(Acceptor) photobleaching

[mmodel]

Dear Colleagues -

 

I found myself confused on a rather basic issue. I always thought that:

 

1. FRET requires that the acceptor only absorbs, not necessarily fluoresces (and that seems to be correct: Ganesan et al, PNAS, 2006, vol 103, 4089-94)

2. Bleaching of fluorophores mostly affects the quantum yield, not the absorption (cannot find any specific information on this subject, other than the molecule gets "damaged")

 

But then it's not clear how the acceptor photobleaching method would work if the acceptor absorption remains intact.  So is it the absorption that gets destroyed in photobleaching? Or both?

 

Thank you

 

Mike Model

Kent State University

[John Runions]

Hi Michael, If the emission from the putative donor increases after bleaching the acceptor, then doesn't the acceptor absorption have to have been decreased? John.

MODEL, MICHAEL wrote:

Dear Colleagues -

 

I found myself confused on a rather basic issue. I always thought that:

 

1. FRET requires that the acceptor only absorbs, not necessarily fluoresces (and that seems to be correct: Ganesan et al, PNAS, 2006, vol 103, 4089-94)

2. Bleaching of fluorophores mostly affects the quantum yield, not the absorption (cannot find any specific information on this subject, other than the molecule gets "damaged")

 

But then it's not clear how the acceptor photobleaching method would work if the acceptor absorption remains intact.  So is it the absorption that gets destroyed in photobleaching? Or both?

 

Thank you

 

Mike Model

Kent State University

[Guillermo Palchik]

Dear Confocalists,

Does anybody have a good working protocol for flash freezing fresh rat  
brains (not perfused) ?
I have been doing flash freezing by cooling Isopentane to -50 C and  
then scooping the fresh brains into it and letting it sit for about  
5-10 seconds, then scooping it into dry ice an letting it sit for a  
few minutes and then into the -80 freezer.
The problem is that I have many cracks (crystals) when I cut the  
brains in the cryostat.
After doing some research I found out that proper flash freezing is  
supposed to prevent that...
I have tried leaving it longer than 20-30 seconds in the Isopentane  
(-50C) but the brains crack open.
I also tried directly placing them into LN2 but it cracks after 5  
seconds...
I have heard that cooler Isopentane (-80 C) might work.
Any ideas?
Thanks
Gil Palchik

--
There are people who fight one day and are good...
There are those who fight one year and are better...
There are people who fight many years and are very good...
But there are those who fight their entire lives: they are  
indispensable...

Bertolt Brecht (1898-1956)


How can it be that mathematics, being after all a product
of human thought which is independent of experience, is so
admirably appropriate to the objects of reality?
Is human reason, then, without experience, merely by taking thought,
able to fathom the properties of real things?

Albert Einstein (1879-1955)

[Grzegorz Tylko]

I think that you will never get good results when you want to freeze rat brain just plunging it to isopentane directly. In my opinion even slash (solid nitrogen, about -210) does not give you proper results. Rat brains are too big to be frozen without ice crystals formation. Only the layer <15 microns can be frozen with negligible sizes of ice crystals when plunge technique is used. All other sections of the tissue (deeper) are cracked. However, it is possible to have tissues properly frozen when cryoprotectant (like 25% sucrose) is introduced into cells, usually with fixative compound.
If you do not want to infiltrate brains with cryoprotectant you can cut out quickly (difficult with nervous tissue) an interesting part of a tissue and plunge a small piece of such biopsy to cold isopentane (cooled by LN2 to -140). It is much easier with biopsy gun (if you have) because the process of cutting and freezing is much faster.
I hope it will help you,
Grzegorz Tylko

-----
Department of Cytology and Histology
Institute of Zoology
Jagiellonian University
Ingardena 6, 30-060 Krakow
tel. [phone]
fax [phone]
mobile: [phone]
e-mail: [hidden email]

----- Original Message -----
From: Guillermo Palchik <[hidden email]>
Date: Monday, December 1, 2008 4:40 pm
Subject: Flash Freezing Protocol for non-perfused rat brains
To: [hidden email]

> Dear Confocalists,
>
> Does anybody have a good working protocol for flash freezing fresh rat
>  
> brains (not perfused) ?
> I have been doing flash freezing by cooling Isopentane to -50 C and  
> then scooping the fresh brains into it and letting it sit for about  
> 5-10 seconds, then scooping it into dry ice an letting it sit for a  
> few minutes and then into the -80 freezer.
> The problem is that I have many cracks (crystals) when I cut the  
> brains in the cryostat.
> After doing some research I found out that proper flash freezing is  
> supposed to prevent that...
> I have tried leaving it longer than 20-30 seconds in the Isopentane  
> (-50C) but the brains crack open.
> I also tried directly placing them into LN2 but it cracks after 5  
> seconds...
> I have heard that cooler Isopentane (-80 C) might work.
> Any ideas?
> Thanks
> Gil Palchik
>
> --
> There are people who fight one day and are good...
> There are those who fight one year and are better...
> There are people who fight many years and are very good...
> But there are those who fight their entire lives: they are  
> indispensable...
>
> Bertolt Brecht (1898-1956)
>
>
> How can it be that mathematics, being after all a product
> of human thought which is independent of experience, is so
> admirably appropriate to the objects of reality?
> Is human reason, then, without experience, merely by taking thought,
> able to fathom the properties of real things?
>
> Albert Einstein (1879-1955)

