Anti-bleaching agents in live-cell microscopy

[Csúcs Gábor-3]

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Dear All,

A user of us uses Oxyrase in her experiments to reduce the amount of
bleaching (tracking YFP labelled spots). Though we are working on
optimizing her experiments also in terms of optics, still the question
came up: what alternatives do exist to Oxyrase and what are your
experiences with those? Clearly we would need something that works with
living cells. By looking on the Internet I have seen the special imaging
biffer of Evrogen. Is anyone using it? Or alternative suggestions?


Thanks    Gabor

Gabor Csucs
ScopeM
Wolgang-Pauli Str. 14
CH-8093 Zurich

Phone: [phone]

[Steffen Dietzel]

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A former colleague used Trolox and was happy about it. It's a
water-solubale Vitamin E derivate.

Steffen

Am 23.01.2015 um 12:03 schrieb Csúcs Gábor:

> *****
> 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.
> *****
>
> Dear All,
>
> A user of us uses Oxyrase in her experiments to reduce the amount of
> bleaching (tracking YFP labelled spots). Though we are working on
> optimizing her experiments also in terms of optics, still the question
> came up: what alternatives do exist to Oxyrase and what are your
> experiences with those? Clearly we would need something that works with
> living cells. By looking on the Internet I have seen the special imaging
> biffer of Evrogen. Is anyone using it? Or alternative suggestions?
>
>
> Thanks    Gabor
>
> Gabor Csucs
> ScopeM
> Wolgang-Pauli Str. 14
> CH-8093 Zurich
>
> Phone: [phone]
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Marchioninistr. 27
D-81377 München

Phone: [phone]
Fax-to-email: +49/89/2180-9976509 skype: steffendietzel
e-mail: [hidden email]

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Marchioninistr. 27
D-81377 München
Germany

[Hallworth, Richard J.]

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Steffen, Gabor - I have never heard of Trolox or any other such agent working for fluorescent proteins, though they work brilliantly for organic fluorophores.  The bleaching mechanisms for fps are unknown, as far as I know.  Anyone know different?

        Rick

Richard Hallworth, Ph.D., Professor
Director, Integrated Biomedical Imaging Facility
Department of Biomedical Sciences
Creighton University School of Medicine
2500 California Plaza
Omaha NE 68178
Ph: [phone]
FAX: [phone]
Email: [hidden email]
http://biomedsci.creighton.edu/hallworth 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: Friday, January 23, 2015 12:03 PM
To: [hidden email]
Subject: Re: Anti-bleaching agents in live-cell microscopy

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A former colleague used Trolox and was happy about it. It's a water-solubale Vitamin E derivate.

Steffen

Am 23.01.2015 um 12:03 schrieb Csúcs Gábor:

> *****
> 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.
> *****
>
> Dear All,
>
> A user of us uses Oxyrase in her experiments to reduce the amount of
> bleaching (tracking YFP labelled spots). Though we are working on
> optimizing her experiments also in terms of optics, still the question
> came up: what alternatives do exist to Oxyrase and what are your
> experiences with those? Clearly we would need something that works
> with living cells. By looking on the Internet I have seen the special
> imaging biffer of Evrogen. Is anyone using it? Or alternative suggestions?
>
>
> Thanks    Gabor
>
> Gabor Csucs
> ScopeM
> Wolgang-Pauli Str. 14
> CH-8093 Zurich
>
> Phone: [phone]
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy

Marchioninistr. 27
D-81377 München

Phone: [phone]
Fax-to-email: +49/89/2180-9976509 skype: steffendietzel
e-mail: [hidden email]

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy

Marchioninistr. 27
D-81377 München
Germany

[WHEELER Ann]

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Hi Gabor

I've tried Oxyrase too, it does work (by black magic) and I definitely would never use it with any leukocytes. I have had some success with Ascorbic acid as well, although thr most ideal solution would be to move to an FP with a higher quantum yeild such as Clover or mNeon green, but cloning is a pain.


Ann

Dr Ann Wheeler

Head of Advanced Imaging / ESRIC@IGMM

MRC IGMM
Crewe Road South
University of Edinburgh
Edinburgh EH4 2XU
UK
________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Hallworth, Richard J. <[hidden email]>
Sent: 23 January 2015 21:16
To: [hidden email]
Subject: Re: Anti-bleaching agents in live-cell microscopy

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Steffen, Gabor - I have never heard of Trolox or any other such agent working for fluorescent proteins, though they work brilliantly for organic fluorophores.  The bleaching mechanisms for fps are unknown, as far as I know.  Anyone know different?

