheck
> >> prepared slides is 0.13-0.16 mm" It´s no ideal
> >> but I think that this is not the problem.
> >
> > M. Teresa
> >
> > SERVICIO de MICROSCOPIA CONFOCAL y CCD
> > Mª Teresa Seisdedos Domínguez
> > Oscar Hidalgo Blanco
> > Amadeo Cazaña Soto
> > [log in to unmask]
> > Centro Investigaciones Biológicas (CIB) CSIC
> > C/Ramiro de Maeztu, 9
> > 28040 Madrid
> > Phone: + 34-91 8373112 ext.4401
> > Fax: + 34-91 536 04 32
George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[log in to unmask]
[log in to unmask]
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the
entire 2000+ spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara
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