[Pertti Panula]

Hi,

We have been doing this for many years for in situ hybridization. We use
a 50 ml plastic tube filled with isopentane. This is placed in alcohol
in a beaker. Alcohol is precooled with pieces of dry ice. The exact
amounts needed are difficult to tell, we follow the freezing speed. We
keep adding dry ice as it melts. Freezing of whole rat and mouse brains
works fine. Do not leave the brains in isopentane too long, remove them
from the tube with forceps as soon as they are frozen. Then keep them at
-20--80 until cutting. Before cutting allow enough time in the cryostat
to get the sample in appropriate cutting temp.

Feel free to write off the list if you need more details.

Best regards Pertti Panula


Guillermo Palchik wrote:

> Dear Confocalists,
>
> Does anybody have a good working protocol for flash freezing fresh rat
> brains (not perfused) ?
> I have been doing flash freezing by cooling Isopentane to -50 C and then
> scooping the fresh brains into it and letting it sit for about 5-10
> seconds, then scooping it into dry ice an letting it sit for a few
> minutes and then into the -80 freezer.
> The problem is that I have many cracks (crystals) when I cut the brains
> in the cryostat.
> After doing some research I found out that proper flash freezing is
> supposed to prevent that...
> I have tried leaving it longer than 20-30 seconds in the Isopentane
> (-50C) but the brains crack open.
> I also tried directly placing them into LN2 but it cracks after 5
> seconds...
> I have heard that cooler Isopentane (-80 C) might work.
> Any ideas?
> Thanks
> Gil Palchik
>

--
Pertti Panula
Professor, Research Director
Neuroscience Center
Institute of Biomedicine/Anatomy
POB 63
00014 University of Helsinki
Finland
Phone: [phone]
Fax: [phone]
Mobile: [phone]
[hidden email]
http://www.helsinki.fi/neurosci/panula.htm

[Stephen Cody-2]

I have not worked with brain tissue but....

 

Don't immerse directly into liquid liquid nitrogen (LN2) it will rapidly boil, the LN2 bubbles will very effectively insulate the tissue, and the cooling rate will be very very slow. Gaseous nitrogen is a very effective insulator.

 

Try using a modification of the method described by Pertti - cool the isopentane in a polyethylene plastic beaker immersed in liquid nitrogen, stir with a wooden stick until some of the isopentane starts to freeze on the bottom of the beaker. This will give rapid cooling, the faster the cooling he smaller the ice-crystals. The smaller the ice crystals at the surface of the tissue, the better the tissue will be deeper in the thick slice. Ice crystals will grow bigger and bigger deeper into thick tissue, so the faster the freezing the better.

 

Also try using a cryoprotectant such as OCT, but just a thin layer. Don't allow the tissue to thaw; always pre-cool the forceps before removing the tissue.

 

Cheers

Stephen Cody 

2008/12/2 Pertti Panula <[hidden email]>

Hi,

We have been doing this for many years for in situ hybridization. We use a 50 ml plastic tube filled with isopentane. This is placed in alcohol in a beaker. Alcohol is precooled with pieces of dry ice. The exact amounts needed are difficult to tell, we follow the freezing speed. We keep adding dry ice as it melts. Freezing of whole rat and mouse brains works fine. Do not leave the brains in isopentane too long, remove them from the tube with forceps as soon as they are frozen. Then keep them at -20--80 until cutting. Before cutting allow enough time in the cryostat to get the sample in appropriate cutting temp.

Feel free to write off the list if you need more details.

Best regards Pertti Panula

Guillermo Palchik wrote:

Dear Confocalists,

Does anybody have a good working protocol for flash freezing fresh rat brains (not perfused) ?
I have been doing flash freezing by cooling Isopentane to -50 C and then scooping the fresh brains into it and letting it sit for about 5-10 seconds, then scooping it into dry ice an letting it sit for a few minutes and then into the -80 freezer.
The problem is that I have many cracks (crystals) when I cut the brains in the cryostat.
After doing some research I found out that proper flash freezing is supposed to prevent that...
I have tried leaving it longer than 20-30 seconds in the Isopentane (-50C) but the brains crack open.
I also tried directly placing them into LN2 but it cracks after 5 seconds...
I have heard that cooler Isopentane (-80 C) might work.
Any ideas?
Thanks
Gil Palchik

--

Pertti Panula
Professor, Research Director
Neuroscience Center
Institute of Biomedicine/Anatomy
POB 63
00014 University of Helsinki
Finland
Phone:  [phone] 
Fax: [phone]
Mobile:  [phone] 
[hidden email]
http://www.helsinki.fi/neurosci/panula.htm

--
Stephen Cody