Laser effects during bleaching

[Michael Schell]

Christian--

I've not tried the RFP you mention.  The bottom line for the "which GFP" question is the old adage that one should *always* sequence any cDNA construct obtained from an outside source.   By the way, the A207K mutation in EGFP that changes it from weakly dimeric to monomeric has no obvious effect on the propensity to form the round, smooth aggregates.

M

Michael J. Schell, Ph.D., CIV, USUHS
Assist. Professor
Dept. of Pharmacology
Uniformed Services University
4301 Jones Bridge Rd.
Bethesda, MD  20814-3220
tel:  [phone]
[hidden email]
>>> Christian <[hidden email]> 11/20/08 5:33 PM >>>
Michael,

What about the "newer" RFP derived from Entacmaea quadricolor?  Have you seen it aggregate at all?

I guess part of my problem is that I often can not get a hold of which "GFP" I'm being handed, so when there are "punctate spots" or "aggregates" in the cytoplasm, I can only shrug as to what they might be (plant cells by the way).

Christian

[Jeremy Adler]

A recent paper by Hell et al argues that bleaching is increased by a second photon hitting a molecule that is in the triplet state.
They demonstrate that pulsed exictation, when the interval between pulses is sufficient for molecules to escape from the triplet state, produces greater fluorescence for a given amount of bleaching.
 
 
 
 
 
Major signal increase in fluorescence microscopy through dark-state relaxation

Gerald Donnert, Christian Eggeling, Stefan W Hell

SUMMARY: We report a substantial signal gain in fluorescence microscopy by ensuring that transient molecular dark states with lifetimes >1 ?s, such as the triplet

Nature Methods 4, 81 - 86 (01 Jan 2007),

 
Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories F5
Stockholm University
Stockholm 106 91
Sweden

________________________________

From: Confocal Microscopy List on behalf of Kevin Braeckmans
Sent: Thu 20-Nov-08 20:37
To: [hidden email]
Subject: Re: Laser effects during bleaching



Hi,

 

Not necessarily. Most photobleaching happens from the long-lived triplet state, at least for a fluorescein, a dye that is frequently used for FRAP experiments.

 

First consider the case where a light dose is delivered at a very short time scale. At high laser powers, fluorophores will be quickly pumped to the triplet state, i.e. the triplet state will be saturated.  At that point, photobleaching reactions will compete with relaxation of the triplet state to the ground state (by intersystem crossing). A limited amount of photobleaching results.

 

Now consider the same light dose being delivered by a much reduced light beam over a longer time interval. In this case only a fraction of the fluorophores will in the triplet state at any point in time, but integrated over time, more molecules will have reached the triplet state resulting in more bleaching.

 

We have shown this theoretically as well as experimentally for fluorescein. Hesitating because of the self advertisement, but since the question was raised ... details can be found in J Biomed Opt 11(4), 044013 (2006). Specifically you could have a look at Fig 8A.

 

Of course this all depends on the reaction rates of the different photochemical processes taking part in the photobleaching process and might be different for other fluorophores.

 

Hope this helps,

 

Best regards,

 

Kevin

 

Van: Confocal Microscopy List [mailto:[hidden email]] Namens Craig Brideau
Verzonden: donderdag 20 november 2008 17:49
Aan: [hidden email]
Onderwerp: Re: Laser effects during bleaching

 

Here's a related question that's been bugging me:
If photobleaching is due to free radicals, high pulse energy should generate more free radicals and thus more bleaching.
If this is the case, if you need to get more signal from your sample, is it better to increase your pulse energy, or just crank up your average power?

Thoughts?

Craig




On Wed, Nov 19, 2008 at 6:32 PM, Stephen Cody <[hidden email]> wrote:

G'day List,

I've never done much in the way of deliberate photobleaching
experiments, I'm usually trying to avoid it. But isn't photobleaching
mediated through reactive oxygen species? And if so how can you limit
damage to just the protein you wish to bleach? Or have I missed the
point somewhere?

Cheers
Stephen Cody

2008/11/19 John Runions <[hidden email]>:

> Hi Tom,
>
> When doing FRAP, we generally try to keep the bleaching lasers as low as
> possible so that a loss of fluorescence results but so that we aren't
> applying enough laser power to damage other proteins in membranes.  If the
> lasers were so hot as to denature proteins the whole procedure would be
> invalidated as we'd be killing the cells (I do often imagine the sound of
> frying bacon during the bleaching phase and hope that it's not really
> happening!).
>
> At normal laser power levels for confocal, cells seem to function properly
> over the relatively long term.  The trick during the bleaching phase is to
> increase the laser power so that a bit of bleaching results.  If the region
> to be studied is bleached completely black, then I feel there really is the
> potential for cellular damage.  I've certainly exploded cells with too much
> laser power so your specimens should be handled gently.  The small amount of
> bleaching attained using reasonable laser power is analyzed
> relativistically, i.e. you normalize post-bleach and pre-bleach intensity
> between 0-100% intensity so that there is no requirement to bleach the
> fluorescence completely away.  The idea of fluorescence recovery is that any
> molecule will move to its equilibrium concentration in time (unless
> constrained).  The species of molecule that we are analyzing the dynamics of
> in FRAP are the bleached and unbleached versions of the fluorochome.  FRAP
> can be done, therefore, with a small population of bleached molecules.
>
> In some recent experiments, we have been FRAPing so that only a very small
> decrease in fluorescence occurs within a region of interest.  Recovery
> curves fit from these experiments seem equivalent to those fit to data sets
> in which much more bleaching of the ROI was visible.  We need to ascertain
> if they are significantly the same, however.  If any physicists of diffusion
> are listening it would be nice to have your take on this.
>
> Regards, John.
>
> Donnelly, Tom wrote:
>
> Just to add to the confusion.
>
> What happens to the non-fluorescent proteins in the cell when you crank up
> the power to bleach or image in live cells?
>
> Tom
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]On
> Behalf Of John Runions
> Sent: Tuesday, November 18, 2008 9:08 AM
> To: [hidden email]
> Subject: Re: broadband excitation vs. narrow band
>
>
>
> Boy, what a stupid question Carl.  I think we should all fail to dignify it
> with a response!
>
> Actually, that kind of question is fundamental to us in the FRAP world and
> it sounds like someone needs to do some good empirical measuring.  When
> bleaching GFP we will often use all of the 458, 477,488 and 514 lines of the
> argon laser at the same time.  It works better that just using the 488 and I
> explain this by saying that it is because we are bleaching with a broader
> spectrum across the excitation range.  I have never been able (or tried) to
> confirm if this is the case or if the higher power at the specimen plays a
> role.
>
> Sorry to be not much help.  John.
>
> Carl Boswell wrote:
>
> Hi all,
> I was told once that there are no stupid questions, so let's test that
> assumption.
>
> The question has to do with photobleaching vs. excitation energy.  To get X
> photons from a fluor, would there be less photobleaching using a single
> wavelength excitation source at peak excitation wavelength, or a broadband
> (20-30nm) light source centered on the peak excitation of the molecule, or
> would there be no difference?  My assumption is that lower "power"
> (brightness?) would be needed for the broadband source, but would the
> overall photon flux be greater to get equivalent output?
>
> To take this one step farther, is there less or more bleaching from
> "inefficient" excitation, i.e. off-peak excitation, to get the same output?
> If a fluor is less efficiently excited, is it less efficiently bleached,
> even though more power may be needed to get equivalent output?
>
> Thanks,
> Carl
>
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> University of Arizona
> 520-954-7053
> FAX [phone]
>
> --
>
> (Sent from my cra%#y non-Blackberry electronic device that still has wires)
>
> *********************************
> C. John Runions, Ph.D.
> School of Life Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email:  [hidden email]
> phone: [phone]
>
> Runions' lab web site
>
>
>
> Visit The Illuminated Plant Cell dot com
> Oxford Brookes Master's in Bioimaging with Molecular Technology
>
> --
>
> (Sent from my cra%#y non-Blackberry electronic device that still has wires)
>
>
>
> *********************************
> C. John Runions, Ph.D.
> School of Life Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email:  [hidden email]
> phone: [phone]
>
> Runions' lab web site
>
>
>
> Visit The Illuminated Plant Cell dot com
> Oxford Brookes Master's in Bioimaging with Molecular Technology

--
Stephen Cody

 

[Rietdorf, Jens]

Dear David,

My 2 cents: Be aware that not only the GFP variant but also the method
by which the gfp is transferred into the cells, as well as expression
levels, may have a strong influence on the appearance of cellular
membranes and lipid metabolism. Adenovirus is nice because of its high
transfection efficiency but exactly that may be your problem at the same
time. It may be (it generally is) worthwhile to take the effort and
produce stable lines or at least try different methods to make the cells
express the FP.

Best, jens

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of David Burk
Sent: Thursday, November 20, 2008 8:31 PM
To: [hidden email]
Subject: GFP and lipid localization?

GFP Experts:

We have a group transfecting cells with adenovirus.  This virus contains
an EGFP sequence to let you know that, yes, the cell was transfected.
We have noticed in some of the transfected cells that the GFP signal
appears to be strongly associated with lipid droplets.  This is new to
me as I wouldn't expect WT-GFP to strongly associate with any particular
subcellular organelle and is confounding their work since they are
primarily interested in - of course - lipid droplets.