        Rick

Richard Hallworth, Ph.D., Professor
Director, Integrated Biomedical Imaging Facility
Department of Biomedical Sciences
Creighton University School of Medicine
2500 California Plaza
Omaha NE 68178
Ph: [phone]
FAX: [phone]
Email: [hidden email]
http://biomedsci.creighton.edu/hallworth


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


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: Friday, January 23, 2015 12:03 PM
To: [hidden email]
Subject: Re: Anti-bleaching agents in live-cell microscopy

*****
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.
*****

A former colleague used Trolox and was happy about it. It's a water-solubale Vitamin E derivate.

Steffen

Am 23.01.2015 um 12:03 schrieb Csúcs Gábor:

> *****
> 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.
> *****
>
> Dear All,
>
> A user of us uses Oxyrase in her experiments to reduce the amount of
> bleaching (tracking YFP labelled spots). Though we are working on
> optimizing her experiments also in terms of optics, still the question
> came up: what alternatives do exist to Oxyrase and what are your
> experiences with those? Clearly we would need something that works
> with living cells. By looking on the Internet I have seen the special
> imaging biffer of Evrogen. Is anyone using it? Or alternative suggestions?
>
>
> Thanks    Gabor
>
> Gabor Csucs
> ScopeM
> Wolgang-Pauli Str. 14
> CH-8093 Zurich
>
> Phone: [phone]
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy

Marchioninistr. 27
D-81377 München

Phone: [phone]
Fax-to-email: +49/89/2180-9976509 skype: steffendietzel
e-mail: [hidden email]

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy

Marchioninistr. 27
D-81377 München
Germany

[George McNamara]

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*****

Hi Gabor,

Evrogen DMEMgfp2 ... note option to add Rutin (also available from
sigma-aldrich) on last page of the pdf.
http://www.evrogen.com/medium_DMEM_gfp-description/DMEMgfp2.pdf

Marker Gene Technologies Opti-Klear Live Cell Imaging Buffer
http://www.markergene.com/ProductDetails.php/M1919
http://www.markergene.com/ProductDetails.php/M1898

since you are in Europe, probably easier for you to get Evrogen's. For
researchers in the US, Evrogen has distributors here.

Some suggestions for everyone:
* EGFP is soooo 1996 ... are you using a 1996 PC, cell phone or car?
...  middle of http://works.bepress.com/gmcnamara/63/  has a list of
state of the art color FPs. See also Adam 2014 paper (ref below) and
Kurt Thorn's FP properties page,
http://nic.ucsf.edu/FPvisualization/
* multimerize! ... Steven Vogel (Nguyen 2012) has published V6 and even
tastier and more powerful engine, unpublished V8.
* "go binary" - see ://works.bepress.com/gmcnamara/65 ... see also
Burgess 2012 (PCNA Chromobodies) and Stasevich 2014 (FabLEM; can use
alternative ways to get into cells)
* don't overexpress! Just trashes your SNR and produces O2 radicals all
over your cells - see "65" FingR references for one way, or TetOn/Off
(though many tetracyclines are fluorescent), Cumate, RheoSwitch, etc
ligand controllable transcription factors.


Adam V. Phototransformable fluorescent proteins: which one for which application?
Histochem Cell Biol. 2014 Jul;142(1):19-41. doi:10.1007/s00418-014-1190-5. PubMed PMID: 24522394.


Nguyen TA, Sarkar P, Veetil JV, Koushik SV, Vogel SS. Fluorescence
polarization and fluctuation analysis monitors subunit proximity, stoichiometry,
and protein complex hydrodynamics. PLoS One. 2012;7(5):e38209. doi:
10.1371/journal.pone.0038209. Epub 2012 May 30. PubMed PMID: 22666486; PubMed
Central PMCID: PMC3364239.

There are also nanobodies/chromobodies to detect or amplify FPs, see
http://www.allelebiotech.com/nab
and the Burgess paper

Burgess A, Lorca T, Castro A. Quantitative live imaging of endogenous DNA
replication in mammalian cells. PLoS One. 2012;7(9):e45726. doi:
10.1371/journal.pone.0045726. Epub 2012 Sep 20. PubMed PMID: 23029203; PubMed
Central PMCID: PMC3447815.

and Fab-dye equivalent

Stasevich TJ, Hayashi-Takanaka Y, Sato Y, Maehara K, Ohkawa Y, Sakata-Sogawa
K, Tokunaga M, Nagase T, Nozaki N, McNally JG, Kimura H. Regulation of RNA
polymerase II activation by histone acetylation in single living cells. Nature.
2014 Dec 11;516(7530):272-5. doi: 10.1038/nature13714. Epub 2014 Sep 21. PubMed
PMID: 25252976.


