Re: CONFOCALMICROSCOPY Digest - 6 Nov 2012 to 7 Nov 2012 (#2012-265)

[Liang, Zhangyi]

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2. Why perform Methanol, Methanol/Acetone fixation at -20C
Methanol/Acetone fixation always provide sharper or clearer immunofluorescence images (it might because of the dehydration).  Formaldehyde always contains some percentage of methanol except you choose a special one.
Paraformaldehyde need to be used freshly, and sometimes to get the "better" pictures, you have to do "pre-extract" before fixation (use PBST to rinse the cells before fixation).

Zhangyi Liang, Ph.D.

Molecular & Cellular Biology, Harvard University
Rm 140, Northwest Building
Cambridge, MA 02138
phone [phone]


________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of CONFOCALMICROSCOPY automatic digest system [[hidden email]]
Sent: Thursday, November 08, 2012 1:02 AM
To: [hidden email]
Subject: CONFOCALMICROSCOPY Digest - 6 Nov 2012 to 7 Nov 2012 (#2012-265)

There are 12 messages totalling 934 lines in this issue.

Topics of the day:

  1. conversion of mEOS2 by 2-photon (3)
  2. Why perform Methanol, Methanol/Acetone fixation at -20C (8)
  3. slidebook file format issues

----------------------------------------------------------------------

Date:    Wed, 7 Nov 2012 09:18:09 +0100
From:    Ekaterina PAPUSHEVA <[hidden email]>
Subject: Re: conversion of mEOS2 by 2-photon

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Hi Sarah,

This protocol worked for us

You have to photoconvert at 800nm with the laser power not more than 0.5 mW=
 at the objective plane.
You need a long time: 3-5min for one z plane  of a square with a size of ha=
lf a cell. To detect the red form you have to do imaging at 970nm.
If the laser power is higher, you bleach immediately the red form.

Play around these lines!

Best regards,
Ekaterina

___

Ekaterina Papusheva

Manager of Bioimaging Facility

Institute of Science and Technology Austria

Phone  [phone];
Mobile [phone]
Fax    [phone]

Am Campus 1

A-3400 Klosterneuburg

Visit our website: www.ist.ac.at


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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On=
 Behalf Of Sarah Richert
Sent: Wednesday, October 31, 2012 3:39 PM
To: [hidden email]
Subject: conversion of mEOS2 by 2-photon

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Dear all,=20
we are working with mEOS2 and would like to photo-convert it by 2-photon.
With 405 nm laser it works but not in 2-photon mode.
Has anybody successfully done that?
Many thanks in advance!
Sarah

------------------------------

Date:    Wed, 7 Nov 2012 09:37:53 +0100
From:    Christophe Leterrier <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

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Something related to this non-complete fixation by aldehydes: Akihiro
Kusumi's lab reported two years ago that lipids and membrane proteins
were still diffusing in the plasma membrane after fixation for up to
90 mintues in 4% PFA. Cold methanol was better in this regard,
although a residual mobility was still observed:
Tanaka, K. A. K., Suzuki, K. G. N., Shirai, Y. M., Shibutani, S. T.,
Miyahara, M. S. H., Tsuboi, H., et al. (2010). Membrane molecules
mobile even after chemical fixation. Nature Methods.
doi:10.1038/nmeth.f.314


Another good & recent article about immunocytochemistry procedures has
been published in Nature Methods:
Schnell, U., Dijk, F., Sjollema, K. A., & Giepmans, B. N. G. (2012).
Immunolabeling artifacts and the need for live-cell imaging. Nature
Methods, 9(2), 152=96158. doi:10.1038/nmeth.1855


And I highly suggest to people that are doing
immunocyto/histochemistry to read R. Burry's book. It covers
everything from fixation to fluorescence imaging and explains a lot of
"chinese whispers", debunking a few ones on the way:
Burry, R. W. (2010). Immunocytochemistry. Springer Verlag.
http://books.google.fr/books?id=3DsvzyJdQVsaEC&printsec=3Dfrontcover&hl=3Df=
r&source=3Dgbs_ge_summary_r&cad=3D0#v=3Donepage&q&f=3Dfalse

Cheers,

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France


On Wed, Nov 7, 2012 at 6:33 AM, Paul Rigby <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>
>
> As an aside, if one looks at the chemistry literature for aldehyde fixati=
on, it is interesting that for complete fixation and stabilisation, times s=
ignificantly longer than 24 hours are required for the cross linking reacti=
ons to go to completion. (For a good explanation see Kiernan, J.A., Microsc=
opy Today, January 2000, pp 8-12.). So, why do we typically fix single cell=
s for only 10 or 30 minutes in formaldehyde solutions before immunolabellin=
g? Probably because it works. Do longer fixation times (24 hours or more) w=
ork better for preservation of single cell structures prior to immunolabell=
ing? I suspect not, but does anyone have direct experience?
>

------------------------------

Date:    Wed, 7 Nov 2012 10:45:03 +0100
From:    "SANJUAN SAMARRA, XAVIER" <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

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Years ago I worked with vascular tissue; we routinely fixed arterial rings
of aprox. 2 mm overnight at the 4=BA room with 4% PFA, but sometimes we lef=
t
the rings in PFA one even two days without a signifficant effect on the
result of the immunostaining. I accidentally forgot a set of arterial rings
for about a month in the fixative (for sure PFA was gradually degraded over
that time)... And the vessels turned into a hard piece of cement totally
impermeable to antibodies.

You can always learn something from mistakes!

Best,

Xavi.

PS: Thanks Christophe for pointing at Burry's book, seems the Bible of
immunolabeling!
___________________________________

*Xavier Sanjuan**
*Advanced Light Microscopy Unit
Parc de Recerca Biom=E8dica de Barcelona
Doctor Aiguader, 88
08003 Barcelona - Spain
Tel: + 34 93 316 0206
Fax: + 34 93 316 09 01
E-mail: [hidden email]
Web:
http://pasteur.crg.es/portal/page/portal/Internet/03_CORES/Core_Facilities/=
Advanced%20Light%20Microscopy%20Unit


2012/11/7 Paul Rigby <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>
> Hi Ke,
> What a great question!! Why don't more of us (biologists/microscopists
> etc) ever question the basic protocols that are in common use. Often thes=
e
> protocols are the result of "chinese wispers" and are passed down and
> modified over time. Eventually, either something stops working or someone
> like yourself actually thinks "outside the box". Well done.
>
> Now to try to answer your question - methanol or methanol/acetone are
> coagulative fixatives most commonly used to preserve cytoskeletal element=
s

> in cells. Fixation is usually for a short time (5 or 10 minutes at -20C).
> It seems they do a "better" job at preserving or exposing some epitopes,
> allowing better antibody access than do cross-linking fixatives like
> formaldehyde or glutaraldehyde. Why? - I have no idea; they just seem to
> work better in some cases. As Guy has suggested, perhaps it is related to
> the strong lipid dissolving properties.
>
> As an aside, if one looks at the chemistry literature for aldehyde
> fixation, it is interesting that for complete fixation and stabilisation,
> times significantly longer than 24 hours are required for the cross linki=

ng
> reactions to go to completion. (For a good explanation see Kiernan, J.A.,
> Microscopy Today, January 2000, pp 8-12.). So, why do we typically fix
> single cells for only 10 or 30 minutes in formaldehyde solutions before
> immunolabelling? Probably because it works. Do longer fixation times (24
> hours or more) work better for preservation of single cell structures pri=
or
> to immunolabelling? I suspect not, but does anyone have direct experience=
?