Do any of you know of a reason we would see wild-type GFP with no
targeting sequence or modifications labeling lipid droplets?  We are
beginning some additional control experiments with a large titration
range of virus to determine if the lipid labeling is related to vector
concentration (~GFP expression level).

Thanks for your help!

David H. Burk

[neeraj Gohad-3]

Dear List,

We are trying to compare open source deconvolution algorithms with the
commercial deconvolution package that we have. So far we have tried the
Iterative Deconvolve 3D for ImageJ. I wanted to know which other open
source algorithms people are using for deconvolution.

Best Regards,

Neeraj.


Neeraj V. Gohad, PhD
Postdoctoral Fellow,
Okeanos Research Group
Department of Biological Sciences
132 Long Hall,Clemson University
Clemson, SC-29634
864-656-3597
[hidden email]

[neeraj Gohad-3]

 

Dear List,

 

We are trying to compare open source deconvolution algorithms with the commercial deconvolution package that we have. So far we have tried the Iterative Deconvolve 3D for ImageJ. I wanted to know which other open source algorithms people are using for deconvolution.

 

Best Regards,

 

Neeraj.

 

 

Neeraj V. Gohad, PhD

Postdoctoral Fellow,

Okeanos Research Group

Department of Biological Sciences

132 Long Hall,Clemson University

Clemson, SC-29634

864-656-3597

[hidden email]

 

 

 

[mahogny]

Neeraj Gohad wrote:
> Dear List,
>
> We are trying to compare open source deconvolution algorithms with the
> commercial deconvolution package that we have. So far we have tried the
> Iterative Deconvolve 3D for ImageJ. I wanted to know which other open
> source algorithms people are using for deconvolution.
>
> Best Regards,
>  

haven't tried yet:
http://piotr.wendykier.googlepages.com/iterativedeconvolution

also used your package there, it failed, and since imagej has problems
with batching I wouldn't bother with IJ plugins for it anyway. I have
used my own code in the past, but for special classes of deconv, not the
normal one in biology. we're writing a new algorithm at the moment.

/Johan

--
--
------------------------------------------------
Johan Henriksson
MSc Engineering
PhD student, Karolinska Institutet
http://mahogny.areta.org http://www.endrov.net

[Aryeh Weiss]

I am wondering if anyone on list has run a *second* copy of Leica LAF (not to
acquire from the confocal, but an offline copy) on 64 bit Windows? It was my
understanding that 32 bit software runs under the 64 bit version, but one never
knows.

Thanks in advance.
--aryeh
--
Aryeh Weiss
School of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]

[Graham Wright-5]

Hello Aryeh,
I'm told by Leica (Singapore) that the LAS AF Lite software cannot be installed on Windows XP 64bit (not sure about other platforms). I had tried and failed which is why i asked them.
Best wishes,
Graham

----- Original Message -----
From: "Aryeh Weiss" <[hidden email]>
To: [hidden email]
Sent: Wednesday, November 26, 2008 5:04:28 PM GMT +08:00 Beijing / Chongqing / Hong Kong / Urumqi
Subject: Leica LAF on 64 bit windows?

I am wondering if anyone on list has run a *second* copy of Leica LAF (not to
acquire from the confocal, but an offline copy) on 64 bit Windows? It was my
understanding that 32 bit software runs under the 64 bit version, but one never
knows.

Thanks in advance.
--aryeh
--
Aryeh Weiss
School of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]

[Zoltan]

I can confirm that, it only works on the 32-bit version of Windows.  Zoltan

On Wed, Nov 26, 2008 at 9:03 AM, Dr Graham Wright <[hidden email]> wrote:

Hello Aryeh,
I'm told by Leica (Singapore) that the LAS AF Lite software cannot be installed on Windows XP 64bit (not sure about other platforms). I had tried and failed which is why i asked them.
Best wishes,
Graham

----- Original Message -----
From: "Aryeh Weiss" <[hidden email]>
To: [hidden email]
Sent: Wednesday, November 26, 2008 5:04:28 PM GMT +08:00 Beijing / Chongqing / Hong Kong / Urumqi
Subject: Leica LAF on 64 bit windows?

I am wondering if anyone on list has run a *second* copy of Leica LAF (not to
acquire from the confocal, but an offline copy) on 64 bit Windows? It was my
understanding that 32 bit software runs under the 64 bit version, but one never
knows.

Thanks in advance.
--aryeh
--
Aryeh Weiss
School of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]

--

Zoltan Cseresnyes
Facility manager, Imaging Suite

[Aryeh Weiss]

Thank you for the rapid replies. Looks like I have to either set up dual boot or
find a separate machine to run it.

