Re: 3D Eroded Object Masks *commercial response*

[Bitplane Support]

Dear Cam,

I'll have to give your question a bit more thought and maybe talk it
over with some others, so I might have more info later, but for now Eric
is right, for the current version of Imaris, at least some of this would
require an ImarisXT "XTension".

For "subtractive image mathematics" there is one that already exists
called "channel arithmetics".  It's actually one of the XTensions that
is included in the Imaris installer.  It requires that you have MatLab
also installed (this is not a general requirement of XTensions, but it
is required for most of the ones that I know of).

I'm not sure you really need channel arithmetics, because I got the
impression you only need to apply a binary mask.  This is done in Imaris
by creating a "Surfaces" object, and then on the "Edit" tab, you'll find
buttons called "Mask All" and "Mask Sel" (use all of the objects or just
selected one(s) )

That still does not solve your 3D erosion problem (which could also be
solved by an XTension, but I don't know of an existing one).  As I said,
I might have more ideas for you later, but for now, since you said all
of the MetaMorph masks worked fairly well except the membrane, you might
try running a Gaussian smoothing filter in Imaris on your "stack of
rubber bands".  Because the Gaussian smoothing in Imaris operates in 3D,
this should lessen the stepping effect along the z direction.  Note that
the smoothing width value is in um, not pixels/voxels, so you may have
to be fairly aggressive with the value if your z-steps are relatively large.

Tip:  you can force the smoothing units to operate in the pixel/voxel
domain by temporarily setting your x-y-z voxel sizes to 1-1-1.  Your
image might look quite distorted, but after you have run the filter, set
the values back to their original size, and viola! your image shape is
normal again, and you have just forced an anisotropic Gaussian
smoothing - because the "real" Z dimension is actually not the same as
XY, you are applying more smoothing along Z (in spatial/object terms,
not in terms of voxels).

You can use ratios other than 1:1:1 if you want to "tune" the anisotropy
in a particular way (decreasing the Z voxel size will cause the Z
dimension to be more smoothed than X and Y - in terms of voxels).  I
suggest experimenting on a cropped version of your dataset so you can
quickly see the results of different variations.
* drastic changes to voxel dimensions will require you to re-center the
dataset with the "100%" button, followed by "fit"

Best regards,
-Kevin

Kevin Frischmann
Head of Technical Support, US & Canada
Bitplane, Inc.
tel: [phone], ext. 11
fax: [phone]

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Eric Scarfone wrote:

> "is it possible in Imaris to do
> subtractive image mathematics?"
>
> not with the standard modules I din't think.
>
> But there is a ImarisXT modulethat lest you access, from Imaris,
> applications developped with other software. That gives also access to a
> whole library of customer developped appications where you could find
> what you are looking for!
>
> Good luck
>
> Eric
>
>
> Eric Scarfone, PhD, CNRS,
> Center for Hearing and communication Research
> Department of Clinical Neuroscience
> Karolinska Institutet
>
> Postal Address:
> CFH, M1:02
> Karolinska Hospital,
> SE-171 76 Stockholm, Sweden
>
> Work: [phone],
> Cell: [phone]
> Fax: [phone]
>
> email: [hidden email]
> http://www.ki.se/cfh/
>
>
> ----- Original Message -----
> From: Cameron Nowell <[hidden email]>
> Date: Wednesday, July 15, 2009 8:15 am
> Subject: 3D Eroded Object Masks
> To: [hidden email]
>
>  > I am trying to segment out membrane, cytoplasm and nuclei from a
>  > confocal image set. This is so we can measure translocation of a
>  > proteinfrom the membrane to the cytoplasm/nucleus over time. I can
>  > do this in
>  > MetaMorph but would like to be able to do it in 3D in Imaris if
>  > possible.
>  >
>  > The sample is stained as follows
>  > - Nuclei - DAPI
>  > - Whole Cell - CellMask (Invitrogen)
>  > - Protein of Interest (Alexa Antibody)
>  >
>  > Since there is not a specific stain from membrane or cytoplasm i have
>  > been doing the following
>  > - Segment out whole cells and create a binary mask
>  > - Erode that mask by 4 or so pixels, this represents the cytoplasm
>  > and nuclei of the cell
>  > - Subtract the eroded mask from the whole cell mask. This leave a
>  > ring that represents the membrane of the cell.
>  > - Segment and subtract the nuclei from the combined cytoplasm and
>  > nuclei mask to give a cytoplasm mask.
>  > - The end result is three masks; one each representing membrane,
>  > nuclei and cytoplasm.
>  >
>  > I can create these masks for each slice of a confocal set and get
>  > intensity etc information out from MetaMorph. I have tried
>  > exporting the
>  > mask stacks out to Imaris to create 3D masks but it doesn't work very
>  > well, especially for the membrane mask, as the slices don't
>  > necessarilyoverlap so there are gaps in the mask. My 3D model of
>  > the membrane masks
>  > looks more like a badly piled up lot of rubber bands. The 3D masks for
>  > the whole cell or nucleus work fine.
>  >
>  > So i guess the main question is: is it possible in Imaris to do
>  > subtractive image mathematics?
>  >
>  >
>  >
>  > Thanks
>  >
>  >
>  > Cam
>  >
>  >
>  >
>  > Cameron J. Nowell
>  > Microscopy Manager
>  > Centre for Advanced Microscopy
>  > Ludwig Institute for Cancer Research
>  > PO Box 2008
>  > Royal Melbourne Hospital
>  > Victoria, 3050
>  > AUSTRALIA
>  > Office: [phone]
>  > Mobile: [phone]
>  > Fax: [phone]
>  > Facility Website
>  >
>

[Cameron Nowell]

Hi List,
 
I have found a solution for my problem. Thanks for all the on and off list responses. One thing i probably should have mentioned in my first post. The images are taken with a 60x NA1.4 Oil lens on samples mounted in DPX. So there is no refractive index mismatch. Also cubic voxels were captured (in most cases 0.25x0.25x0.25 um).
 
As to why not use imageJ or other free programs. We have MetaMorph that i am much more familar with and we have Imaris as well. So it woudl be best to use the programs that we have paid a lot of money for if possible.
 
Kevin- I tried the Gaussian filtering and it didn't really work very well, it always made things a bit too fuzzy.
 
So onto the solution. Basically i had to tell Imaris to stop making things pretty and smooth. My masks from MetaMorph were fine, it was just imaris trying to fill in gaps that was causing all my problems. Once i disabled smoothing when generating my 3D surfaces based on my Metamorph binary masks it worked like a dream. I can now create a 3D version of what i could do in 2D in MetaMorph. And better yet the data it seems to be producing frmo our samples is exactly what we were looking for.
 