>
> Cheers
> Paul
>
> Assoc. Prof. Paul Rigby
> Centre for Microscopy, Characterisation & Analysis (M510)
> The University of Western Australia
> 35 Stirling Highway
> Crawley  WA  6007
> Australia
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of aropro
> Sent: Tuesday, 6 November 2012 7:52 PM
> To: [hidden email]
> Subject: Why perform Methanol, Methanol/Acetone fixation at -20C
>
>  *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>
> Hello Everyone:
>
> Sorry to bother you with a naive/entry level question: why the fixation
> with methanol or methanol/acetone are normally performed at -20C? I tried
> to look it up on the internet but except for finding a number of protocol=

s
> describing this I have not found one explaining this so far.
>
> Thanks a lot for your help in advance.
>
> Ke
>

------------------------------

Date:    Wed, 7 Nov 2012 11:22:20 +0000
From:    Mark Cannell <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

*****
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I think the paper by Schnell et al. shows that live cell imaging of =
constructs is NOT a gold standard at all. This is made clearer by =
inspection of the supp. images!. It seems to me that the major =
conclusion of their paper is that permeabilisation can remove soluble =
proteins?  Hardly a revelation=85 As for the reliability of =
immunocytochemistry. I think we are all aware of the problems=85

Cheers


On 7/11/2012, at 8:37 AM, Christophe Leterrier =
<[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>=20
> Something related to this non-complete fixation by aldehydes: Akihiro
> Kusumi's lab reported two years ago that lipids and membrane proteins
> were still diffusing in the plasma membrane after fixation for up to
> 90 mintues in 4% PFA. Cold methanol was better in this regard,
> although a residual mobility was still observed:
> Tanaka, K. A. K., Suzuki, K. G. N., Shirai, Y. M., Shibutani, S. T.,
> Miyahara, M. S. H., Tsuboi, H., et al. (2010). Membrane molecules
> mobile even after chemical fixation. Nature Methods.
> doi:10.1038/nmeth.f.314
>=20
>=20
> Another good & recent article about immunocytochemistry procedures has
> been published in Nature Methods:
> Schnell, U., Dijk, F., Sjollema, K. A., & Giepmans, B. N. G. (2012).
> Immunolabeling artifacts and the need for live-cell imaging. Nature
> Methods, 9(2), 152=96158. doi:10.1038/nmeth.1855
>=20
>=20
> And I highly suggest to people that are doing
> immunocyto/histochemistry to read R. Burry's book. It covers
> everything from fixation to fluorescence imaging and explains a lot of
> "chinese whispers", debunking a few ones on the way:
> Burry, R. W. (2010). Immunocytochemistry. Springer Verlag.
> =

http://books.google.fr/books?id=3DsvzyJdQVsaEC&printsec=3Dfrontcover&hl=3D=
fr&source=3Dgbs_ge_summary_r&cad=3D0#v=3Donepage&q&f=3Dfalse

>=20
> Cheers,
>=20
> --
> Christophe Leterrier
> Researcher
> Axonal Domains Architecture Team
> CRN2M CNRS UMR 7286
> Aix Marseille University, France
>=20
>=20
> On Wed, Nov 7, 2012 at 6:33 AM, Paul Rigby <[hidden email]> =

wrote:
>>=20
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>> *****
>>=20
>>=20
>> As an aside, if one looks at the chemistry literature for aldehyde =
fixation, it is interesting that for complete fixation and =
stabilisation, times significantly longer than 24 hours are required for =
the cross linking reactions to go to completion. (For a good explanation =
see Kiernan, J.A., Microscopy Today, January 2000, pp 8-12.). So, why do =
we typically fix single cells for only 10 or 30 minutes in formaldehyde =
solutions before immunolabelling? Probably because it works. Do longer =
fixation times (24 hours or more) work better for preservation of single =
cell structures prior to immunolabelling? I suspect not, but does anyone =
have direct experience?
>>=20

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology&  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

------------------------------

Date:    Wed, 7 Nov 2012 13:35:41 +0100
From:    Christophe Leterrier <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
*****

I agree that the message of the Schnell et al. paper is somewhat
skewed to promote live-cell imaging instead of immunocytochemistry.
Also the fact that fixation can alter the distribution of proteins is
hardly new, see the Burry book, or Brock et al. 1999 Cytometry part A
35(4), 353=96362 (and I'm sure there are others).

However, there are some good data nuggets, including live imaging of
what happens during fixation, and the fact that 4% PFA already has a
very high osmolarity (~1300 mOsm), so that adding sucrose to adjust
osmolarity isn't really necessary.

Cheers,

Christophe


On Wed, Nov 7, 2012 at 12:22 PM, Mark Cannell
<[hidden email]> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>
> I think the paper by Schnell et al. shows that live cell imaging of const=
ructs is NOT a gold standard at all. This is made clearer by inspection of =
the supp. images!. It seems to me that the major conclusion of their paper =
is that permeabilisation can remove soluble proteins?  Hardly a revelation=
=85 As for the reliability of immunocytochemistry. I think we are all aware=
 of the problems=85
>
> Cheers

------------------------------

Date:    Wed, 7 Nov 2012 07:12:52 -0600
From:    Sarah Richert <[hidden email]>
Subject: Re: conversion of mEOS2 by 2-photon

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*****

Many thanks for your answers and suggestions!
Good to hear that photoconversion of mEOS by 2P seems to work with mEOS-F=
P
at 810 nm. It would be great to know, how many scans it takes and ideally=

which laser power should be used. Would you be so kind to share these
details, Chen?=20

Unfortunately, AG York has stated it did not work (efficiently) at 800 nm=

with mEOS2.
I am wondering if this could be due to differences between mEOS-FP and
mEOS2. Or if it is a difference between 800 versus 810 nm excitation?

Thanks a lot,=20
Sarah

------------------------------

Date:    Wed, 7 Nov 2012 13:36:18 +0000
From:    "Phillips, Thomas E." <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

*****
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*****

4% PFA might have a high osmolarity but since it crosses the membrane, but =
its tonicity is another question that has been debated extensively over the=
 years.=20

Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
573-882-4712 (office)
573-882-0123 (fax)
[hidden email]

http://www.biology.missouri.edu/faculty/phillips.html
http://www.biotech.missouri.edu/mcc/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On=
 Behalf Of Christophe Leterrier
Sent: Wednesday, November 07, 2012 6:36 AM
To: [hidden email]
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
*****

I agree that the message of the Schnell et al. paper is somewhat skewed to =
promote live-cell imaging instead of immunocytochemistry.
Also the fact that fixation can alter the distribution of proteins is hardl=
y new, see the Burry book, or Brock et al. 1999 Cytometry part A 35(4), 353=
-362 (and I'm sure there are others).