--aryeh


Zoltan Cseresnyes wrote:

> I can confirm that, it only works on the 32-bit version of Windows.  Zoltan
>
> On Wed, Nov 26, 2008 at 9:03 AM, Dr Graham Wright <[hidden email]
> <mailto:[hidden email]>> wrote:
>
>     Hello Aryeh,
>     I'm told by Leica (Singapore) that the LAS AF Lite software cannot
>     be installed on Windows XP 64bit (not sure about other platforms). I
>     had tried and failed which is why i asked them.
>     Best wishes,
>     Graham
>
>     ----- Original Message -----
>     From: "Aryeh Weiss" <[hidden email] <mailto:[hidden email]>>
>     To: [hidden email]
>     <mailto:[hidden email]>
>     Sent: Wednesday, November 26, 2008 5:04:28 PM GMT +08:00 Beijing /
>     Chongqing / Hong Kong / Urumqi
>     Subject: Leica LAF on 64 bit windows?
>
>     I am wondering if anyone on list has run a *second* copy of Leica
>     LAF (not to
>     acquire from the confocal, but an offline copy) on 64 bit Windows?
>     It was my
>     understanding that 32 bit software runs under the 64 bit version,
>     but one never
>     knows.
>
>     Thanks in advance.
>     --aryeh
>     --
>     Aryeh Weiss
>     School of Engineering
>     Bar Ilan University
>     Ramat Gan 52900 Israel
>
>     Ph:  972-3-5317638
>     FAX: [phone]
>
>
>
>
> --
>
> Zoltan Cseresnyes
> Facility manager, Imaging Suite

[Torsten.Fregin]

Moin,

Maybe you can just install a virtual environment on your computer? Then you can use the
Software anytime on your XP64-pc (without dual boot or else).

http://www.virtualbox.org/wiki/Downloads

;-) Torsten

On 26 Nov 2008 at 13:03, Aryeh Weiss wrote:

> Thank you for the rapid replies. Looks like I have to either set up dual boot or
> find a separate machine to run it.
>
> --aryeh
>
>
> Zoltan Cseresnyes wrote:
> > I can confirm that, it only works on the 32-bit version of Windows.  Zoltan
> >
> > On Wed, Nov 26, 2008 at 9:03 AM, Dr Graham Wright <[hidden email]
> > <mailto:[hidden email]>> wrote:
> >
> >     Hello Aryeh,
> >     I'm told by Leica (Singapore) that the LAS AF Lite software cannot
> >     be installed on Windows XP 64bit (not sure about other platforms). I
> >     had tried and failed which is why i asked them.
> >     Best wishes,
> >     Graham
> >
> >     ----- Original Message -----
> >     From: "Aryeh Weiss" <[hidden email] <mailto:[hidden email]>>
> >     To: [hidden email]
> >     <mailto:[hidden email]>
> >     Sent: Wednesday, November 26, 2008 5:04:28 PM GMT +08:00 Beijing /
> >     Chongqing / Hong Kong / Urumqi
> >     Subject: Leica LAF on 64 bit windows?
> >
> >     I am wondering if anyone on list has run a *second* copy of Leica
> >     LAF (not to
> >     acquire from the confocal, but an offline copy) on 64 bit Windows?
> >     It was my
> >     understanding that 32 bit software runs under the 64 bit version,
> >     but one never
> >     knows.
> >
> >     Thanks in advance.
> >     --aryeh
> >     --
> >     Aryeh Weiss
> >     School of Engineering
> >     Bar Ilan University
> >     Ramat Gan 52900 Israel
> >
> >     Ph:  972-3-5317638
> >     FAX: [phone]
> >
> >
> >
> >
> > --
> >
> > Zoltan Cseresnyes
> > Facility manager, Imaging Suite

[Aryeh Weiss]

Thanks for the pointer. That looks very interesting, but I wonder if it will
support the USB dongle that comes with the program.

--aryeh

[hidden email] wrote:

> Moin,
>
> Maybe you can just install a virtual environment on your computer? Then you can use the
> Software anytime on your XP64-pc (without dual boot or else).
>
> http://www.virtualbox.org/wiki/Downloads
>
> ;-) Torsten
>
> On 26 Nov 2008 at 13:03, Aryeh Weiss wrote:
>
>> Thank you for the rapid replies. Looks like I have to either set up dual boot or
>> find a separate machine to run it.
>>
>> --aryeh
>>
>>
>> Zoltan Cseresnyes wrote:
>>> I can confirm that, it only works on the 32-bit version of Windows.  Zoltan
>>>
>>> On Wed, Nov 26, 2008 at 9:03 AM, Dr Graham Wright <[hidden email]
>>> <mailto:[hidden email]>> wrote:
>>>
>>>     Hello Aryeh,
>>>     I'm told by Leica (Singapore) that the LAS AF Lite software cannot
>>>     be installed on Windows XP 64bit (not sure about other platforms). I
>>>     had tried and failed which is why i asked them.
>>>     Best wishes,
>>>     Graham
>>>
>>>     ----- Original Message -----
>>>     From: "Aryeh Weiss" <[hidden email] <mailto:[hidden email]>>
>>>     To: [hidden email]
>>>     <mailto:[hidden email]>
>>>     Sent: Wednesday, November 26, 2008 5:04:28 PM GMT +08:00 Beijing /
>>>     Chongqing / Hong Kong / Urumqi
>>>     Subject: Leica LAF on 64 bit windows?
>>>
>>>     I am wondering if anyone on list has run a *second* copy of Leica
>>>     LAF (not to
>>>     acquire from the confocal, but an offline copy) on 64 bit Windows?
>>>     It was my
>>>     understanding that 32 bit software runs under the 64 bit version,
>>>     but one never
>>>     knows.
>>>
>>>     Thanks in advance.
>>>     --aryeh
>>>     --
>>>     Aryeh Weiss
>>>     School of Engineering
>>>     Bar Ilan University
>>>     Ramat Gan 52900 Israel
>>>
>>>     Ph:  972-3-5317638
>>>     FAX: [phone]
>>>
>>>
>>>
>>>
>>> --
>>>
>>> Zoltan Cseresnyes
>>> Facility manager, Imaging Suite
>
>

[davek604]

Hello Aryeh

It is possible to run LAS on 64bit XP but its not easy. When installing
you run the program from the link below. This spoofs LAS into thinking
its running on a 32 bit system. The problem is that everytime you
upgrade your confocal software you need to upgrade your workstation
software and this spoofing doesn't always work. So if you have the time
and the inclination you can do it but unless you are dealing with very
big image stacks and need the extra memory I wouldn't bother

http://www.microsoft.com/downloads/details.aspx?familyid=bd02c19c-1250-433c-8c1b-2619bd93b3a2&displaylang=en 


When this doesn't work there are other utilities that also spoof XP
version numbers with varying degrees of success

Hope this helps

Dave

Aryeh Weiss wrote:
> I am wondering if anyone on list has run a *second* copy of Leica LAF
> (not to acquire from the confocal, but an offline copy) on 64 bit
> Windows? It was my understanding that 32 bit software runs under the
> 64 bit version, but one never knows.
>
> Thanks in advance.
> --aryeh

--
David Kelly
University of Edinburgh
Wellcome Trust Centre for Cell Biology
Michael Swann Building
Mayfield Road
EH9 3JR

Tel: [phone]
E-Mail: [hidden email]
Website: http://www.wcb.ed.ac.uk/COIL


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

[Michael Weber-4]

Hey Dave,

interesting solution, thanks. But regarding the memory handling, the
LAS-AF program is still not able to handle more than 2 GB even on x64 XP
since it remains a 32 bit program, or am I missing something?

Michael


David Kelly wrote:

> Hello Aryeh
>
> It is possible to run LAS on 64bit XP but its not easy. When installing
> you run the program from the link below. This spoofs LAS into thinking
> its running on a 32 bit system. The problem is that everytime you
> upgrade your confocal software you need to upgrade your workstation
> software and this spoofing doesn't always work. So if you have the time
> and the inclination you can do it but unless you are dealing with very
> big image stacks and need the extra memory I wouldn't bother
>
> http://www.microsoft.com/downloads/details.aspx?familyid=bd02c19c-1250-433c-8c1b-2619bd93b3a2&displaylang=en 
>
>
> When this doesn't work there are other utilities that also spoof XP
> version numbers with varying degrees of success
>
> Hope this helps
>
> Dave
>
> Aryeh Weiss wrote:
>> I am wondering if anyone on list has run a *second* copy of Leica LAF
>> (not to acquire from the confocal, but an offline copy) on 64 bit
>> Windows? It was my understanding that 32 bit software runs under the
>> 64 bit version, but one never knows.
>>
>> Thanks in advance.
>> --aryeh

[davek604]

Hello Michael

As I understand it LAS leaves the memory handling to Windows so if you
are using 64 bit then it should allow you to address more than the 4GB
limit on 32 bit systems. I know Leica people watch this list so can any
of them clear this up as its quite a hassle to get it working on 64 bit xp.