 
Once again thaks to all for their suggestions.
 
 
 
Cheers
 
 
Cam
 
 
 
Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
 
Office: [phone]
Mobile: [phone]
Fax: [phone]
 
http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
 

[Kozlenkov, Alexey]

Dear all,


here is a question from a newbie venturing into the field of 2photon
FLIM-FRET measurements.

I'm considering using the Becker-Hickl time-domain FLIM / LSM NLO system
to measure FRET between some membrane proteins, fused to CFP and YFP.
The FLIM-based approach looked like an attractive option since it should
allow for analysing cells with not too highly expressed proteins of
interest, thus reducing the risk of obtaining FRET due only to membrane
overcrowding.
However, since my proteins are partially present in a highly motile pool
of vesicles, I intended to use fixed cell samples (as FLIM would require
some tens of seconds for one measurement).

Now, to my question:
The Becker-Hickl TCSPC handbook by Wolfgang Becker makes a strong point
of NOT using fixed samples for FLIM-FRET, due to changes in lifetimes
and strongly double-exponential decay profiles. However, other
publications, such as a protocol in the Molecular Cloning "Bible", do
use fixed samples for FLIM-FRET. Thus, I would welcome any comments or
advice from the community about this matter. Is fixed sample FLIM-FRET
really not recommended, and if it is not true, what would be the best
methodology to use (and pitfalls to avoid). How important would be the
choice of particular fluorescent protein, fixation methods and mounting
media? Obviously, I would also be grateful for links to good reviews and
experimental publications that I might have missed.


Thanks in advance,

Alex

=============================

Alexey Kozlenkov, PhD
Molecular Physiology of Somatic Sensation
Max-Delbruck Centrum for Molecular Medicine
13125 Berlin
Germany
[phone]

[Periasamy, Ammasi (ap3t)]

Hi Alexey
Yes, Dr. Wolfgang Becker is right and the fixative produce additional problems in the lifetime measurements.
Unfortunately for some of the biological experiment it is difficult to use the live samples. SO, currently we are working on it...how to overcome or correct the issues involved in lifetime measurement using the fixed samples versus live.
We will post the results soon.
Best,
Ammasi


Ammasi Periasamy, Ph.D.
Director, Keck Center for Cellular Imaging (KCCI)
Professor of Biology and Biomedical Engineering
Biology, Gilmer Hall (064), McCormick Rd
University of Virginia
Charlottesville, VA 22904
Voice: 434-243-7602 (Office); 982-4869 (lab)
Fax:[phone]; Email:[hidden email]
http//:www.kcci.virginia.edu
************************
Workshop on FRET Microscopy, March 9-13, 2010
http://www.kcci.virginia.edu/workshop/workshop2010/index.php
*************************


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kozlenkov, Alexey
Sent: Monday, July 20, 2009 8:03 AM
To: [hidden email]
Subject: time-domain FLIM-FRET with fixed samples

Dear all,


here is a question from a newbie venturing into the field of 2photon
FLIM-FRET measurements.

I'm considering using the Becker-Hickl time-domain FLIM / LSM NLO system
to measure FRET between some membrane proteins, fused to CFP and YFP.
The FLIM-based approach looked like an attractive option since it should
allow for analysing cells with not too highly expressed proteins of
interest, thus reducing the risk of obtaining FRET due only to membrane
overcrowding.
However, since my proteins are partially present in a highly motile pool
of vesicles, I intended to use fixed cell samples (as FLIM would require
some tens of seconds for one measurement).

Now, to my question:
The Becker-Hickl TCSPC handbook by Wolfgang Becker makes a strong point
of NOT using fixed samples for FLIM-FRET, due to changes in lifetimes
and strongly double-exponential decay profiles. However, other
publications, such as a protocol in the Molecular Cloning "Bible", do
use fixed samples for FLIM-FRET. Thus, I would welcome any comments or
advice from the community about this matter. Is fixed sample FLIM-FRET
really not recommended, and if it is not true, what would be the best
methodology to use (and pitfalls to avoid). How important would be the
choice of particular fluorescent protein, fixation methods and mounting
media? Obviously, I would also be grateful for links to good reviews and
experimental publications that I might have missed.


Thanks in advance,

Alex

=============================

Alexey Kozlenkov, PhD
Molecular Physiology of Somatic Sensation
Max-Delbruck Centrum for Molecular Medicine
13125 Berlin
Germany
[phone]

[Guy Cox]

You should consider that there are much faster FLIM systems than the Becker & Hickl.  Of course there is always a tradeoff, and typically you will trade some degree of lifetime resolution for the extra speed.  In the time-domain realm the Nikon (Europe) Limo system is much faster than the B&H (but collects into only 4 gates rather than 256).  It has a much higher photon efficiency and so can give good results at speeds compatible with live cell imaging, and our Limo system is routinely used on live cells.  (No connection other than as a customer).

You can probably get even higher speeds with wide-field frequency-domain systems such as the Lambert LIFA.

                                          Guy



Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Periasamy, Ammasi (ap3t)
Sent: Tuesday, 21 July 2009 7:45 AM
To: [hidden email]
Subject: Re: time-domain FLIM-FRET with fixed samples

Hi Alexey
Yes, Dr. Wolfgang Becker is right and the fixative produce additional problems in the lifetime measurements.
Unfortunately for some of the biological experiment it is difficult to use the live samples. SO, currently we are working on it...how to overcome or correct the issues involved in lifetime measurement using the fixed samples versus live.
We will post the results soon.
Best,
Ammasi


Ammasi Periasamy, Ph.D.
Director, Keck Center for Cellular Imaging (KCCI) Professor of Biology and Biomedical Engineering Biology, Gilmer Hall (064), McCormick Rd University of Virginia Charlottesville, VA 22904
Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:[phone]; Email:[hidden email] http//:www.kcci.virginia.edu
************************
Workshop on FRET Microscopy, March 9-13, 2010 http://www.kcci.virginia.edu/workshop/workshop2010/index.php
*************************


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kozlenkov, Alexey
Sent: Monday, July 20, 2009 8:03 AM
To: [hidden email]
Subject: time-domain FLIM-FRET with fixed samples

Dear all,


here is a question from a newbie venturing into the field of 2photon FLIM-FRET measurements.