However, there are some good data nuggets, including live imaging of what h=
appens during fixation, and the fact that 4% PFA already has a very high os=
molarity (~1300 mOsm), so that adding sucrose to adjust osmolarity isn't re=
ally necessary.

Cheers,

Christophe


On Wed, Nov 7, 2012 at 12:22 PM, Mark Cannell <[hidden email]> =
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>
> I think the paper by Schnell et al. shows that live cell imaging of=20
> constructs is NOT a gold standard at all. This is made clearer by=20
> inspection of the supp. images!. It seems to me that the major=20
> conclusion of their paper is that permeabilisation can remove soluble=20
> proteins?  Hardly a revelation... As for the reliability of immunocytoche=

mistry. I think we are all aware of the problems...
>
> Cheers

------------------------------

Date:    Wed, 7 Nov 2012 08:50:55 -0500
From:    "Lemasters, John J." <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

*****
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*****

Formaldehyde and glutaraldehyde cross membranes essentially as readily as w=
ater, as well established over the years. Thus, fixatives must be osmotical=
ly balanced with sucrose, salines, or buffers with phosphate and arsenate (=
cacodylate) ignoring the contribution of the aldehydes to osmotic strength.=
 Without this osmotic adjustment, cells and organelles (e.g., mitochondria)=
 will swell.

--
John J. Lemasters, MD, PhD
Professor and GlaxoSmithKline Distinguished Endowed Chair
Director, Center for Cell Death, Injury & Regeneration
Departments of Drug Discovery & Biomedical Sciences and Biochemistry & Mole=
cular Biology
Medical University of South Carolina
DD504 Drug Discovery Building
70 President Street, MSC 140
Charleston, SC 29425

Office: 843-876-2360
Lab: 843-876-2354
Fax: [phone]
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On=
 Behalf Of Christophe Leterrier
Sent: Wednesday, November 07, 2012 7:36 AM
To: [hidden email]
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
*****

I agree that the message of the Schnell et al. paper is somewhat skewed to =
promote live-cell imaging instead of immunocytochemistry.
Also the fact that fixation can alter the distribution of proteins is hardl=
y new, see the Burry book, or Brock et al. 1999 Cytometry part A 35(4), 353=
-362 (and I'm sure there are others).

However, there are some good data nuggets, including live imaging of what h=
appens during fixation, and the fact that 4% PFA already has a very high os=
molarity (~1300 mOsm), so that adding sucrose to adjust osmolarity isn't re=
ally necessary.

Cheers,

Christophe


On Wed, Nov 7, 2012 at 12:22 PM, Mark Cannell <[hidden email]> =
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>
> I think the paper by Schnell et al. shows that live cell imaging of=20
> constructs is NOT a gold standard at all. This is made clearer by=20
> inspection of the supp. images!. It seems to me that the major=20
> conclusion of their paper is that permeabilisation can remove soluble=20
> proteins?  Hardly a revelation... As for the reliability of immunocytoche=

mistry. I think we are all aware of the problems...
>
> Cheers

------------------------------

Date:    Wed, 7 Nov 2012 15:13:29 +0100
From:    Christophe Leterrier <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
*****

Thanks for the correction, I had read that sucrose was for osmolarity
balance and observed a lot of people don't use it when fixing cultured
cells, so I inferred this was the reason. Very happy to learn the real
reason why people use sucrose in their PFA fixative! This is why I
love this list.

Cheers,

Christophe

On Wed, Nov 7, 2012 at 2:50 PM, Lemasters, John J. <[hidden email]> wrote=
:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>
> Formaldehyde and glutaraldehyde cross membranes essentially as readily as=
 water, as well established over the years. Thus, fixatives must be osmotic=
ally balanced with sucrose, salines, or buffers with phosphate and arsenate=
 (cacodylate) ignoring the contribution of the aldehydes to osmotic strengt=
h. Without this osmotic adjustment, cells and organelles (e.g., mitochondri=
a) will swell.
>
> --
> John J. Lemasters, MD, PhD
> Professor and GlaxoSmithKline Distinguished Endowed Chair
> Director, Center for Cell Death, Injury & Regeneration
> Departments of Drug Discovery & Biomedical Sciences and Biochemistry & Mo=
lecular Biology

> Medical University of South Carolina
> DD504 Drug Discovery Building
> 70 President Street, MSC 140
> Charleston, SC 29425
>
> Office: 843-876-2360
> Lab: 843-876-2354
> Fax: [phone]
> Email: [hidden email]
> http://academicdepartments.musc.edu/ccdir
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] =

On Behalf Of Christophe Leterrier

> Sent: Wednesday, November 07, 2012 7:36 AM
> To: [hidden email]
> Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>
> I agree that the message of the Schnell et al. paper is somewhat skewed t=

o promote live-cell imaging instead of immunocytochemistry.
> Also the fact that fixation can alter the distribution of proteins is har=
dly new, see the Burry book, or Brock et al. 1999 Cytometry part A 35(4), 3=
53-362 (and I'm sure there are others).
>
> However, there are some good data nuggets, including live imaging of what=
 happens during fixation, and the fact that 4% PFA already has a very high =
osmolarity (~1300 mOsm), so that adding sucrose to adjust osmolarity isn't =
really necessary.

>
> Cheers,
>
> Christophe
>
>
> On Wed, Nov 7, 2012 at 12:22 PM, Mark Cannell <[hidden email]=
> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>> *****
>>
>> I think the paper by Schnell et al. shows that live cell imaging of
>> constructs is NOT a gold standard at all. This is made clearer by
>> inspection of the supp. images!. It seems to me that the major
>> conclusion of their paper is that permeabilisation can remove soluble
>> proteins?  Hardly a revelation... As for the reliability of immunocytoch=

emistry. I think we are all aware of the problems...
>>
>> Cheers

------------------------------

Date:    Wed, 7 Nov 2012 16:01:30 +0100
From:    Patric Van Oostveldt <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Dear,

The use of methanol/aceton at minus 20 is infact a simple
cryosubstitution approach. The mixture should be water free and used
in great volume. If e.g. using a mixture that was out of the freezer
for some times and put back in the freezer will give bad tubuline
fixation because it attracts water on the bench and if the bottle is
not filled, even more water from the air will dissolve in the mixture.
A solution to this is to include some water attracting resins in the
solute, this will bind the water and you fixative will stay "water
free".
Changing from -20 to room temperature will disolve lipids but at that
moment the proteins are nearly completely dehydrated and will stay in
a good or natural shape.

