eGFP slides?

[Jean-Yves Tinevez-3]

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Dear friends and colleagues,

For a microscope calibration assay, I am trying to image a thin layer  
of eGFP-like fluorophore.

I aim at imaging a thin layer of fluorophore deposited on a microscopy  
slide. I turned to the Murray and colleague solution (1), but they  
image fluorescein, which spectra are quite different from eGFP.

I have been looking for eGFP preparation commercially available, but  
unfruitfully so far.

The closest I could find was the BD Living Colors EGFP Calibration  
Beads, but these beads are 7-10 µm in diameter, larger than the depth  
of focus of the systems I will use.

Would you know of any solution to get or purchase eGFP on small beads?

Thank you very much
Best regards
jyt




(1) Murray et al. Evaluating performance in three-dimensional  
fluorescence microscopy. Journal of microscopy (2007) vol. 228 (Pt 3)  
pp. 390-405



--
Jean-Yves Tinevez
PFID - Imagopole
Institut Pasteur
25-28, rue du Docteur Roux
75724 Paris cedex 15
France
tel: [phone]

[mmodel]

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Hi Jean-Yves,
Are you talking about wide-field or confocal? If you are using WF, the intensity collected from a thin layer will depend on the depth of the layer, and  one way to do measure the number of GFP molecules is to prepare a layer of GFP solution of controlled depth
Cytometry Part A
Volume 75A, Issue 10, pages 874–881, October 2009

Mike




________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Jean-Yves Tinevez [[hidden email]]
Sent: Monday, September 20, 2010 4:03 AM
To: [hidden email]
Subject: eGFP slides?

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Dear friends and colleagues,

For a microscope calibration assay, I am trying to image a thin layer
of eGFP-like fluorophore.

I aim at imaging a thin layer of fluorophore deposited on a microscopy
slide. I turned to the Murray and colleague solution (1), but they
image fluorescein, which spectra are quite different from eGFP.

I have been looking for eGFP preparation commercially available, but
unfruitfully so far.

The closest I could find was the BD Living Colors EGFP Calibration
Beads, but these beads are 7-10 µm in diameter, larger than the depth
of focus of the systems I will use.

Would you know of any solution to get or purchase eGFP on small beads?

Thank you very much
Best regards
jyt




(1) Murray et al. Evaluating performance in three-dimensional
fluorescence microscopy. Journal of microscopy (2007) vol. 228 (Pt 3)
pp. 390-405



--
Jean-Yves Tinevez
PFID - Imagopole
Institut Pasteur
25-28, rue du Docteur Roux
75724 Paris cedex 15
France
tel: [phone]

[Michael Weber-4]

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Dear JY,

since you wrote "eGFP-like", I suppose another fluorophore with comparable
excitation/emission properties will do the job? So you need a thin layer
of fluorescence? One way is to use FITC in solution. You can put a drop
between slide and coverslip. Or you spin-coat a coverslip with the
solution, this would result in a thinner layer. FITC is very bright, but
bleaching will be a problem, so maybe there are more stable dyes available
in solution.

Michael

> ============================================================
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> ============================================================
>
> Dear friends and colleagues,
>
> For a microscope calibration assay, I am trying to image a thin layer
> of eGFP-like fluorophore.
>
> I aim at imaging a thin layer of fluorophore deposited on a microscopy
> slide. I turned to the Murray and colleague solution (1), but they
> image fluorescein, which spectra are quite different from eGFP.
>
> I have been looking for eGFP preparation commercially available, but
> unfruitfully so far.
>
> The closest I could find was the BD Living Colors EGFP Calibration
> Beads, but these beads are 7-10 µm in diameter, larger than the depth
> of focus of the systems I will use.
>
> Would you know of any solution to get or purchase eGFP on small beads?
>
> Thank you very much
> Best regards
> jyt
>
>
>
>
> (1) Murray et al. Evaluating performance in three-dimensional
> fluorescence microscopy. Journal of microscopy (2007) vol. 228 (Pt 3)
> pp. 390-405
>
>
>
> --
> Jean-Yves Tinevez
> PFID - Imagopole
> Institut Pasteur
> 25-28, rue du Docteur Roux
> 75724 Paris cedex 15
> France
> tel: [phone]

[Johannes-P. Koch]

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  Dear Jean-Yves,

similarly, since you wrote "eGFP-like" and "small beads", why don't you try:

PS-Speck Beads from Molecular Probes, Catno P-7220 - they are
subresolution beads, very bleach-resistant to my knowledge (at least for
"normal" measurements...)

http://products.invitrogen.com/ivgn/product/P7220?ICID=search-p7220

Just let them air-dry on your CS, embed it, and that's it...