Cheers

Dave

Michael Weber wrote:

> Hey Dave,
>
> interesting solution, thanks. But regarding the memory handling, the
> LAS-AF program is still not able to handle more than 2 GB even on x64
> XP since it remains a 32 bit program, or am I missing something?
>
> Michael
>
>
> David Kelly wrote:
>> Hello Aryeh
>>
>> It is possible to run LAS on 64bit XP but its not easy. When
>> installing you run the program from the link below. This spoofs LAS
>> into thinking its running on a 32 bit system. The problem is that
>> everytime you upgrade your confocal software you need to upgrade your
>> workstation software and this spoofing doesn't always work. So if you
>> have the time and the inclination you can do it but unless you are
>> dealing with very big image stacks and need the extra memory I
>> wouldn't bother
>>
>> http://www.microsoft.com/downloads/details.aspx?familyid=bd02c19c-1250-433c-8c1b-2619bd93b3a2&displaylang=en 
>>
>>
>> When this doesn't work there are other utilities that also spoof XP
>> version numbers with varying degrees of success
>>
>> Hope this helps
>>
>> Dave
>>
>> Aryeh Weiss wrote:
>>> I am wondering if anyone on list has run a *second* copy of Leica
>>> LAF (not to acquire from the confocal, but an offline copy) on 64
>>> bit Windows? It was my understanding that 32 bit software runs under
>>> the 64 bit version, but one never knows.
>>>
>>> Thanks in advance.
>>> --aryeh
>

--
David Kelly
University of Edinburgh
Wellcome Trust Centre for Cell Biology
Michael Swann Building
Mayfield Road
EH9 3JR

Tel: [phone]
E-Mail: [hidden email]
Website: http://www.wcb.ed.ac.uk/COIL


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

[mahogny]

David Kelly wrote:
> Hello Michael
>
this level of memory handling is not something you can hand to the OS.
32-bit applications are limited to 2GB on old mac and windows, 3GB on
linux. the rest can only be used by windows. in fact, the limit is not
4GB but much higher (extensions available on newer 32-bit CPUs)

the limitations are in the variables used internally and can only by
fixed by recompilation. that is, if the source code was well-written.
maybe time to consider alternatives?

/Johan

> As I understand it LAS leaves the memory handling to Windows so if you
> are using 64 bit then it should allow you to address more than the 4GB
> limit on 32 bit systems. I know Leica people watch this list so can
> any of them clear this up as its quite a hassle to get it working on
> 64 bit xp.
>
> Cheers
>
> Dave
>
> Michael Weber wrote:
>> Hey Dave,
>>
>> interesting solution, thanks. But regarding the memory handling, the
>> LAS-AF program is still not able to handle more than 2 GB even on x64
>> XP since it remains a 32 bit program, or am I missing something?
>>
>> Michael
>>
>>
>> David Kelly wrote:
>>> Hello Aryeh
>>>
>>> It is possible to run LAS on 64bit XP but its not easy. When
>>> installing you run the program from the link below. This spoofs LAS
>>> into thinking its running on a 32 bit system. The problem is that
>>> everytime you upgrade your confocal software you need to upgrade
>>> your workstation software and this spoofing doesn't always work. So
>>> if you have the time and the inclination you can do it but unless
>>> you are dealing with very big image stacks and need the extra memory
>>> I wouldn't bother
>>>
>>> http://www.microsoft.com/downloads/details.aspx?familyid=bd02c19c-1250-433c-8c1b-2619bd93b3a2&displaylang=en
>>>
>>>
>>> When this doesn't work there are other utilities that also spoof XP
>>> version numbers with varying degrees of success
>>>
>>> Hope this helps
>>>
>>> Dave
>>>
>>> Aryeh Weiss wrote:
>>>> I am wondering if anyone on list has run a *second* copy of Leica
>>>> LAF (not to acquire from the confocal, but an offline copy) on 64
>>>> bit Windows? It was my understanding that 32 bit software runs
>>>> under the 64 bit version, but one never knows.
>>>>
>>>> Thanks in advance.
>>>> --aryeh
>>
>

--
--
------------------------------------------------
Johan Henriksson
MSc Engineering
PhD student, Karolinska Institutet
http://mahogny.areta.org http://www.endrov.net

[Aryeh Weiss]

I appreciate the replies that my inquiry is getting.
I more or less ruled out dual boot, because we already have XP64 installed, and
the dual boot solutions I found all advise installing the 32 bit OS first, or
else it is a hassle.

I am not worried about having LAF use more the 4GB. The reason for installing on
the machine with XP64 is that we dont have any machines with a 32 bit OS nearby.

I installed VirtualBox, and it came up smoothly. I still have to figure out how
to get the USB support to work right. Once that is solved, I will install LAF
and see if it works. This appears to be the least complicated way.

I will post my results to the list.