Patrick
--
Dep. Moleculaire Biotechnologie
Coupure links 653
B 9000 GENT

tel at lab: 09 264 5969
priv tel: [phone]
fax [phone]
GSM [phone]



Citeren Christophe Leterrier <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Thanks for the correction, I had read that sucrose was for osmolarity
> balance and observed a lot of people don't use it when fixing cultured
> cells, so I inferred this was the reason. Very happy to learn the real
> reason why people use sucrose in their PFA fixative! This is why I
> love this list.
>
> Cheers,
>
> Christophe
>
> On Wed, Nov 7, 2012 at 2:50 PM, Lemasters, John J. <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
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>> *****
>>
>> Formaldehyde and glutaraldehyde cross membranes essentially as
>> readily as water, as well established over the years. Thus,
>> fixatives must be osmotically balanced with sucrose, salines, or
>> buffers with phosphate and arsenate (cacodylate) ignoring the
>> contribution of the aldehydes to osmotic strength. Without this
>> osmotic adjustment, cells and organelles (e.g., mitochondria) will
>> swell.
>>
>> --
>> John J. Lemasters, MD, PhD
>> Professor and GlaxoSmithKline Distinguished Endowed Chair
>> Director, Center for Cell Death, Injury & Regeneration
>> Departments of Drug Discovery & Biomedical Sciences and
>> Biochemistry & Molecular Biology
>> Medical University of South Carolina
>> DD504 Drug Discovery Building
>> 70 President Street, MSC 140
>> Charleston, SC 29425
>>
>> Office: 843-876-2360
>> Lab: 843-876-2354
>> Fax: [phone]
>> Email: [hidden email]
>> http://academicdepartments.musc.edu/ccdir
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Christophe
>> Leterrier
>> Sent: Wednesday, November 07, 2012 7:36 AM
>> To: [hidden email]
>> Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I agree that the message of the Schnell et al. paper is somewhat
>> skewed to promote live-cell imaging instead of immunocytochemistry.
>> Also the fact that fixation can alter the distribution of proteins
>> is hardly new, see the Burry book, or Brock et al. 1999 Cytometry
>> part A 35(4), 353-362 (and I'm sure there are others).
>>
>> However, there are some good data nuggets, including live imaging
>> of what happens during fixation, and the fact that 4% PFA already
>> has a very high osmolarity (~1300 mOsm), so that adding sucrose to
>> adjust osmolarity isn't really necessary.
>>
>> Cheers,
>>
>> Christophe
>>
>>
>> On Wed, Nov 7, 2012 at 12:22 PM, Mark Cannell
>> <[hidden email]> wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> I think the paper by Schnell et al. shows that live cell imaging of
>>> constructs is NOT a gold standard at all. This is made clearer by
>>> inspection of the supp. images!. It seems to me that the major
>>> conclusion of their paper is that permeabilisation can remove soluble
>>> proteins?  Hardly a revelation... As for the reliability of
>>> immunocytochemistry. I think we are all aware of the problems...
>>>
>>> Cheers

------------------------------

Date:    Wed, 7 Nov 2012 16:24:26 +0000
From:    alexia ferrand <[hidden email]>
Subject: slidebook file format issues

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Dear all,=0A=A0=0AIn our facility in Basel, we are frequently using a 3i=0A=
spinning disk microscope. While the system itself performs extremely well a=
nd=0Aour users are happy with the acquisition process, things become much m=
ore=0Acomplex as we get into photomanipulation analysis.=0A=A0=0ASo far, we=
 have advised our users to save their data in=0Athe proprietary slidebook f=
ile format (that's the software shipped by 3i for=0Aacquisition and analysi=
s), which could be opened with the latest versions of=0AImaris and more rec=
ently with Fiji. However, since last month we cannot use=0AFiji anymore, th=
e slidebook file format is not read properly, even using the=0Alatest stabl=
e version of the LOCI plugin. The issue has been reported and will=0Abe cha=
nged for the subsequent release. The LOCI team advised us to use the=0AOME-=
TIFF export function in the slidebook software. This file format is=0Ahelpf=
ul, by keeping the metadata and the properties of the initial data.=0A=A0=
=0AHowever, when we tried to save our files as OME-TIFF and=0Aopen them in =
Fiji, we end up with individual files for each channel and=0Atimepoint... =
=0AJust like usual TIFF files, but with an extra text file=0Acontaining the=
 metadata.=0A=A0=0AWhen using Imaris to read the proprietary slidebook=0Afo=
rmat, the files open as expected, but it cannot process the ROIs stored in=
=0Athere. We are aware of the community's recognition of the problem with s=
uch=0Awork by the SciJava group and their effort to provide a common set of=
 ROI types=0Awhich will be usable in all image analysis programs. (See http=
://www.scijava.org/roi-model/roi.html#model-toplevel).=0ABut unfortunately =
this is not available yet.=0A=A0=0AWe could use the OME-TIFF and reorder th=
e=0Achannels/timepoints to reconstruct the files as movies, but we have the=
 feeling=0Athat we will still lose the information for the ROIs anyway. The=
 ROIs could be=0Amanually re-created, and the data analysed in Fiji, but th=
is is adding extra steps=0Aand introduces inaccuracy by the manual part.=0A=
=A0=0AThe other option will be to use the slidebook software to=0Ado the wh=
ole analysis. It is easy to have the FRAP curves visualization and the=0Aan=
alysis done there. However, with the slidebook software we have then only=
=0Abeen able to save the intensity values. This means that the analysis has=
 to be=0Aredone from scratch, which is extremely frustrating... We are cons=
idering=0Awriting a matlab script for it, but we were wondering if such a s=
cript already=0Aexists somewhere?=0A=A0=0AWe are interested to know if anyo=
ne has encountered such=0Aissues and managed to find any other suitable wor=
karounds?=0A=A0=0ABest,=0AAlexia=0A=A0=0A----------------------------------=
--=0ADr Alexia Ferrand=0AImaging Core Facility=0AKragenbau,=0ARoom G1055=0A=
Klingelbergstrasse=0A50 / 70=0ACH-4056=0ABasel =0A=A0=0AOffice:=A0 +41 (61)=
 267 22 50=0AEmail: [hidden email]

------------------------------

Date:    Wed, 7 Nov 2012 13:46:53 -0500
From:    Andrew York <[hidden email]>
Subject: Re: conversion of mEOS2 by 2-photon

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We never tried the long exposures Ekaterina Papusheva describes. Worth a
try for sure.

One caveat: We used temporal focusing (lower peak power, higher average
power), not point-scanning. I suspect EP's description is for a
point-scanning system. For a nonlinear process like this, it's possible
that's an important difference.