I'm considering using the Becker-Hickl time-domain FLIM / LSM NLO system to measure FRET between some membrane proteins, fused to CFP and YFP.
The FLIM-based approach looked like an attractive option since it should allow for analysing cells with not too highly expressed proteins of interest, thus reducing the risk of obtaining FRET due only to membrane overcrowding.
However, since my proteins are partially present in a highly motile pool of vesicles, I intended to use fixed cell samples (as FLIM would require some tens of seconds for one measurement).

Now, to my question:
The Becker-Hickl TCSPC handbook by Wolfgang Becker makes a strong point of NOT using fixed samples for FLIM-FRET, due to changes in lifetimes and strongly double-exponential decay profiles. However, other publications, such as a protocol in the Molecular Cloning "Bible", do use fixed samples for FLIM-FRET. Thus, I would welcome any comments or advice from the community about this matter. Is fixed sample FLIM-FRET really not recommended, and if it is not true, what would be the best methodology to use (and pitfalls to avoid). How important would be the choice of particular fluorescent protein, fixation methods and mounting media? Obviously, I would also be grateful for links to good reviews and experimental publications that I might have missed.


Thanks in advance,

Alex

=============================

Alexey Kozlenkov, PhD
Molecular Physiology of Somatic Sensation Max-Delbruck Centrum for Molecular Medicine
13125 Berlin
Germany
[phone]

Internal Virus Database is out-of-date.
Checked by AVG.
Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date: 15/05/2009 6:16 AM
 

Internal Virus Database is out-of-date.
Checked by AVG.
Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date: 15/05/2009 6:16 AM
 

[Colin Rickman-2]

It is important to be careful when comparing different systems and their
relative speed of acquisition. Few groups have more than one system at
any one time to compare, using the same sample, and reported speeds and
accuracies are rarely from the sample (high speed and high accuracy are
normally achieved through differing acquisition settings).

We routinely use  a Becker & Hickl system for mono and bi-exponential
FLIM recordings (both from live cells and fixed samples). Recently we
trialled a Lambert LIFA system for our wide-field microscope (we were
hoping to use it under TIRF illumination). On the face of it this system
should be able to acquire lifetime data far faster. With low temporal
accuracy and a single exponential fit the system is faster using
widefield illumination than our Becker and Hickl system (normally around
30s). However, with comparable accuracy requirements and a
bi-exponential fit the acquisition time was far longer on the Lambert
system. The problem turned out to be the way the system works -  by
modulating the voltage on the MCP. To achieve a modulation the voltage
oscillates around 0V (ie off) meaning the MCP is very insensitive.

The main limitation for all FLIM recordings is the number of detected
photons used for the fitting. More photons = longer acquisition = higher
temporal accuracy and vice versa. Depends what you want to measure.

Colin

--
Dr Colin Rickman
Centre for Integrative Physiology
School of Biomedical Sciences
University of Edinburgh
Hugh Robson Building
George Square
Edinburgh
EH8 9XD

Tel: [phone]
Fax: [phone]



Guy Cox wrote:

> You should consider that there are much faster FLIM systems than the Becker & Hickl.  Of course there is always a tradeoff, and typically you will trade some degree of lifetime resolution for the extra speed.  In the time-domain realm the Nikon (Europe) Limo system is much faster than the B&H (but collects into only 4 gates rather than 256).  It has a much higher photon efficiency and so can give good results at speeds compatible with live cell imaging, and our Limo system is routinely used on live cells.  (No connection other than as a customer).
>
> You can probably get even higher speeds with wide-field frequency-domain systems such as the Lambert LIFA.
>
>                                           Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Electron Microscope Unit, Madsen Building F09,
> University of Sydney, NSW 2006
> ______________________________________________
> Phone [phone]     Fax [phone]
> Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Periasamy, Ammasi (ap3t)
> Sent: Tuesday, 21 July 2009 7:45 AM
> To: [hidden email]
> Subject: Re: time-domain FLIM-FRET with fixed samples
>
> Hi Alexey
> Yes, Dr. Wolfgang Becker is right and the fixative produce additional problems in the lifetime measurements.
> Unfortunately for some of the biological experiment it is difficult to use the live samples. SO, currently we are working on it...how to overcome or correct the issues involved in lifetime measurement using the fixed samples versus live.
> We will post the results soon.
> Best,
> Ammasi
>
>
> Ammasi Periasamy, Ph.D.
> Director, Keck Center for Cellular Imaging (KCCI) Professor of Biology and Biomedical Engineering Biology, Gilmer Hall (064), McCormick Rd University of Virginia Charlottesville, VA 22904
> Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:[phone]; Email:[hidden email] http//:www.kcci.virginia.edu
> ************************
> Workshop on FRET Microscopy, March 9-13, 2010 http://www.kcci.virginia.edu/workshop/workshop2010/index.php
> *************************
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kozlenkov, Alexey
> Sent: Monday, July 20, 2009 8:03 AM
> To: [hidden email]
> Subject: time-domain FLIM-FRET with fixed samples
>
> Dear all,
>
>
> here is a question from a newbie venturing into the field of 2photon FLIM-FRET measurements.
>
> I'm considering using the Becker-Hickl time-domain FLIM / LSM NLO system to measure FRET between some membrane proteins, fused to CFP and YFP.
> The FLIM-based approach looked like an attractive option since it should allow for analysing cells with not too highly expressed proteins of interest, thus reducing the risk of obtaining FRET due only to membrane overcrowding.
> However, since my proteins are partially present in a highly motile pool of vesicles, I intended to use fixed cell samples (as FLIM would require some tens of seconds for one measurement).
>
> Now, to my question:
> The Becker-Hickl TCSPC handbook by Wolfgang Becker makes a strong point of NOT using fixed samples for FLIM-FRET, due to changes in lifetimes and strongly double-exponential decay profiles. However, other publications, such as a protocol in the Molecular Cloning "Bible", do use fixed samples for FLIM-FRET. Thus, I would welcome any comments or advice from the community about this matter. Is fixed sample FLIM-FRET really not recommended, and if it is not true, what would be the best methodology to use (and pitfalls to avoid). How important would be the choice of particular fluorescent protein, fixation methods and mounting media? Obviously, I would also be grateful for links to good reviews and experimental publications that I might have missed.
>
>
> Thanks in advance,
>
> Alex
>
> =============================
>
> Alexey Kozlenkov, PhD
> Molecular Physiology of Somatic Sensation Max-Delbruck Centrum for Molecular Medicine
> 13125 Berlin
> Germany
> [phone]
>
> Internal Virus Database is out-of-date.
> Checked by AVG.
> Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date: 15/05/2009 6:16 AM
>  
>
> Internal Virus Database is out-of-date.
> Checked by AVG.
> Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date: 15/05/2009 6:16 AM
>  
>
>  

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

[Adrian Smith-6]

Hi Guy,

What sort of speeds/specifications are you talking about for the Nikon  
Limo system?

