Deposit FluoSpheres (carboxylate-modified) for calibration measurements

[lechristophe]

***** 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,

I have some small FluoSpheres (20 nm far-red, carboxylate-modified) that I would like to deposit on polylysine-coated glass coverslips (or non-coated ones if better) in order to calibrate my STORM microscope (similar to PSF measurement, but denser to calibrate Z astigmatism).

I usually do it with 100 nm tetraspeck beads, which readily adhere to PLL-coated coverslips (I use 1/50-1/500 depending on the density I want to obtain). With the FluoSphere I don't get any specific signal. Should I use them less diluted (they are "2% solid"), or is there an adhesion problem (my understanding is that carboxylate-modified are negatively charged and won't adhere to PLL)? Do you have a specific protocol for these FluoSpheres?

Thanks,

Christophe

[Jens]

***** 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 Christophe,

carboxylated beads typically bind very well to just glass if in PBS. However if used in water they do not stick.

Best,

Jens

On 28 Sep 2016, at 14:44, Christophe Leterrier <[hidden email]> 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,

I have some small FluoSpheres (20 nm far-red, carboxylate-modified) that I would like to deposit on polylysine-coated glass coverslips (or non-coated ones if better) in order to calibrate my STORM microscope (similar to PSF measurement, but denser to calibrate Z astigmatism).

I usually do it with 100 nm tetraspeck beads, which readily adhere to PLL-coated coverslips (I use 1/50-1/500 depending on the density I want to obtain). With the FluoSphere I don't get any specific signal. Should I use them less diluted (they are "2% solid"), or is there an adhesion problem (my understanding is that carboxylate-modified are negatively charged and won't adhere to PLL)? Do you have a specific protocol for these FluoSpheres?

Thanks,

Christophe

[mmodel]

***** 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. *****

Would it be possible to simply dry them on a coverslip? You don’t need liquid above the coverslip to image what is on its surface

 

Mike Model

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Wednesday, September 28, 2016 8:45 AM
To: [hidden email]
Subject: Deposit FluoSpheres (carboxylate-modified) for calibration measurements

 

***** 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,

 

I have some small FluoSpheres (20 nm far-red, carboxylate-modified) that I would like to deposit on polylysine-coated glass coverslips (or non-coated ones if better) in order to calibrate my STORM microscope (similar to PSF measurement, but denser to calibrate Z astigmatism).

 

I usually do it with 100 nm tetraspeck beads, which readily adhere to PLL-coated coverslips (I use 1/50-1/500 depending on the density I want to obtain). With the FluoSphere I don't get any specific signal. Should I use them less diluted (they are "2% solid"), or is there an adhesion problem (my understanding is that carboxylate-modified are negatively charged and won't adhere to PLL)? Do you have a specific protocol for these FluoSpheres?

 

Thanks,

 

Christophe

[Ewers, Helge]

***** 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 Christophe, 

the problem with the very small spheres is that they are extremely strongly charged. For example they are useless for SPT as well. We never got anything to work with them. I would suggest using bigger ones, or maybe the new ones from Gattaquant. I have ordered some, but not received them yet, I’ll let you know if they work well.

For the far red region, also gold rods work well. And they are in the 20 nm range.

Cheers,

Helge

--
 
 
Dr. Helge Ewers
Senior Lecturer
King's College London
Randall Division of Cell and Molecular Biophysics
 2nd Floor, Hodgkin Building
Guy's Campus
London SE1 1UL
United Kingdom
 
[hidden email]
[phone] tel

On 28 Sep 2016, at 14:44, Christophe Leterrier <[hidden email]> 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,

I have some small FluoSpheres (20 nm far-red, carboxylate-modified) that I would like to deposit on polylysine-coated glass coverslips (or non-coated ones if better) in order to calibrate my STORM microscope (similar to PSF measurement, but denser to calibrate Z astigmatism).

I usually do it with 100 nm tetraspeck beads, which readily adhere to PLL-coated coverslips (I use 1/50-1/500 depending on the density I want to obtain). With the FluoSphere I don't get any specific signal. Should I use them less diluted (they are "2% solid"), or is there an adhesion problem (my understanding is that carboxylate-modified are negatively charged and won't adhere to PLL)? Do you have a specific protocol for these FluoSpheres?

Thanks,

Christophe

[Ann-W]

***** 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 Christophe

 

I’m playing catch up here, however I’ve spent quite a lot of time on this very question. I discovered that drying the spheres onto the coverslip (which as Michael suggests works well) has an issue for 3D. The spheres are literally on the coverslip but for good 3D calibration you would want the spheres slightly above the coverslip. I’ve tried a variety of ways of doing this with success.

 

1.       Dilute the spheres in low mounting point agarose which is transparent and mount these on the coverslip.

2.       Mix the spheres in gelatin. I used fluorescent gelatin (Oregon green 488) which was useful for finding focus.

3.       Dilute the spheres in PDMS and cure the PDMS. This test sample will then likely last a really long time. I tried this sometime ago and it worked and I want to go back to this procedure now as we’ll have a sample we can return to time and again for alignment

4.       If all else fails Gattaquant Nanoruler are also good for this job.

 

Another trick which you may find useful – we certainly did for setting up 2 colour STORM – is mixing the tetraspeck beads with the much smaller beads (maybe a 1:5 ratio?). Its very difficult to find 20nm beads by eye and its useful to find a good place in the bead lawn for setting up the system with.

 

Hope this helps

Ann

 

Dr Ann Wheeler
Advanced Imaging Resource, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU

E: [hidden email]
T: 0131 651 8665
W: http://www.igmm.ac.uk/imaging.htm

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: 28 September 2016 13:45
To: [hidden email]
Subject: Deposit FluoSpheres (carboxylate-modified) for calibration measurements

 

***** 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,

 

I have some small FluoSpheres (20 nm far-red, carboxylate-modified) that I would like to deposit on polylysine-coated glass coverslips (or non-coated ones if better) in order to calibrate my STORM microscope (similar to PSF measurement, but denser to calibrate Z astigmatism).

 

I usually do it with 100 nm tetraspeck beads, which readily adhere to PLL-coated coverslips (I use 1/50-1/500 depending on the density I want to obtain). With the FluoSphere I don't get any specific signal. Should I use them less diluted (they are "2% solid"), or is there an adhesion problem (my understanding is that carboxylate-modified are negatively charged and won't adhere to PLL)? Do you have a specific protocol for these FluoSpheres?

 

Thanks,

 

Christophe

*****
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.
*****

The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.