On Wed, Nov 7, 2012 at 8:12 AM, Sarah Richert <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Many thanks for your answers and suggestions!
> Good to hear that photoconversion of mEOS by 2P seems to work with mEOS-FP
> at 810 nm. It would be great to know, how many scans it takes and ideally
> which laser power should be used. Would you be so kind to share these
> details, Chen?
>
> Unfortunately, AG York has stated it did not work (efficiently) at 800 nm
> with mEOS2.
> I am wondering if this could be due to differences between mEOS-FP and
> mEOS2. Or if it is a difference between 800 versus 810 nm excitation?
>
> Thanks a lot,
> Sarah
>

------------------------------

End of CONFOCALMICROSCOPY Digest - 6 Nov 2012 to 7 Nov 2012 (#2012-265)
***********************************************************************

[Knecht, David]

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On Nov 8, 2012, at 10:15 AM, Liang, Zhangyi wrote:

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2. Why perform Methanol, Methanol/Acetone fixation at -20C
Methanol/Acetone fixation always provide sharper or clearer immunofluorescence images (it might because of the dehydration).
We find that -80 is better than -20 and -20 is much better than higher temperatures.  There are actually two issues.  The localization by immunofluorescence is sharper as the temperature goes down. We have one protein that we cannot fix by any means other than -80 methanol. But morphology of the residual transmitted light image is also an issue.   This is one of the big downsides of alcohol fixation is that morphology changes much more compared to aldehyde fixation, where they look very much like live cells after fixation, so it depends on whether that matters for your application.
Formaldehyde always contains some percentage of methanol except you choose a special one.
We always use pure formaldehyde diluted from ampules (Polysciences) that contains no methanol to get around this problem.
Paraformaldehyde need to be used freshly, and sometimes to get the "better" pictures, you have to do "pre-extract" before fixation (use PBST to rinse the cells before fixation).
I have no idea what that means.  We never pre-extract before fixation and always fix in the media that the cells are in when we aldehyde fix.

Zhangyi Liang, Ph.D.

Molecular & Cellular Biology, Harvard University
Rm 140, Northwest Building
Cambridge, MA 02138
phone [phone]


________________________________________
From: Confocal Microscopy List [[hidden email]<mailto:[hidden email]>] On Behalf Of CONFOCALMICROSCOPY automatic digest system [[hidden email]<mailto:[hidden email]>]
Sent: Thursday, November 08, 2012 1:02 AM
To: [hidden email]<mailto:[hidden email]>
Subject: CONFOCALMICROSCOPY Digest - 6 Nov 2012 to 7 Nov 2012 (#2012-265)

There are 12 messages totalling 934 lines in this issue.

Topics of the day:

 1. conversion of mEOS2 by 2-photon (3)
 2. Why perform Methanol, Methanol/Acetone fixation at -20C (8)
 3. slidebook file format issues

----------------------------------------------------------------------

Date:    Wed, 7 Nov 2012 09:18:09 +0100
From:    Ekaterina PAPUSHEVA <[hidden email]<mailto:[hidden email]>>
Subject: Re: conversion of mEOS2 by 2-photon

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Hi Sarah,

This protocol worked for us

You have to photoconvert at 800nm with the laser power not more than 0.5 mW=
at the objective plane.
You need a long time: 3-5min for one z plane  of a square with a size of ha=
lf a cell. To detect the red form you have to do imaging at 970nm.
If the laser power is higher, you bleach immediately the red form.

Play around these lines!

Best regards,
Ekaterina

___

Ekaterina Papusheva

Manager of Bioimaging Facility

Institute of Science and Technology Austria

Phone  [phone];
Mobile [phone]
Fax    [phone]

Am Campus 1

A-3400 Klosterneuburg

Visit our website: www.ist.ac.at


This message and any attachment (together "the Message") are confidential a=
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On=
Behalf Of Sarah Richert
Sent: Wednesday, October 31, 2012 3:39 PM
To: [hidden email]
Subject: conversion of mEOS2 by 2-photon

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Dear all,=20
we are working with mEOS2 and would like to photo-convert it by 2-photon.
With 405 nm laser it works but not in 2-photon mode.
Has anybody successfully done that?
Many thanks in advance!
Sarah

------------------------------

Date:    Wed, 7 Nov 2012 09:37:53 +0100
From:    Christophe Leterrier <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

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Something related to this non-complete fixation by aldehydes: Akihiro
Kusumi's lab reported two years ago that lipids and membrane proteins
were still diffusing in the plasma membrane after fixation for up to
90 mintues in 4% PFA. Cold methanol was better in this regard,
although a residual mobility was still observed:
Tanaka, K. A. K., Suzuki, K. G. N., Shirai, Y. M., Shibutani, S. T.,
Miyahara, M. S. H., Tsuboi, H., et al. (2010). Membrane molecules
mobile even after chemical fixation. Nature Methods.
doi:10.1038/nmeth.f.314


Another good & recent article about immunocytochemistry procedures has
been published in Nature Methods:
Schnell, U., Dijk, F., Sjollema, K. A., & Giepmans, B. N. G. (2012).
Immunolabeling artifacts and the need for live-cell imaging. Nature
Methods, 9(2), 152=96158. doi:10.1038/nmeth.1855


And I highly suggest to people that are doing
immunocyto/histochemistry to read R. Burry's book. It covers
everything from fixation to fluorescence imaging and explains a lot of
"chinese whispers", debunking a few ones on the way:
Burry, R. W. (2010). Immunocytochemistry. Springer Verlag.
http://books.google.fr/books?id=3DsvzyJdQVsaEC&printsec=3Dfrontcover&hl=3Df=
r&source=3Dgbs_ge_summary_r&cad=3D0#v=3Donepage&q&f=3Dfalse

Cheers,

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France


On Wed, Nov 7, 2012 at 6:33 AM, Paul Rigby <[hidden email]> wrote:

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As an aside, if one looks at the chemistry literature for aldehyde fixati=
on, it is interesting that for complete fixation and stabilisation, times s=
ignificantly longer than 24 hours are required for the cross linking reacti=
ons to go to completion. (For a good explanation see Kiernan, J.A., Microsc=
opy Today, January 2000, pp 8-12.). So, why do we typically fix single cell=
s for only 10 or 30 minutes in formaldehyde solutions before immunolabellin=
g? Probably because it works. Do longer fixation times (24 hours or more) w=
ork better for preservation of single cell structures prior to immunolabell=
ing? I suspect not, but does anyone have direct experience?


------------------------------

Date:    Wed, 7 Nov 2012 10:45:03 +0100
From:    "SANJUAN SAMARRA, XAVIER" <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

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Years ago I worked with vascular tissue; we routinely fixed arterial rings
of aprox. 2 mm overnight at the 4=BA room with 4% PFA, but sometimes we lef=
t
the rings in PFA one even two days without a signifficant effect on the
result of the immunostaining. I accidentally forgot a set of arterial rings
for about a month in the fixative (for sure PFA was gradually degraded over
that time)... And the vessels turned into a hard piece of cement totally
impermeable to antibodies.

You can always learn something from mistakes!

Best,

Xavi.

PS: Thanks Christophe for pointing at Burry's book, seems the Bible of
immunolabeling!
___________________________________

*Xavier Sanjuan**
*Advanced Light Microscopy Unit
Parc de Recerca Biom=E8dica de Barcelona
Doctor Aiguader, 88
08003 Barcelona - Spain
Tel: + 34 93 316 0206
Fax: + 34 93 316 09 01
E-mail: [hidden email]
Web:
http://pasteur.crg.es/portal/page/portal/Internet/03_CORES/Core_Facilities/=
Advanced%20Light%20Microscopy%20Unit


2012/11/7 Paul Rigby <[hidden email]>

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Hi Ke,
What a great question!! Why don't more of us (biologists/microscopists
etc) ever question the basic protocols that are in common use. Often thes=
e
protocols are the result of "chinese wispers" and are passed down and
modified over time. Eventually, either something stops working or someone
like yourself actually thinks "outside the box". Well done.