I've been looking at LaVision Biotec's new high-speed TCSPC FLIM  
detector but I'm not clear how it compares in principle with systems  
like the Nikon (or the Becker & Hickl).

http://www.lavisionbiotec.com/en/microscopy-products/fluorescence-microscopy/

Regards,

Adrian Smith
Centenary Institute, Sydney, Australia



On 21/07/2009, at 10:29 PM, Guy Cox wrote:

> You should consider that there are much faster FLIM systems than the  
> Becker & Hickl.  Of course there is always a tradeoff, and typically  
> you will trade some degree of lifetime resolution for the extra  
> speed.  In the time-domain realm the Nikon (Europe) Limo system is  
> much faster than the B&H (but collects into only 4 gates rather than  
> 256).  It has a much higher photon efficiency and so can give good  
> results at speeds compatible with live cell imaging, and our Limo  
> system is routinely used on live cells.  (No connection other than  
> as a customer).
>
> You can probably get even higher speeds with wide-field frequency-
> domain systems such as the Lambert LIFA.
>
>                                          Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>    http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Electron Microscope Unit, Madsen Building F09,
> University of Sydney, NSW 2006
> ______________________________________________
> Phone [phone]     Fax [phone]
> Mobile [phone]
> ______________________________________________
>     http://www.guycox.net
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ] On Behalf Of Periasamy, Ammasi (ap3t)
> Sent: Tuesday, 21 July 2009 7:45 AM
> To: [hidden email]
> Subject: Re: time-domain FLIM-FRET with fixed samples
>
> Hi Alexey
> Yes, Dr. Wolfgang Becker is right and the fixative produce  
> additional problems in the lifetime measurements.
> Unfortunately for some of the biological experiment it is difficult  
> to use the live samples. SO, currently we are working on it...how to  
> overcome or correct the issues involved in lifetime measurement  
> using the fixed samples versus live.
> We will post the results soon.
> Best,
> Ammasi
>
>
> Ammasi Periasamy, Ph.D.
> Director, Keck Center for Cellular Imaging (KCCI) Professor of  
> Biology and Biomedical Engineering Biology, Gilmer Hall (064),  
> McCormick Rd University of Virginia Charlottesville, VA 22904
> Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:[phone]; Email:[hidden email]
>  http//:www.kcci.virginia.edu
> ************************
> Workshop on FRET Microscopy, March 9-13, 2010 http://www.kcci.virginia.edu/workshop/workshop2010/index.php
> *************************
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ] On Behalf Of Kozlenkov, Alexey
> Sent: Monday, July 20, 2009 8:03 AM
> To: [hidden email]
> Subject: time-domain FLIM-FRET with fixed samples
>
> Dear all,
>
>
> here is a question from a newbie venturing into the field of 2photon  
> FLIM-FRET measurements.
>
> I'm considering using the Becker-Hickl time-domain FLIM / LSM NLO  
> system to measure FRET between some membrane proteins, fused to CFP  
> and YFP.
> The FLIM-based approach looked like an attractive option since it  
> should allow for analysing cells with not too highly expressed  
> proteins of interest, thus reducing the risk of obtaining FRET due  
> only to membrane overcrowding.
> However, since my proteins are partially present in a highly motile  
> pool of vesicles, I intended to use fixed cell samples (as FLIM  
> would require some tens of seconds for one measurement).
>
> Now, to my question:
> The Becker-Hickl TCSPC handbook by Wolfgang Becker makes a strong  
> point of NOT using fixed samples for FLIM-FRET, due to changes in  
> lifetimes and strongly double-exponential decay profiles. However,  
> other publications, such as a protocol in the Molecular Cloning  
> "Bible", do use fixed samples for FLIM-FRET. Thus, I would welcome  
> any comments or advice from the community about this matter. Is  
> fixed sample FLIM-FRET really not recommended, and if it is not  
> true, what would be the best methodology to use (and pitfalls to  
> avoid). How important would be the choice of particular fluorescent  
> protein, fixation methods and mounting media? Obviously, I would  
> also be grateful for links to good reviews and experimental  
> publications that I might have missed.
>
>
> Thanks in advance,
>
> Alex
>
> =============================
>
> Alexey Kozlenkov, PhD
> Molecular Physiology of Somatic Sensation Max-Delbruck Centrum for  
> Molecular Medicine
> 13125 Berlin
> Germany
> [phone]
>
> Internal Virus Database is out-of-date.
> Checked by AVG.
> Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date:  
> 15/05/2009 6:16 AM
>
>
> Internal Virus Database is out-of-date.
> Checked by AVG.
> Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date:  
> 15/05/2009 6:16 AM
>

[Guy Cox]

I have access to Becker & Hickl and Nikon LIMO systems.  I have used both on comparable samples.  

See: Guy Cox, Mikhail Matz and Anya Salih, 2007.  Fluorescence lifetime imaging of coral fluorescent proteins.  Microscopy Research & Technique 70, 243-251.

The LIMO system will give useful data with very much shorter acquisition times than the B&H - but the downside is that you cannot isolate the different exponential components as you can in the B&H.  So both have their place.  

As you say, the number of photons collected is critical - and Wolfgang Becker himself admitted to me that the LIMO is about 10 times as photon efficient as the B&H system.  (Obviously the exact figure will depend on the hardware, since both have many options.)

My views on the Lambert LIFA were based only on first principles and a quick play.  So I'm sorry if I misled people and the expected speed is not in fact available.  But I do think that if you are looking for a bi-exponential fit this isn't the correct tool for the job.  Horses for courses.

                                     Guy  



Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Colin Rickman
Sent: Tuesday, 21 July 2009 10:52 PM
To: [hidden email]
Subject: Re: time-domain FLIM-FRET with fixed samples

It is important to be careful when comparing different systems and their relative speed of acquisition. Few groups have more than one system at any one time to compare, using the same sample, and reported speeds and accuracies are rarely from the sample (high speed and high accuracy are normally achieved through differing acquisition settings).

We routinely use  a Becker & Hickl system for mono and bi-exponential FLIM recordings (both from live cells and fixed samples). Recently we trialled a Lambert LIFA system for our wide-field microscope (we were hoping to use it under TIRF illumination). On the face of it this system should be able to acquire lifetime data far faster. With low temporal accuracy and a single exponential fit the system is faster using widefield illumination than our Becker and Hickl system (normally around 30s). However, with comparable accuracy requirements and a bi-exponential fit the acquisition time was far longer on the Lambert system. The problem turned out to be the way the system works -  by modulating the voltage on the MCP. To achieve a modulation the voltage oscillates around 0V (ie off) meaning the MCP is very insensitive.

