superresolution imaging buffers

[Knecht, David]

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We are setting up a STORM system and I have been reading lots of papers on the topic, especially with regard to imaging buffers.  There are lots of papers examining various buffers in terms of deoxygenation and thiol effects for optimal blinking and probe stability.  The PCA/PCD deoxygenation system seems to crop up often in recent literature and appears to have advantages of pH stability over glucose oxidase/catalase.  However, the reagents are quite expensive.  The pyranose oxidase system is much more reasonable, cost wise, and appears to have the same pH stability advantage, but I am having a hard time telling if there are unstated disadvantages since it appears less commonly used.  Can anyone comment on whether there is any reason not to use pyranose oxidase?  Dave

Dr. David Knecht
Professor of Molecular and Cell Biology
Core Microscopy Facility Director
University of Connecticut
91 N. Eagleville Rd.
Storrs, CT 06269
860-486-2200

[lechristophe]

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Hi David,

We use the pyranose oxidase buffer (standard MEA/Glucose/catalase buffer)
with Alexa Fluor 647 on a Nikon N-STORM microscope and it's working great,
as good as the glucose oxidase one. It is definitely more stable, we
usually prepare it in the morning and using throughout the day, can last
several hours on the same coverslip in a semi-closed chamber, buffer drying
down usually occurs before any drop in efficiency.

Christophe

On Sat, Oct 10, 2015 at 1:31 AM, Knecht, David <[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.
> *****
>
> We are setting up a STORM system and I have been reading lots of papers on
> the topic, especially with regard to imaging buffers.  There are lots of
> papers examining various buffers in terms of deoxygenation and thiol
> effects for optimal blinking and probe stability.  The PCA/PCD
> deoxygenation system seems to crop up often in recent literature and
> appears to have advantages of pH stability over glucose oxidase/catalase.
> However, the reagents are quite expensive.  The pyranose oxidase system is
> much more reasonable, cost wise, and appears to have the same pH stability
> advantage, but I am having a hard time telling if there are unstated
> disadvantages since it appears less commonly used.  Can anyone comment on
> whether there is any reason not to use pyranose oxidase?  Dave
>
> Dr. David Knecht
> Professor of Molecular and Cell Biology
> Core Microscopy Facility Director
> University of Connecticut
> 91 N. Eagleville Rd.
> Storrs, CT 06269
> 860-486-2200
>

[Dan Metcalf]

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Hi David,

The glucose oxidase with MEA-HCl recipe works well and can be make much more stable
if you fill to the top of the chamber and then put another coverslip over the top. To avoid
having to make large volumes of the buffer this is most convenient in the Ibidi or Labtek
8 well type chambers. This will work for 4-6 hours in my experience. I presume this is
because the oxygen exchange is slowed down significantly so the enzymes don't deplete
the substrates and change the pH. I would always use fresh MEA-HCL but I make and
freeze aliquots of the enzymes and glucose. I published the recipe in JOVE (Test Samples
for Optimizing STORM Super-Resolution Microscopy).

If you search for the Bioimaging UK wiki training and documents page you should find this
guide about the glass:
http://www.bioimaginguk.org/images/a/a3/Glass.pdf

Regards,

Dan

=====================
Dan Metcalf
Advanced Imaging Specialist
Instruments
Nikon UK Limited
Nikon House, 380 Richmond Road, Kingston, Surrey, KT2 5PR
Mob: [phone]
http://www.nikoninstruments.com/

[Ann-W]

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Hi Dave

I shouldn't see why not although I find the non hard setting vectasheild gives just as good results, is cost effective and easier to handle. I've considered putting some kind of reducing agent into the vectasheild to see if this gives even better blinking but haven't got round to doing it yet.

All the best!

Ann


Dr Ann Wheeler
ESRIC + 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, www.esric.org




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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Knecht, David
Sent: 10 October 2015 00:31
To: [hidden email]
Subject: superresolution imaging buffers

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*****

We are setting up a STORM system and I have been reading lots of papers on the topic, especially with regard to imaging buffers.  There are lots of papers examining various buffers in terms of deoxygenation and thiol effects for optimal blinking and probe stability.  The PCA/PCD deoxygenation system seems to crop up often in recent literature and appears to have advantages of pH stability over glucose oxidase/catalase.  However, the reagents are quite expensive.  The pyranose oxidase system is much more reasonable, cost wise, and appears to have the same pH stability advantage, but I am having a hard time telling if there are unstated disadvantages since it appears less commonly used.  Can anyone comment on whether there is any reason not to use pyranose oxidase?  Dave

Dr. David Knecht
Professor of Molecular and Cell Biology
Core Microscopy Facility Director
University of Connecticut
91 N. Eagleville Rd.
Storrs, CT 06269
860-486-2200