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Hi Pete and list,
Zeiss states their coverglasses are refractive index = 1.5255 +/-
0.0001 (this is probably for 546 nm light).
Note: the micro-shop web site (
<a href="https://www.micro-shop.zeiss.com/?s=57145921c59652&l=en&p=us&f=a&i=400000002150&o=&h=25&n=0&sd=474030-9000-000#474030-9000-000 " eudora="autourl">
https://www.micro-shop.zeiss.com/?s=57145921c59652&l=en&p=us&f=a&i=400000002150&o=&h=25&n=0&sd=474030-9000-000#474030-9000-000
) states "tolerance D=0.17 mm +/- 0.005 mm " so 170
um +/- 5 um, not the 0.5 um in my previous subject line. If you search
the micro-shop web site, if 474030-9000-000 does not work, use 474030 as
the search term.
Order Number: 474030-9000-000
Size 18x18 mm² type 1 1/2 H as per ISO 8255-1 with restricted
thickness-related tolerance D=0.17 mm +/- 0.005 mm refractive index ...
{GM notes: so these are just the same tolerance as everyone
else's}
Cover glasses, high performance, D=0.17mm, box with 1000
pc.
[go to the zeiss website to see the picture that would go here - the
listserv rejects posts with attachments]
Description:
Size 18x18 mm² type 1 1/2 H as per ISO 8255-1 with restricted
thickness-related tolerance D=0.17 mm +/- 0.005 mm refractive index =
1.5255 +/- 0.00015, Abbe number = 56 +/-2 recommended for applications
with high numerical aperture objectives
As for ISO 8255-1, the ISO is charging CHF 66,00 for the PDF (see
http://www.iso.org/iso/iso_catalogue/catalogue_ics/catalogue_detail_ics.htm?csnumber=51961&ICS1=37&ICS2=20
... available for $37 at
http://www.techstreet.com/standards/ISO/8255_1_1986?product_id=39567 and
other sites), so I am not going to buy it.
Microscopes -- Cover glasses -- Part 1: Dimensional tolerances,
thickness and optical properties
A web page about a draft of the document,
http://www.techstreet.com/standards/din_iso/8255_1_draft?product_id=1077464
, stated the draft was 5 pages.
Further details on a compliant coverglass is at
http://www.proscitech.com.au/cataloguex/get_notes.asp?COVERGLASS
thought the last column of the table at bottom, "flatness in
mm" touting "typically <5.0 mm" looks a bit
silly.
By the way, the thickness ranges for each type of coverglass
are:
#0 ... 85 to 115 um
#1 ... 130 to 160 um
#1.5 ... 160 to 190 um
#2 ... 190 to 230 um.
Enjoy,
George
At 01:23 PM 3/28/2010, Peter Gabriel Pitrone wrote:
Hello George,
You wrote:
"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."
This is exactly what I was implying when I said "They (meaning
Molecular
Probes, from here on MP) should be made with correct coverslips chosen
for
their thickness", however I didn't want to tell MP who to buy
these
coverslips from (to be fair there are other companies making high
precision glass cover slips - Karl. Hecht KG, Sondheim/Rhön, Germany
for
example). It is preposterous to me that MP hasn't done so already. We
are
talking about high quality "Focal" bead check here. How can one
trust the
PSF when the sample was slapped together with what ever was lying
around?
Pete
P.S. What is the R.I. of the common coverslip? After a Google search
for
another coverslip manufacturer, I came across a paper stating "The
ASTM
requires that coverslips have a refractive index of 1.523 + .0052".
Is
this true?
> 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
>>[
[hidden email]] On Behalf Of Peter
Pitrone
>>Sent: Friday, March 26, 2010 6:03 AM
>>To: [hidden email]
>>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
>> [
[hidden email]] On
>> Behalf Of MICROSCOPIA CONFOCAL y CCD
>> > Sent: Thursday, 25 March 2010 10:15 PM
>> > To: [hidden email]
>> > 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
>> > [hidden email]
>> > 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
> [hidden email]
> [hidden email]
> 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
>
Peter Gabriel Pitrone
Light Microscopy Facility
Max Planck Institute for
Molecular Biology and Genetics
Pfotenhauerstr. 108
01307 Dresden
George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
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
George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
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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