The main limitation for all FLIM recordings is the number of detected photons used for the fitting. More photons = longer acquisition = higher temporal accuracy and vice versa. Depends what you want to measure.

Colin

--
Dr Colin Rickman
Centre for Integrative Physiology
School of Biomedical Sciences
University of Edinburgh
Hugh Robson Building
George Square
Edinburgh
EH8 9XD

Tel: [phone]
Fax: [phone]



Guy Cox wrote:

> You should consider that there are much faster FLIM systems than the Becker & Hickl.  Of course there is always a tradeoff, and typically you will trade some degree of lifetime resolution for the extra speed.  In the time-domain realm the Nikon (Europe) Limo system is much faster than the B&H (but collects into only 4 gates rather than 256).  It has a much higher photon efficiency and so can give good results at speeds compatible with live cell imaging, and our Limo system is routinely used on live cells.  (No connection other than as a customer).
>
> You can probably get even higher speeds with wide-field frequency-domain systems such as the Lambert LIFA.
>
>                                           Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit,
> Madsen Building F09, University of Sydney, NSW 2006
> ______________________________________________
> Phone [phone]     Fax [phone]
> Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Periasamy,
> Ammasi (ap3t)
> Sent: Tuesday, 21 July 2009 7:45 AM
> To: [hidden email]
> Subject: Re: time-domain FLIM-FRET with fixed samples
>
> Hi Alexey
> Yes, Dr. Wolfgang Becker is right and the fixative produce additional problems in the lifetime measurements.
> Unfortunately for some of the biological experiment it is difficult to use the live samples. SO, currently we are working on it...how to overcome or correct the issues involved in lifetime measurement using the fixed samples versus live.
> We will post the results soon.
> Best,
> Ammasi
>
>
> Ammasi Periasamy, Ph.D.
> Director, Keck Center for Cellular Imaging (KCCI) Professor of Biology
> and Biomedical Engineering Biology, Gilmer Hall (064), McCormick Rd
> University of Virginia Charlottesville, VA 22904
> Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:[phone];
> Email:[hidden email] http//:www.kcci.virginia.edu
> ************************
> Workshop on FRET Microscopy, March 9-13, 2010
> http://www.kcci.virginia.edu/workshop/workshop2010/index.php
> *************************
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Kozlenkov,
> Alexey
> Sent: Monday, July 20, 2009 8:03 AM
> To: [hidden email]
> Subject: time-domain FLIM-FRET with fixed samples
>
> Dear all,
>
>
> here is a question from a newbie venturing into the field of 2photon FLIM-FRET measurements.
>
> I'm considering using the Becker-Hickl time-domain FLIM / LSM NLO system to measure FRET between some membrane proteins, fused to CFP and YFP.
> The FLIM-based approach looked like an attractive option since it should allow for analysing cells with not too highly expressed proteins of interest, thus reducing the risk of obtaining FRET due only to membrane overcrowding.
> However, since my proteins are partially present in a highly motile pool of vesicles, I intended to use fixed cell samples (as FLIM would require some tens of seconds for one measurement).
>
> Now, to my question:
> The Becker-Hickl TCSPC handbook by Wolfgang Becker makes a strong point of NOT using fixed samples for FLIM-FRET, due to changes in lifetimes and strongly double-exponential decay profiles. However, other publications, such as a protocol in the Molecular Cloning "Bible", do use fixed samples for FLIM-FRET. Thus, I would welcome any comments or advice from the community about this matter. Is fixed sample FLIM-FRET really not recommended, and if it is not true, what would be the best methodology to use (and pitfalls to avoid). How important would be the choice of particular fluorescent protein, fixation methods and mounting media? Obviously, I would also be grateful for links to good reviews and experimental publications that I might have missed.
>
>
> Thanks in advance,
>
> Alex
>
> =============================
>
> Alexey Kozlenkov, PhD
> Molecular Physiology of Somatic Sensation Max-Delbruck Centrum for
> Molecular Medicine
> 13125 Berlin
> Germany
> [phone]
>
> Internal Virus Database is out-of-date.
> Checked by AVG.
> Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date:
> 15/05/2009 6:16 AM
>  
>
> Internal Virus Database is out-of-date.
> Checked by AVG.
> Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date:
> 15/05/2009 6:16 AM
>  
>
>  

Internal Virus Database is out-of-date.
Checked by AVG.
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[Guy Cox]

I've no experience with the LaVision system - I know their Trimscope but not their FLIM detector.  It certainly sounds interesting.  

The LIMO will typically give robust lifetime images in 1 - 4s acquisition time (but I do work with rather bright samples).  You can always (Adrian, that is, not the List) walk over and have a play!

                               Guy


Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Adrian Smith
Sent: Tuesday, 21 July 2009 10:54 PM
To: [hidden email]
Subject: Re: time-domain FLIM-FRET with fixed samples

Hi Guy,

What sort of speeds/specifications are you talking about for the Nikon Limo system?

I've been looking at LaVision Biotec's new high-speed TCSPC FLIM detector but I'm not clear how it compares in principle with systems like the Nikon (or the Becker & Hickl).

http://www.lavisionbiotec.com/en/microscopy-products/fluorescence-microscopy/

Regards,

Adrian Smith
Centenary Institute, Sydney, Australia



On 21/07/2009, at 10:29 PM, Guy Cox wrote:

> You should consider that there are much faster FLIM systems than the
> Becker & Hickl.  Of course there is always a tradeoff, and typically
> you will trade some degree of lifetime resolution for the extra speed.  
> In the time-domain realm the Nikon (Europe) Limo system is much faster
> than the B&H (but collects into only 4 gates rather than 256).  It has
> a much higher photon efficiency and so can give good results at speeds
> compatible with live cell imaging, and our Limo system is routinely
> used on live cells.  (No connection other than as a customer).
>
> You can probably get even higher speeds with wide-field frequency-
> domain systems such as the Lambert LIFA.
>
>                                          Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>    http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit,
> Madsen Building F09, University of Sydney, NSW 2006
> ______________________________________________
> Phone [phone]     Fax [phone]
> Mobile [phone]
> ______________________________________________
>     http://www.guycox.net
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]
> ] On Behalf Of Periasamy, Ammasi (ap3t)
> Sent: Tuesday, 21 July 2009 7:45 AM
> To: [hidden email]
> Subject: Re: time-domain FLIM-FRET with fixed samples
>
> Hi Alexey
> Yes, Dr. Wolfgang Becker is right and the fixative produce additional
> problems in the lifetime measurements.
> Unfortunately for some of the biological experiment it is difficult to
> use the live samples. SO, currently we are working on it...how to
> overcome or correct the issues involved in lifetime measurement using
> the fixed samples versus live.
> We will post the results soon.
> Best,
> Ammasi
>
>
> Ammasi Periasamy, Ph.D.
> Director, Keck Center for Cellular Imaging (KCCI) Professor of Biology
> and Biomedical Engineering Biology, Gilmer Hall (064), McCormick Rd
> University of Virginia Charlottesville, VA 22904
> Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:[phone];
> Email:[hidden email]  http//:www.kcci.virginia.edu
> ************************
> Workshop on FRET Microscopy, March 9-13, 2010
> http://www.kcci.virginia.edu/workshop/workshop2010/index.php
> *************************
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]
> ] On Behalf Of Kozlenkov, Alexey
> Sent: Monday, July 20, 2009 8:03 AM
> To: [hidden email]
> Subject: time-domain FLIM-FRET with fixed samples
>
> Dear all,
>
>
> here is a question from a newbie venturing into the field of 2photon
> FLIM-FRET measurements.
>
> I'm considering using the Becker-Hickl time-domain FLIM / LSM NLO
> system to measure FRET between some membrane proteins, fused to CFP
> and YFP.
> The FLIM-based approach looked like an attractive option since it
> should allow for analysing cells with not too highly expressed
> proteins of interest, thus reducing the risk of obtaining FRET due
> only to membrane overcrowding.
> However, since my proteins are partially present in a highly motile
> pool of vesicles, I intended to use fixed cell samples (as FLIM would
> require some tens of seconds for one measurement).
>
> Now, to my question:
> The Becker-Hickl TCSPC handbook by Wolfgang Becker makes a strong
> point of NOT using fixed samples for FLIM-FRET, due to changes in
> lifetimes and strongly double-exponential decay profiles. However,
> other publications, such as a protocol in the Molecular Cloning
> "Bible", do use fixed samples for FLIM-FRET. Thus, I would welcome any
> comments or advice from the community about this matter. Is fixed
> sample FLIM-FRET really not recommended, and if it is not true, what
> would be the best methodology to use (and pitfalls to avoid). How
> important would be the choice of particular fluorescent protein,
> fixation methods and mounting media? Obviously, I would also be
> grateful for links to good reviews and experimental publications that
> I might have missed.
>
>
> Thanks in advance,
>
> Alex
>
> =============================
>
> Alexey Kozlenkov, PhD
> Molecular Physiology of Somatic Sensation Max-Delbruck Centrum for
> Molecular Medicine
> 13125 Berlin
> Germany
> [phone]
>
> Internal Virus Database is out-of-date.
> Checked by AVG.
> Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date:  
> 15/05/2009 6:16 AM
>
>
> Internal Virus Database is out-of-date.
> Checked by AVG.
> Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date:  
> 15/05/2009 6:16 AM
>

Internal Virus Database is out-of-date.
Checked by AVG.
Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date: 15/05/2009 6:16 AM
 

Internal Virus Database is out-of-date.
Checked by AVG.
Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date: 15/05/2009 6:16 AM
 

[vb-2]

Dear List,

Thank you Guy for mentioning the Lambert LIFA system.

I would really appreciate your feedback on the Lambert LIFA system including
options/flexibility/upgrades along the long-term use.

Best,

Vitaly
NCI-Frederick,
301-846-6575


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Guy Cox
Sent: Tuesday, July 21, 2009 7:29 AM
To: [hidden email]
Subject: Re: time-domain FLIM-FRET with fixed samples

You should consider that there are much faster FLIM systems than the Becker
& Hickl.  Of course there is always a tradeoff, and typically you will trade
some degree of lifetime resolution for the extra speed.  In the time-domain
realm the Nikon (Europe) Limo system is much faster than the B&H (but
collects into only 4 gates rather than 256).  It has a much higher photon
efficiency and so can give good results at speeds compatible with live cell
imaging, and our Limo system is routinely used on live cells.  (No
connection other than as a customer).

You can probably get even higher speeds with wide-field frequency-domain
systems such as the Lambert LIFA.

                                          Guy



Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Periasamy, Ammasi (ap3t)
Sent: Tuesday, 21 July 2009 7:45 AM
To: [hidden email]
Subject: Re: time-domain FLIM-FRET with fixed samples

Hi Alexey
Yes, Dr. Wolfgang Becker is right and the fixative produce additional
problems in the lifetime measurements.
Unfortunately for some of the biological experiment it is difficult to use
the live samples. SO, currently we are working on it...how to overcome or
correct the issues involved in lifetime measurement using the fixed samples
versus live.
We will post the results soon.
Best,
Ammasi


Ammasi Periasamy, Ph.D.
Director, Keck Center for Cellular Imaging (KCCI) Professor of Biology and
Biomedical Engineering Biology, Gilmer Hall (064), McCormick Rd University
of Virginia Charlottesville, VA 22904
Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:[phone];
Email:[hidden email] http//:www.kcci.virginia.edu
************************
Workshop on FRET Microscopy, March 9-13, 2010
http://www.kcci.virginia.edu/workshop/workshop2010/index.php
*************************


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Kozlenkov, Alexey
Sent: Monday, July 20, 2009 8:03 AM
To: [hidden email]
Subject: time-domain FLIM-FRET with fixed samples

Dear all,


here is a question from a newbie venturing into the field of 2photon
FLIM-FRET measurements.

I'm considering using the Becker-Hickl time-domain FLIM / LSM NLO system to
measure FRET between some membrane proteins, fused to CFP and YFP.
The FLIM-based approach looked like an attractive option since it should
allow for analysing cells with not too highly expressed proteins of
interest, thus reducing the risk of obtaining FRET due only to membrane
overcrowding.
However, since my proteins are partially present in a highly motile pool of
vesicles, I intended to use fixed cell samples (as FLIM would require some
tens of seconds for one measurement).

Now, to my question:
The Becker-Hickl TCSPC handbook by Wolfgang Becker makes a strong point of
NOT using fixed samples for FLIM-FRET, due to changes in lifetimes and
strongly double-exponential decay profiles. However, other publications,
such as a protocol in the Molecular Cloning "Bible", do use fixed samples
for FLIM-FRET. Thus, I would welcome any comments or advice from the
community about this matter. Is fixed sample FLIM-FRET really not
recommended, and if it is not true, what would be the best methodology to
use (and pitfalls to avoid). How important would be the choice of particular
fluorescent protein, fixation methods and mounting media? Obviously, I would
also be grateful for links to good reviews and experimental publications
that I might have missed.