Best,
Johannes




Am 20.09.2010 15:54, schrieb Michael Weber:

> ============================================================
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> ============================================================
>
> Dear JY,
>
> since you wrote "eGFP-like", I suppose another fluorophore with comparable
> excitation/emission properties will do the job? So you need a thin layer
> of fluorescence? One way is to use FITC in solution. You can put a drop
> between slide and coverslip. Or you spin-coat a coverslip with the
> solution, this would result in a thinner layer. FITC is very bright, but
> bleaching will be a problem, so maybe there are more stable dyes available
> in solution.
>
> Michael
>
>
>> ============================================================
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> ============================================================
>>
>> Dear friends and colleagues,
>>
>> For a microscope calibration assay, I am trying to image a thin layer
>> of eGFP-like fluorophore.
>>
>> I aim at imaging a thin layer of fluorophore deposited on a microscopy
>> slide. I turned to the Murray and colleague solution (1), but they
>> image fluorescein, which spectra are quite different from eGFP.
>>
>> I have been looking for eGFP preparation commercially available, but
>> unfruitfully so far.
>>
>> The closest I could find was the BD Living Colors EGFP Calibration
>> Beads, but these beads are 7-10 µm in diameter, larger than the depth
>> of focus of the systems I will use.
>>
>> Would you know of any solution to get or purchase eGFP on small beads?
>>
>> Thank you very much
>> Best regards
>> jyt
>>
>>
>>
>>
>> (1) Murray et al. Evaluating performance in three-dimensional
>> fluorescence microscopy. Journal of microscopy (2007) vol. 228 (Pt 3)
>> pp. 390-405
>>
>>
>>
>> --
>> Jean-Yves Tinevez
>> PFID - Imagopole
>> Institut Pasteur
>> 25-28, rue du Docteur Roux
>> 75724 Paris cedex 15
>> France
>> tel: [phone]

--
Mag. Johannes-P. KOCH
Department of Biochemistry and Cell Biology
MFPL, University of Vienna
Dr. Bohrgasse 9/5
A-1030 Vienna
Austria

phone: [phone]
fax: [phone]

mail to: [hidden email]

[Boswell, Carl A - (cboswell)]

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While eGFP and FITC do have different spectra, the differences are
relatively slight (~5nm difference in EM max)
http://www.mcb.arizona.edu/ipc/fret/index.html,
http://www.mcb.arizona.edu/IPC/spectra_page.htm.  What are you hoping to do
and why will FITC not work for you?

Cheers,
c

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]
----- Original Message -----
From: "Jean-Yves Tinevez" <[hidden email]>
To: <[hidden email]>
Sent: Monday, September 20, 2010 1:03 AM
Subject: eGFP slides?


============================================================
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
============================================================

Dear friends and colleagues,

For a microscope calibration assay, I am trying to image a thin layer
of eGFP-like fluorophore.

I aim at imaging a thin layer of fluorophore deposited on a microscopy
slide. I turned to the Murray and colleague solution (1), but they
image fluorescein, which spectra are quite different from eGFP.

I have been looking for eGFP preparation commercially available, but
unfruitfully so far.

The closest I could find was the BD Living Colors EGFP Calibration
Beads, but these beads are 7-10 µm in diameter, larger than the depth
of focus of the systems I will use.

Would you know of any solution to get or purchase eGFP on small beads?

Thank you very much
Best regards
jyt




(1) Murray et al. Evaluating performance in three-dimensional
fluorescence microscopy. Journal of microscopy (2007) vol. 228 (Pt 3)
pp. 390-405



--
Jean-Yves Tinevez
PFID - Imagopole
Institut Pasteur
25-28, rue du Docteur Roux
75724 Paris cedex 15
France
tel: [phone]

[Jean-Yves Tinevez-3]

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  First, thank you all for the fantastic replies.

On 20/09/2010 19:20, Carl Boswell wrote:
>
> While eGFP and FITC do have different spectra, the differences are
> relatively slight (~5nm difference in EM max)
> http://www.mcb.arizona.edu/ipc/fret/index.html,
> http://www.mcb.arizona.edu/IPC/spectra_page.htm.  What are you hoping
> to do and why will FITC not work for you?

Then, it is true that I am quite picky when it comes to spectra.
I am trying to relate a measure made on a eGFP specimen to another
measure made on a thin layer of fluorophore, ideally eGFP.
Because the systems I compare have different excitation wavelength (e.g.
the 470 nm of the Colibri system vs the typical 488 nm laser line), the
measure made on the other fluorophore will not reflect what it would
have been for eGFP...

Best regards
jyt