Happy IYL2015 http://www.light2015.org/Home.html


George

On 1/23/2015 5:03 AM, Csúcs  Gábor 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.
> *****
>
> Dear All,
>
> A user of us uses Oxyrase in her experiments to reduce the amount of
> bleaching (tracking YFP labelled spots). Though we are working on
> optimizing her experiments also in terms of optics, still the question
> came up: what alternatives do exist to Oxyrase and what are your
> experiences with those? Clearly we would need something that works with
> living cells. By looking on the Internet I have seen the special imaging
> biffer of Evrogen. Is anyone using it? Or alternative suggestions?
>
>
> Thanks    Gabor
>
> Gabor Csucs
> ScopeM
> Wolgang-Pauli Str. 14
> CH-8093 Zurich
>
> Phone: [phone]
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42

[Cole, Richard W (HEALTH)]

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Just to throw another option out there:

LiveLight http://www.cellgs.com/Shop/Cell-Culture-Systems/LiveLight.html

Rich



Richard Cole
Research Scientist V
Director: Advanced Light Microscopy & Image Analysis Core
Wadsworth Center
 
Research Assistant Professor
Dept. of Biomedical Sciences
School of Public Health State University of New York

P.O. Box 509 Albany N.Y. 12201-0509
518-474-7048 Phone
518-473-2895 Fax

Website www.wadsworth.org/cores/alm/index.htm



From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of CONFOCALMICROSCOPY automatic digest system
Sent: Monday, January 26, 2015 1:04 AM
To: [hidden email]
Subject: CONFOCALMICROSCOPY Digest - 23 Jan 2015 to 25 Jan 2015 (#2015-22)



 

CONFOCALMICROSCOPY Digest - 23 Jan 2015 to 25 Jan 2015 (#2015-22)
Table of contents:
. Anti-bleaching agents in live-cell microscopy
1. Anti-bleaching agents in live-cell microscopy
o Re: Anti-bleaching agents in live-cell microscopy (01/25)
From: George McNamara <[hidden email]>


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[Vitaly Boyko]

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Junior Digital Microscopy Technician Opening at Memorial Sloan-Kettering Cancer Center’s Molecular Cytology Core Facility


 
Molecular Cytology Core Facility (MCCF) at the Memorial Sloan-Kettering Cancer Center is seeking an entry-level Junior Digital Microscopy Technician to support our daily operation.  The MCCF is equipped with state of the art optical imaging systems, including Leica SP5 and SP8 confocal microscopes, a spinning disk and a Zeiss line-scanning confocals, and several widefield microscopes, digital slide scanners and also an Atomic Force Microscope.  A number of widefield and confocal systems are equipped for live imaging. The image analysis software used includes Metamorph, Amira, Imaris, AutoDeblur, FIJI, MATLAB, Voloom. 
 
Responsibilities:·        Train and assist users with imaging equipment·        Assist in post-acquisition processing and analysis of the data, writing macros and scripts for image acquisition, processing and analysis·        Maintain, calibrate, and monitor the performance of imaging systems·        Stay informed with the hardware and software advances in optical microscopy·        Work in collaborative settingsQualifications and Skills:·        Bachelor’s degree in Biology or a related field with very strong computational background·        Experience with advanced fluorescent microscopy is preferred, but training will be provided·        Prior experience with image analysis would be significant plus·        Excellent communication skills and ability to work in a team environment are very important·        Strong organizational skills, flexibility in working hours and ability to multi-task·        Interest in biomedical research and working with students, post-doctoral and clinical fellows, and faculty performing research requiring generation of 'high end' and state- of-the-art light microscopy imaging dataMemorial Sloan-Kettering Cancer Center is one of the top ranked leading cancer research and teaching centers, located in Upper East Side of Manhattan. We offer a competitive salary, comprehensive benefits and an excellent working environment.        Please e-mail your CV and cover letter to Madeline Leung, [hidden email]  Yevgeniy RominDigital Microscopy SpecialistMolecular Cytology Core FacilityMemorial Sloan-Kettering Cancer Center415 East 68th St. ZRC-1834New York, NY 10065(646) 888-2186, x2186