Thanks in advance,

Alex

=============================

Alexey Kozlenkov, PhD
Molecular Physiology of Somatic Sensation Max-Delbruck Centrum for Molecular
Medicine
13125 Berlin
Germany
[phone]

Internal Virus Database is out-of-date.
Checked by AVG.
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6:16 AM
 

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Checked by AVG.
Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date: 15/05/2009
6:16 AM
 

[Alessandro Esposito]

Dear all, dear Vitaly,
    I used B&H with a PMT or with a MCP-PMT, a LIFA, a LiMo and a custom
biult FD-FLIM based on LaVision MCP-based (Kantech) camera.

As other mentioned is very difficult to compare systems in a precise manner,
however, some trends are quite evident. There is a very good comparison TD
vs FD done by Gratton a few years ago:  

http://dx.doi.org/10.1117/1.1586704 

and we published a work comparing acquisition throughput of various
techniques from a theoretical point of view:

http://www.opticsinfobase.org/abstract.cfm?URI=josaa-24-10-3261

Concerning the mentioned systems, B&H sells a variaty of systems that can
provide very slow or quite fast options. With same electronics, if one will use
an MCP-PMT as detector will get a very good IRF (<60ps), but comparatevly
slow acquisition times. I remember waiting 10 mins to get data with enough
photons/pixel to detect in a reliable manner FRET between Cerulean/Venus.
If one uses PMT (probably also the new hybrid PMT, but I still did not use
them), will get IRFs 200ps broad, but much faster acquisition times. With a
bright sample, 1 min acquisition time start to be possible.

The BIG problem with these systems is pulse-pile up (linked to the dead time
of detector/electronics) and the requirement to measure, in average, only one
photon per laser pulse (typical for TCSPC). B&H solved the latter providing
multi-detector systems, though this will come at a cost.

I do not have direct experience with the LaVision Biotech systems, but it
seems interesting and, if I remember correctly, may provide a very good
dynamic range because of the implementation of how they split light on their
multi-detector system. With a "conventional" TCSPC system you will have to
reduce the excitation light intensity at the level where no pixel in the image
suffer of pulse pile-up. I think the new implementation by LaVision Biotech may
overcome this, but I hope they could post here to explain that.

Now the LiMo system. Its limiting factor is still the dead time of the detector,
but it can detect more photons/laser pulse and this is the reason why can be
faster. Having only 4 gates it provides a lower photon-efficiency and less
access to heterogeneous decays, but overall performs fast and is a simple and
cost-effective system. Guy: is it possible that the comment on photon-
efficiency refers to the lack of the requirement of detecting max 1
photon/pulse?

Among the scanning systems I would like to remind there is also picoQuant,
but I do not have direct experience with their systems.

Wide-fields: LaVision sells a time-gated MCP-based system and Lambert
Instruments the FDFLIM LIFA. Wide-field is faster because collects more light
from the sample. However, both existing TD and FD wide-field systems are
based on gating/modulating an MCP which causes large losses. Still they are
fast and the original limitations of FD have been quite overcome in recent
times. LIFA is relatevely fast, cost effective and user friendly, but if you have
a dim sample for which you need higher spatial resolution, TCSPC could be the
way to go.

I'll post a different message concerning fixed samples, but allow me a bit of
advertizement :) and a last note on wide-field systems.
There is a time- and space- correlated MCP system available by
Europhoton.de. It is a wide-field detector, though all photons are acquired
sequentially and therefore is quite slow, but sensitive.

Also, a new genertation of wide-field systems could be available in a few years
based on solid-state technologies (http://dx.doi.org/10.1117/1.2208999,
http://www.opticsinfobase.org/oe/abstract.cfm?id=86274) which should
overcome problems that current WF systems suffer from.

That is all for now, I hope this long post was somehow more useful than boring!

Alessandro Esposito
www.quantitative-microscopy.org

[Guy Cox]

Alessandro wrote:

"Now the LiMo system. Its limiting factor is still the dead time of the detector, but it can detect more photons/laser pulse and this is the reason why can be faster. Having only 4 gates it provides a lower photon-efficiency and less access to heterogeneous decays, but overall performs fast and is a simple and cost-effective system. Guy: is it possible that the comment on photon- efficiency refers to the lack of the requirement of detecting max 1 photon/pulse? "

Well, Alessandro comes from the lab where the LIMO was developed, so it may seem impertinent for a mere paying customer to comment!  But the ability to detect more photons per pulse - limited only by detector speed - is a key point.  Having only 4 gates means that any photons arriving after about 9ns are lost but unless you have very long-lifetime fluorophores that is relatively trivial.  But the other key factor is that the LIMO collects after every pulse, whereas the B&H versions around at the time my discussions took place simply didn't.  A lot of laser pulses were missed.  This may have improved - but I still don't find people saying they can get good images with 1-4s frame time on a B&H system.

I do emphasize that I'm not denigrating the B&H - I've used it, and published results from it.  The LIMO will not do multi-exponential fits, and these can be very useful.  (Actually - Nikon NB) my mathematically-inclined colleagues tell me that with 4 points you should be able to distinguish single and dual exponential decays, so maybe the software could be upgraded?

                                        Guy




Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alessandro Esposito
Sent: Wednesday, 22 July 2009 6:01 PM
To: [hidden email]
Subject: Re: time-domain FLIM-FRET with fixed samples

Dear all, dear Vitaly,
    I used B&H with a PMT or with a MCP-PMT, a LIFA, a LiMo and a custom biult FD-FLIM based on LaVision MCP-based (Kantech) camera.

As other mentioned is very difficult to compare systems in a precise manner, however, some trends are quite evident. There is a very good comparison TD vs FD done by Gratton a few years ago:  

http://dx.doi.org/10.1117/1.1586704 

and we published a work comparing acquisition throughput of various techniques from a theoretical point of view:

http://www.opticsinfobase.org/abstract.cfm?URI=josaa-24-10-3261

Concerning the mentioned systems, B&H sells a variaty of systems that can provide very slow or quite fast options. With same electronics, if one will use an MCP-PMT as detector will get a very good IRF (<60ps), but comparatevly slow acquisition times. I remember waiting 10 mins to get data with enough photons/pixel to detect in a reliable manner FRET between Cerulean/Venus.
If one uses PMT (probably also the new hybrid PMT, but I still did not use them), will get IRFs 200ps broad, but much faster acquisition times. With a bright sample, 1 min acquisition time start to be possible.