Now to try to answer your question - methanol or methanol/acetone are
coagulative fixatives most commonly used to preserve cytoskeletal element=
s
in cells. Fixation is usually for a short time (5 or 10 minutes at -20C).
It seems they do a "better" job at preserving or exposing some epitopes,
allowing better antibody access than do cross-linking fixatives like
formaldehyde or glutaraldehyde. Why? - I have no idea; they just seem to
work better in some cases. As Guy has suggested, perhaps it is related to
the strong lipid dissolving properties.

As an aside, if one looks at the chemistry literature for aldehyde
fixation, it is interesting that for complete fixation and stabilisation,
times significantly longer than 24 hours are required for the cross linki=
ng
reactions to go to completion. (For a good explanation see Kiernan, J.A.,
Microscopy Today, January 2000, pp 8-12.). So, why do we typically fix
single cells for only 10 or 30 minutes in formaldehyde solutions before
immunolabelling? Probably because it works. Do longer fixation times (24
hours or more) work better for preservation of single cell structures pri=
or
to immunolabelling? I suspect not, but does anyone have direct experience=
?

Cheers
Paul

Assoc. Prof. Paul Rigby
Centre for Microscopy, Characterisation & Analysis (M510)
The University of Western Australia
35 Stirling Highway
Crawley  WA  6007
Australia

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of aropro
Sent: Tuesday, 6 November 2012 7:52 PM
To: [hidden email]
Subject: Why perform Methanol, Methanol/Acetone fixation at -20C

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Hello Everyone:

Sorry to bother you with a naive/entry level question: why the fixation
with methanol or methanol/acetone are normally performed at -20C? I tried
to look it up on the internet but except for finding a number of protocol=
s
describing this I have not found one explaining this so far.

Thanks a lot for your help in advance.

Ke


------------------------------

Date:    Wed, 7 Nov 2012 11:22:20 +0000
From:    Mark Cannell <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

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I think the paper by Schnell et al. shows that live cell imaging of =
constructs is NOT a gold standard at all. This is made clearer by =
inspection of the supp. images!. It seems to me that the major =
conclusion of their paper is that permeabilisation can remove soluble =
proteins?  Hardly a revelation=85 As for the reliability of =
immunocytochemistry. I think we are all aware of the problems=85

Cheers


On 7/11/2012, at 8:37 AM, Christophe Leterrier =
<[hidden email]> wrote:

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=20
Something related to this non-complete fixation by aldehydes: Akihiro
Kusumi's lab reported two years ago that lipids and membrane proteins
were still diffusing in the plasma membrane after fixation for up to
90 mintues in 4% PFA. Cold methanol was better in this regard,
although a residual mobility was still observed:
Tanaka, K. A. K., Suzuki, K. G. N., Shirai, Y. M., Shibutani, S. T.,
Miyahara, M. S. H., Tsuboi, H., et al. (2010). Membrane molecules
mobile even after chemical fixation. Nature Methods.
doi:10.1038/nmeth.f.314
=20
=20
Another good & recent article about immunocytochemistry procedures has
been published in Nature Methods:
Schnell, U., Dijk, F., Sjollema, K. A., & Giepmans, B. N. G. (2012).
Immunolabeling artifacts and the need for live-cell imaging. Nature
Methods, 9(2), 152=96158. doi:10.1038/nmeth.1855
=20
=20
And I highly suggest to people that are doing
immunocyto/histochemistry to read R. Burry's book. It covers
everything from fixation to fluorescence imaging and explains a lot of
"chinese whispers", debunking a few ones on the way:
Burry, R. W. (2010). Immunocytochemistry. Springer Verlag.
=
http://books.google.fr/books?id=3DsvzyJdQVsaEC&printsec=3Dfrontcover&hl=3D=
fr&source=3Dgbs_ge_summary_r&cad=3D0#v=3Donepage&q&f=3Dfalse
=20
Cheers,
=20
--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France
=20
=20
On Wed, Nov 7, 2012 at 6:33 AM, Paul Rigby <[hidden email]> =
wrote:
=20
*****
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=20
=20
As an aside, if one looks at the chemistry literature for aldehyde =
fixation, it is interesting that for complete fixation and =
stabilisation, times significantly longer than 24 hours are required for =
the cross linking reactions to go to completion. (For a good explanation =
see Kiernan, J.A., Microscopy Today, January 2000, pp 8-12.). So, why do =
we typically fix single cells for only 10 or 30 minutes in formaldehyde =
solutions before immunolabelling? Probably because it works. Do longer =
fixation times (24 hours or more) work better for preservation of single =
cell structures prior to immunolabelling? I suspect not, but does anyone =
have direct experience?
=20

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology&  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

------------------------------

Date:    Wed, 7 Nov 2012 13:35:41 +0100
From:    Christophe Leterrier <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

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I agree that the message of the Schnell et al. paper is somewhat
skewed to promote live-cell imaging instead of immunocytochemistry.
Also the fact that fixation can alter the distribution of proteins is
hardly new, see the Burry book, or Brock et al. 1999 Cytometry part A
35(4), 353=96362 (and I'm sure there are others).

However, there are some good data nuggets, including live imaging of
what happens during fixation, and the fact that 4% PFA already has a
very high osmolarity (~1300 mOsm), so that adding sucrose to adjust
osmolarity isn't really necessary.

Cheers,

Christophe


On Wed, Nov 7, 2012 at 12:22 PM, Mark Cannell
<[hidden email]> wrote:
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I think the paper by Schnell et al. shows that live cell imaging of const=
ructs is NOT a gold standard at all. This is made clearer by inspection of =
the supp. images!. It seems to me that the major conclusion of their paper =
is that permeabilisation can remove soluble proteins?  Hardly a revelation=
=85 As for the reliability of immunocytochemistry. I think we are all aware=
of the problems=85

Cheers

------------------------------

Date:    Wed, 7 Nov 2012 07:12:52 -0600
From:    Sarah Richert <[hidden email]>
Subject: Re: conversion of mEOS2 by 2-photon

*****
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*****

Many thanks for your answers and suggestions!
Good to hear that photoconversion of mEOS by 2P seems to work with mEOS-F=
P
at 810 nm. It would be great to know, how many scans it takes and ideally=

which laser power should be used. Would you be so kind to share these
details, Chen?=20

Unfortunately, AG York has stated it did not work (efficiently) at 800 nm=

with mEOS2.
I am wondering if this could be due to differences between mEOS-FP and
mEOS2. Or if it is a difference between 800 versus 810 nm excitation?