--aryeh


Johan Henriksson wrote:

> David Kelly wrote:
>> Hello Michael
>>
> this level of memory handling is not something you can hand to the OS.
> 32-bit applications are limited to 2GB on old mac and windows, 3GB on
> linux. the rest can only be used by windows. in fact, the limit is not
> 4GB but much higher (extensions available on newer 32-bit CPUs)
>
> the limitations are in the variables used internally and can only by
> fixed by recompilation. that is, if the source code was well-written.
> maybe time to consider alternatives?
>
> /Johan
>
>> As I understand it LAS leaves the memory handling to Windows so if you
>> are using 64 bit then it should allow you to address more than the 4GB
>> limit on 32 bit systems. I know Leica people watch this list so can
>> any of them clear this up as its quite a hassle to get it working on
>> 64 bit xp.
>>
>> Cheers
>>
>> Dave
>>
>> Michael Weber wrote:
>>> Hey Dave,
>>>
>>> interesting solution, thanks. But regarding the memory handling, the
>>> LAS-AF program is still not able to handle more than 2 GB even on x64
>>> XP since it remains a 32 bit program, or am I missing something?
>>>
>>> Michael
>>>
>>>
>>> David Kelly wrote:
>>>> Hello Aryeh
>>>>
>>>> It is possible to run LAS on 64bit XP but its not easy. When
>>>> installing you run the program from the link below. This spoofs LAS
>>>> into thinking its running on a 32 bit system. The problem is that
>>>> everytime you upgrade your confocal software you need to upgrade
>>>> your workstation software and this spoofing doesn't always work. So
>>>> if you have the time and the inclination you can do it but unless
>>>> you are dealing with very big image stacks and need the extra memory
>>>> I wouldn't bother
>>>>
>>>> http://www.microsoft.com/downloads/details.aspx?familyid=bd02c19c-1250-433c-8c1b-2619bd93b3a2&displaylang=en
>>>>
>>>>
>>>> When this doesn't work there are other utilities that also spoof XP
>>>> version numbers with varying degrees of success
>>>>
>>>> Hope this helps
>>>>
>>>> Dave
>>>>
>>>> Aryeh Weiss wrote:
>>>>> I am wondering if anyone on list has run a *second* copy of Leica
>>>>> LAF (not to acquire from the confocal, but an offline copy) on 64
>>>>> bit Windows? It was my understanding that 32 bit software runs
>>>>> under the 64 bit version, but one never knows.
>>>>>
>>>>> Thanks in advance.
>>>>> --aryeh
>
>

[Stanislav Vitha]

Some time ago I used XCOSM   http://www.essrl.wustl.edu/~preza/xcosm/
under linux (with graphical user interface), and also in command-line mode on
a Windows machine with cygwin installed.
I did not do any extensive tests, for my few samples I was quite satisfied with
the results. The best algorithm (Expectation Maximization) has very slow
convergence, so you need hundreds or thousands of iterations (= a lot of
time).
I liked the results better than those from the commercial AutoDeblur v. 9.2
package (Blind deconvolution), but on the other hand, there the deconvolution
took about 1/10th of the time.

Sincerely,
Stan Vitha

Microscopy and Imaging Center,
Texas A&M University
 
:
On Wed, 26 Nov 2008 00:35:13 +0100, Johan Henriksson
<[hidden email]> wrote:

>Neeraj Gohad wrote:
>> Dear List,
>>
>> We are trying to compare open source deconvolution algorithms with the
>> commercial deconvolution package that we have. So far we have tried the
>> Iterative Deconvolve 3D for ImageJ. I wanted to know which other open
>> source algorithms people are using for deconvolution.
>>
>> Best Regards,
>>
>
>haven't tried yet:
>http://piotr.wendykier.googlepages.com/iterativedeconvolution
>
>also used your package there, it failed, and since imagej has problems
>with batching I wouldn't bother with IJ plugins for it anyway. I have
>used my own code in the past, but for special classes of deconv, not the
>normal one in biology. we're writing a new algorithm at the moment.
>
>/Johan
>
>--
>--
>------------------------------------------------
>Johan Henriksson
>MSc Engineering
>PhD student, Karolinska Institutet
>http://mahogny.areta.org http://www.endrov.net

[Steve Baxter-2]

Hi Michael,

I don't know about the LAS software specifically, however in general  
the per-application address space is limited to:

- 2 GB for a vanilla 32-bit Windows XP install
- 3 GB for 32-bit Windows XP with the /3GB switch in boot.ini (less  
if /userva is used as well)
- 4 GB for a 32-bit application running on Windows XP x64
- 8 TB for a 64-bit application running on Windows XP x64 (in practice  
this probably means available disk space for the backing store)

Cheers,

Steve

On 26 Nov 2008, at 17:00, Michael Weber wrote:

> Hey Dave,
>
> interesting solution, thanks. But regarding the memory handling, the  
> LAS-AF program is still not able to handle more than 2 GB even on  
> x64 XP since it remains a 32 bit program, or am I missing something?
>
> Michael
>
>
> David Kelly wrote:
>> Hello Aryeh
>> It is possible to run LAS on 64bit XP but its not easy. When  
>> installing you run the program from the link below. This spoofs LAS  
>> into thinking its running on a 32 bit system. The problem is that  
>> everytime you upgrade your confocal software you need to upgrade  
>> your workstation software and this spoofing doesn't always work. So  
>> if you have the time and the inclination you can do it but unless  
>> you are dealing with very big image stacks and need the extra  
>> memory I wouldn't bother
>> http://www.microsoft.com/downloads/details.aspx?familyid=bd02c19c-1250-433c-8c1b-2619bd93b3a2&displaylang=en 
>>  When this doesn't work there are other utilities that also spoof  
>> XP version numbers with varying degrees of success
>> Hope this helps
>> Dave
>> Aryeh Weiss wrote:
>>> I am wondering if anyone on list has run a *second* copy of Leica  
>>> LAF (not to acquire from the confocal, but an offline copy) on 64  
>>> bit Windows? It was my understanding that 32 bit software runs  
>>> under the 64 bit version, but one never knows.
>>>
>>> Thanks in advance.
>>> --aryeh

Steve Baxter
R&D Leader
Improvision, a PerkinElmer Company
[hidden email]
[hidden email]
[phone]

Image Processing and Vision Company Limited (trading as Improvision)  
is a company registered in England and Wales with company number  
2505778.  VAT number GB536188722.

[Manu Dubin, FB18 Genetik]

Hi Neeraj,

I've also used the XCOSM (Expectation Maximization) algorithm in the past I
found it gave very nice results, at least on my Images.

I never managed to get it original implementation to run properly on either
linux or a mac (http://bigwww.epfl.ch/vandeville/XCOSM/). So what i used instead
was a program called ImageTrak (developed by Peter Stys)  
which includes the XCOSM algorithm for deconvolution of images.
ImageTrak comes packaged as a typical mac .dmg install and is reasonably user
friendly. Unfortunately 1: the program only works on power-pc macs (e.g. G5's)
and 2: the original website is down and the program seems not to be available
any more on the internet.  

If you have a PPC mac available and want to give it a try, contact me of list, I
should be able to dig out a copy of the program.

Recently I've mostly switched over to using the ImageJ Parallel Iterative
Deconvolution plugin from Piotr Wendykier mentioned by Johan, which at least for
me gives much  better results than some of the earlier ImageJ Deconvolution
plugins. It also has the advantages of 1: quite fast on multi-cpu machines, I
don't bother cropping my images anymore.  
2: the latest version can be called from a macro, so I can write a macro to
batch-process my images.

in my hands the FFT preconditioner causes problems, so i disable it, and I
usually use the HyBR algorithm (although they all give my pretty similar
results, as you would hope!).

I hope this helps,

manu





Quoting Stanislav Vitha <[hidden email]>:

> Some time ago I used XCOSM   http://www.essrl.wustl.edu/~preza/xcosm/
> under linux (with graphical user interface), and also in command-line mode on
>
> a Windows machine with cygwin installed.
> I did not do any extensive tests, for my few samples I was quite satisfied
> with
> the results. The best algorithm (Expectation Maximization) has very slow
> convergence, so you need hundreds or thousands of iterations (= a lot of
> time).
> I liked the results better than those from the commercial AutoDeblur v. 9.2
> package (Blind deconvolution), but on the other hand, there the deconvolution
>
> took about 1/10th of the time.
>
> Sincerely,
> Stan Vitha
>
> Microscopy and Imaging Center,
> Texas A&M University
>  
> :
> On Wed, 26 Nov 2008 00:35:13 +0100, Johan Henriksson
> <[hidden email]> wrote:
>
> >Neeraj Gohad wrote:
> >> Dear List,
> >>
> >> We are trying to compare open source deconvolution algorithms with the
> >> commercial deconvolution package that we have. So far we have tried the
> >> Iterative Deconvolve 3D for ImageJ. I wanted to know which other open
> >> source algorithms people are using for deconvolution.
> >>
> >> Best Regards,
> >>
> >
> >haven't tried yet:
> >http://piotr.wendykier.googlepages.com/iterativedeconvolution
> >
> >also used your package there, it failed, and since imagej has problems
> >with batching I wouldn't bother with IJ plugins for it anyway. I have
> >used my own code in the past, but for special classes of deconv, not the
> >normal one in biology. we're writing a new algorithm at the moment.
> >
> >/Johan
> >
> >--
> >--
> >------------------------------------------------
> >Johan Henriksson
> >MSc Engineering
> >PhD student, Karolinska Institutet
> >http://mahogny.areta.org http://www.endrov.net
>

--
Manu Dubin

Abt. Genetik
Univ. Kassel
Heinrich-Plett-Str. 40
34132 Kassel, Germany

phone: (++49) 561 8044178
fax: (++49) 561 8044800

[hidden email]

http://www.biologie.uni-kassel.de/genetics/

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