Thanks a lot,=20
Sarah

------------------------------

Date:    Wed, 7 Nov 2012 13:36:18 +0000
From:    "Phillips, Thomas E." <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

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4% PFA might have a high osmolarity but since it crosses the membrane, but =
its tonicity is another question that has been debated extensively over the=
years.=20

Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
573-882-4712 (office)
573-882-0123 (fax)
[hidden email]

http://www.biology.missouri.edu/faculty/phillips.html
http://www.biotech.missouri.edu/mcc/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On=
Behalf Of Christophe Leterrier
Sent: Wednesday, November 07, 2012 6:36 AM
To: [hidden email]
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

*****
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I agree that the message of the Schnell et al. paper is somewhat skewed to =
promote live-cell imaging instead of immunocytochemistry.
Also the fact that fixation can alter the distribution of proteins is hardl=
y new, see the Burry book, or Brock et al. 1999 Cytometry part A 35(4), 353=
-362 (and I'm sure there are others).

However, there are some good data nuggets, including live imaging of what h=
appens during fixation, and the fact that 4% PFA already has a very high os=
molarity (~1300 mOsm), so that adding sucrose to adjust osmolarity isn't re=
ally necessary.

Cheers,

Christophe


On Wed, Nov 7, 2012 at 12:22 PM, Mark Cannell <[hidden email]> =
wrote:
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
*****

I think the paper by Schnell et al. shows that live cell imaging of=20
constructs is NOT a gold standard at all. This is made clearer by=20
inspection of the supp. images!. It seems to me that the major=20
conclusion of their paper is that permeabilisation can remove soluble=20
proteins?  Hardly a revelation... As for the reliability of immunocytoche=
mistry. I think we are all aware of the problems...

Cheers

------------------------------

Date:    Wed, 7 Nov 2012 08:50:55 -0500
From:    "Lemasters, John J." <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
*****

Formaldehyde and glutaraldehyde cross membranes essentially as readily as w=
ater, as well established over the years. Thus, fixatives must be osmotical=
ly balanced with sucrose, salines, or buffers with phosphate and arsenate (=
cacodylate) ignoring the contribution of the aldehydes to osmotic strength.=
Without this osmotic adjustment, cells and organelles (e.g., mitochondria)=
will swell.

--
John J. Lemasters, MD, PhD
Professor and GlaxoSmithKline Distinguished Endowed Chair
Director, Center for Cell Death, Injury & Regeneration
Departments of Drug Discovery & Biomedical Sciences and Biochemistry & Mole=
cular Biology
Medical University of South Carolina
DD504 Drug Discovery Building
70 President Street, MSC 140
Charleston, SC 29425

Office: 843-876-2360
Lab: 843-876-2354
Fax: [phone]
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On=
Behalf Of Christophe Leterrier
Sent: Wednesday, November 07, 2012 7:36 AM
To: [hidden email]
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
*****

I agree that the message of the Schnell et al. paper is somewhat skewed to =
promote live-cell imaging instead of immunocytochemistry.
Also the fact that fixation can alter the distribution of proteins is hardl=
y new, see the Burry book, or Brock et al. 1999 Cytometry part A 35(4), 353=
-362 (and I'm sure there are others).

However, there are some good data nuggets, including live imaging of what h=
appens during fixation, and the fact that 4% PFA already has a very high os=
molarity (~1300 mOsm), so that adding sucrose to adjust osmolarity isn't re=
ally necessary.

Cheers,

Christophe


On Wed, Nov 7, 2012 at 12:22 PM, Mark Cannell <[hidden email]> =
wrote:
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
*****

I think the paper by Schnell et al. shows that live cell imaging of=20
constructs is NOT a gold standard at all. This is made clearer by=20
inspection of the supp. images!. It seems to me that the major=20
conclusion of their paper is that permeabilisation can remove soluble=20
proteins?  Hardly a revelation... As for the reliability of immunocytoche=
mistry. I think we are all aware of the problems...

Cheers

------------------------------

Date:    Wed, 7 Nov 2012 15:13:29 +0100
From:    Christophe Leterrier <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
*****

Thanks for the correction, I had read that sucrose was for osmolarity
balance and observed a lot of people don't use it when fixing cultured
cells, so I inferred this was the reason. Very happy to learn the real
reason why people use sucrose in their PFA fixative! This is why I
love this list.

Cheers,

Christophe

On Wed, Nov 7, 2012 at 2:50 PM, Lemasters, John J. <[hidden email]> wrote=
:
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
*****

Formaldehyde and glutaraldehyde cross membranes essentially as readily as=
water, as well established over the years. Thus, fixatives must be osmotic=
ally balanced with sucrose, salines, or buffers with phosphate and arsenate=
(cacodylate) ignoring the contribution of the aldehydes to osmotic strengt=
h. Without this osmotic adjustment, cells and organelles (e.g., mitochondri=
a) will swell.

--
John J. Lemasters, MD, PhD
Professor and GlaxoSmithKline Distinguished Endowed Chair
Director, Center for Cell Death, Injury & Regeneration
Departments of Drug Discovery & Biomedical Sciences and Biochemistry & Mo=
lecular Biology
Medical University of South Carolina
DD504 Drug Discovery Building
70 President Street, MSC 140
Charleston, SC 29425

Office: 843-876-2360
Lab: 843-876-2354
Fax: [phone]
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] =
On Behalf Of Christophe Leterrier
Sent: Wednesday, November 07, 2012 7:36 AM
To: [hidden email]
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
*****

I agree that the message of the Schnell et al. paper is somewhat skewed t=
o promote live-cell imaging instead of immunocytochemistry.
Also the fact that fixation can alter the distribution of proteins is har=
dly new, see the Burry book, or Brock et al. 1999 Cytometry part A 35(4), 3=
53-362 (and I'm sure there are others).

However, there are some good data nuggets, including live imaging of what=
happens during fixation, and the fact that 4% PFA already has a very high =
osmolarity (~1300 mOsm), so that adding sucrose to adjust osmolarity isn't =
really necessary.

Cheers,

Christophe


On Wed, Nov 7, 2012 at 12:22 PM, Mark Cannell <[hidden email]=
wrote:
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
*****

I think the paper by Schnell et al. shows that live cell imaging of
constructs is NOT a gold standard at all. This is made clearer by
inspection of the supp. images!. It seems to me that the major
conclusion of their paper is that permeabilisation can remove soluble
proteins?  Hardly a revelation... As for the reliability of immunocytoch=
emistry. I think we are all aware of the problems...

Cheers

------------------------------

Date:    Wed, 7 Nov 2012 16:01:30 +0100
From:    Patric Van Oostveldt <[hidden email]>
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

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

Dear,

The use of methanol/aceton at minus 20 is infact a simple
cryosubstitution approach. The mixture should be water free and used
in great volume. If e.g. using a mixture that was out of the freezer
for some times and put back in the freezer will give bad tubuline
fixation because it attracts water on the bench and if the bottle is
not filled, even more water from the air will dissolve in the mixture.
A solution to this is to include some water attracting resins in the
solute, this will bind the water and you fixative will stay "water
free".
Changing from -20 to room temperature will disolve lipids but at that
moment the proteins are nearly completely dehydrated and will stay in
a good or natural shape.

Patrick
--
Dep. Moleculaire Biotechnologie
Coupure links 653
B 9000 GENT

tel at lab: 09 264 5969
priv tel: [phone]
fax [phone]
GSM [phone]



Citeren Christophe Leterrier <[hidden email]>:

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

Thanks for the correction, I had read that sucrose was for osmolarity
balance and observed a lot of people don't use it when fixing cultured
cells, so I inferred this was the reason. Very happy to learn the real
reason why people use sucrose in their PFA fixative! This is why I
love this list.

Cheers,

Christophe

On Wed, Nov 7, 2012 at 2:50 PM, Lemasters, John J. <[hidden email]> wrote:
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Formaldehyde and glutaraldehyde cross membranes essentially as
readily as water, as well established over the years. Thus,
fixatives must be osmotically balanced with sucrose, salines, or
buffers with phosphate and arsenate (cacodylate) ignoring the
contribution of the aldehydes to osmotic strength. Without this
osmotic adjustment, cells and organelles (e.g., mitochondria) will
swell.

--
John J. Lemasters, MD, PhD
Professor and GlaxoSmithKline Distinguished Endowed Chair
Director, Center for Cell Death, Injury & Regeneration
Departments of Drug Discovery & Biomedical Sciences and
Biochemistry & Molecular Biology
Medical University of South Carolina
DD504 Drug Discovery Building
70 President Street, MSC 140
Charleston, SC 29425

Office: 843-876-2360
Lab: 843-876-2354
Fax: [phone]
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir


-----Original Message-----
From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Christophe
Leterrier
Sent: Wednesday, November 07, 2012 7:36 AM
To: [hidden email]
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

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

I agree that the message of the Schnell et al. paper is somewhat
skewed to promote live-cell imaging instead of immunocytochemistry.
Also the fact that fixation can alter the distribution of proteins
is hardly new, see the Burry book, or Brock et al. 1999 Cytometry
part A 35(4), 353-362 (and I'm sure there are others).

However, there are some good data nuggets, including live imaging
of what happens during fixation, and the fact that 4% PFA already
has a very high osmolarity (~1300 mOsm), so that adding sucrose to
adjust osmolarity isn't really necessary.

Cheers,

Christophe


On Wed, Nov 7, 2012 at 12:22 PM, Mark Cannell
<[hidden email]> wrote:
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I think the paper by Schnell et al. shows that live cell imaging of
constructs is NOT a gold standard at all. This is made clearer by
inspection of the supp. images!. It seems to me that the major
conclusion of their paper is that permeabilisation can remove soluble
proteins?  Hardly a revelation... As for the reliability of
immunocytochemistry. I think we are all aware of the problems...

Cheers

------------------------------

Date:    Wed, 7 Nov 2012 16:24:26 +0000
From:    alexia ferrand <[hidden email]>
Subject: slidebook file format issues

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
*****

Dear all,=0A=A0=0AIn our facility in Basel, we are frequently using a 3i=0A=
spinning disk microscope. While the system itself performs extremely well a=
nd=0Aour users are happy with the acquisition process, things become much m=
ore=0Acomplex as we get into photomanipulation analysis.=0A=A0=0ASo far, we=
have advised our users to save their data in=0Athe proprietary slidebook f=
ile format (that's the software shipped by 3i for=0Aacquisition and analysi=
s), which could be opened with the latest versions of=0AImaris and more rec=
ently with Fiji. However, since last month we cannot use=0AFiji anymore, th=
e slidebook file format is not read properly, even using the=0Alatest stabl=
e version of the LOCI plugin. The issue has been reported and will=0Abe cha=
nged for the subsequent release. The LOCI team advised us to use the=0AOME-=
TIFF export function in the slidebook software. This file format is=0Ahelpf=
ul, by keeping the metadata and the properties of the initial data.=0A=A0=
=0AHowever, when we tried to save our files as OME-TIFF and=0Aopen them in =
Fiji, we end up with individual files for each channel and=0Atimepoint... =
=0AJust like usual TIFF files, but with an extra text file=0Acontaining the=
metadata.=0A=A0=0AWhen using Imaris to read the proprietary slidebook=0Afo=
rmat, the files open as expected, but it cannot process the ROIs stored in=
=0Athere. We are aware of the community's recognition of the problem with s=
uch=0Awork by the SciJava group and their effort to provide a common set of=
ROI types=0Awhich will be usable in all image analysis programs. (See http=
://www.scijava.org/roi-model/roi.html#model-toplevel).=0ABut unfortunately =
this is not available yet.=0A=A0=0AWe could use the OME-TIFF and reorder th=
e=0Achannels/timepoints to reconstruct the files as movies, but we have the=
feeling=0Athat we will still lose the information for the ROIs anyway. The=
ROIs could be=0Amanually re-created, and the data analysed in Fiji, but th=
is is adding extra steps=0Aand introduces inaccuracy by the manual part.=0A=
=A0=0AThe other option will be to use the slidebook software to=0Ado the wh=
ole analysis. It is easy to have the FRAP curves visualization and the=0Aan=
alysis done there. However, with the slidebook software we have then only=
=0Abeen able to save the intensity values. This means that the analysis has=
to be=0Aredone from scratch, which is extremely frustrating... We are cons=
idering=0Awriting a matlab script for it, but we were wondering if such a s=
cript already=0Aexists somewhere?=0A=A0=0AWe are interested to know if anyo=
ne has encountered such=0Aissues and managed to find any other suitable wor=
karounds?=0A=A0=0ABest,=0AAlexia=0A=A0=0A----------------------------------=
--=0ADr Alexia Ferrand=0AImaging Core Facility=0AKragenbau,=0ARoom G1055=0A=
Klingelbergstrasse=0A50 / 70=0ACH-4056=0ABasel =0A=A0=0AOffice:=A0 +41 (61)=
267 22 50=0AEmail: [hidden email]

------------------------------

Date:    Wed, 7 Nov 2012 13:46:53 -0500
From:    Andrew York <[hidden email]>
Subject: Re: conversion of mEOS2 by 2-photon

*****
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*****

We never tried the long exposures Ekaterina Papusheva describes. Worth a
try for sure.

One caveat: We used temporal focusing (lower peak power, higher average
power), not point-scanning. I suspect EP's description is for a
point-scanning system. For a nonlinear process like this, it's possible
that's an important difference.



On Wed, Nov 7, 2012 at 8:12 AM, Sarah Richert <[hidden email]>wrote:

*****
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*****

Many thanks for your answers and suggestions!
Good to hear that photoconversion of mEOS by 2P seems to work with mEOS-FP
at 810 nm. It would be great to know, how many scans it takes and ideally
which laser power should be used. Would you be so kind to share these
details, Chen?

Unfortunately, AG York has stated it did not work (efficiently) at 800 nm
with mEOS2.
I am wondering if this could be due to differences between mEOS-FP and
mEOS2. Or if it is a difference between 800 versus 810 nm excitation?

Thanks a lot,
Sarah


------------------------------

End of CONFOCALMICROSCOPY Digest - 6 Nov 2012 to 7 Nov 2012 (#2012-265)
***********************************************************************

Dr. David Knecht
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)