The BIG problem with these systems is pulse-pile up (linked to the dead time of detector/electronics) and the requirement to measure, in average, only one photon per laser pulse (typical for TCSPC). B&H solved the latter providing multi-detector systems, though this will come at a cost.

I do not have direct experience with the LaVision Biotech systems, but it seems interesting and, if I remember correctly, may provide a very good dynamic range because of the implementation of how they split light on their multi-detector system. With a "conventional" TCSPC system you will have to reduce the excitation light intensity at the level where no pixel in the image suffer of pulse pile-up. I think the new implementation by LaVision Biotech may overcome this, but I hope they could post here to explain that.

Now the LiMo system. Its limiting factor is still the dead time of the detector, but it can detect more photons/laser pulse and this is the reason why can be faster. Having only 4 gates it provides a lower photon-efficiency and less access to heterogeneous decays, but overall performs fast and is a simple and cost-effective system. Guy: is it possible that the comment on photon- efficiency refers to the lack of the requirement of detecting max 1 photon/pulse?

Among the scanning systems I would like to remind there is also picoQuant, but I do not have direct experience with their systems.

Wide-fields: LaVision sells a time-gated MCP-based system and Lambert Instruments the FDFLIM LIFA. Wide-field is faster because collects more light from the sample. However, both existing TD and FD wide-field systems are based on gating/modulating an MCP which causes large losses. Still they are fast and the original limitations of FD have been quite overcome in recent times. LIFA is relatevely fast, cost effective and user friendly, but if you have a dim sample for which you need higher spatial resolution, TCSPC could be the way to go.

I'll post a different message concerning fixed samples, but allow me a bit of advertizement :) and a last note on wide-field systems.
There is a time- and space- correlated MCP system available by Europhoton.de. It is a wide-field detector, though all photons are acquired sequentially and therefore is quite slow, but sensitive.

Also, a new genertation of wide-field systems could be available in a few years based on solid-state technologies (http://dx.doi.org/10.1117/1.2208999,
http://www.opticsinfobase.org/oe/abstract.cfm?id=86274) which should overcome problems that current WF systems suffer from.

That is all for now, I hope this long post was somehow more useful than boring!

Alessandro Esposito
www.quantitative-microscopy.org

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[Alessandro Esposito]

Dear Alexey,
     it depends a lot on what you would like to measure. If you are planning to
make very accurate measurements to detect either photophysical properties
of the fluorophores or intermolecular distances, additional heterogeneities in
the lifetime decay are a problem.

If you would like to measure FRET / no FRET (interaction / no interaction, just
simplifying) more complex decays are not a big problem (within limits). Notably,
in a typical FRET experiment you would anyway measure multi-exponential
decays because of a mixture of non-interacting donors and may be also donor-
acceptor pairs that are interacting in different ways. On the top of this, there
is also the possibility of non purely single exponential decays of the
unperturbed fluorophore. Consider that often we are not capable to detect
multi exponential decays because we do not have enough signal-to-noise ratio
to start with.

Occasionally, fixing and mounting could be even beneficial because of the
improved optical properties of the sample and, when using some anti-fading
agent, less photobleaching. However, fixing and mounting will introduce
differences in the biochemical environment of the fluorophore which could, in
principle, even suppress any FRET: this is not a rule.

In general, FRET on fixed samples does work (there is no physical reason why
it should not), but could add some nuances that can/should be dealt with.

Last remark. Fixing and mounting can alter the localization of proteins and
cause false positive or false negative FRET: good control experiments are
always necessary (in any case).

Cheers,

Alessandro Esposito
University of Cambridge
www.quantitative-microscopy.org

[Alessandro Esposito]

Dear Guy,
   we fully agree, I may have explained myself not correctly. I was just
mentioning that time-gating with a few time windows is slightly less photon
efficient, but can provide faster acquisition times. It provide less information
content per counted photons, but can count a higher number of photons.

It is a couple of years that B&H claims thay can detect lifetimes that fast, but
I guess this can happen only with their multi-detector system and I personally
never met anybody that got a TCSPC (good) image in a few seconds.

Cheers,

Alessandro

[Kozlenkov, Alexey]

Thanks a lot to all you people replying.

At the moment, the B-n-H time domain FLIM system is the only option
easily available for me, and it seems unlikely that our Microscope Core
Facility makes any big purchases this year. However in case a new FLIM
system is considered, I'll definitely come back to this thread to review
your kind replies and suggestions.  

Other people who use the system at our institute, do live cell
measurements, as their samples are better suited for that, and they told
me that fixation decreased the lifetimes of ECFP in their constructs.
With my very preliminary data so far, I also do see a decrease in
lifetime (amplitude-weighted means of 2-exponential fit) for fixed
SCFP3A (kind gift of Dorus Gadella); within 1 day after fixation the
non-fused protein had T = 2.5 (that actually was nearly ok), but the
fused variant had T = 2.1, which in one week after fixation became 1.6.
The latter would obviously preclude any meaningful FRET measurements,
though I might still play with freshly fixed samples.

One last question I have for this thread is - what are the
recommendations for the CFP variants to use in (time-domain) FLIM: for
example, whether Cerulean is preferred over ECFP? Or do people switch
now to novel CFP variants?

Thanks again,

Best,
Alex

=============================

Alexey Kozlenkov, PhD
Molecular Physiology of Somatic Sensation
Max-Delbruck Centrum for Molecular Medicine
13125 Berlin
Germany
[phone]




-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Kozlenkov, Alexey
Sent: Montag, 20. Juli 2009 14:03
To: [hidden email]
Subject: time-domain FLIM-FRET with fixed samples

Dear all,


here is a question from a newbie venturing into the field of 2photon
FLIM-FRET measurements.

I'm considering using the Becker-Hickl time-domain FLIM / LSM NLO system
to measure FRET between some membrane proteins, fused to CFP and YFP.
The FLIM-based approach looked like an attractive option since it should
allow for analysing cells with not too highly expressed proteins of
interest, thus reducing the risk of obtaining FRET due only to membrane
overcrowding.
However, since my proteins are partially present in a highly motile pool
of vesicles, I intended to use fixed cell samples (as FLIM would require
some tens of seconds for one measurement).

Now, to my question:
The Becker-Hickl TCSPC handbook by Wolfgang Becker makes a strong point
of NOT using fixed samples for FLIM-FRET, due to changes in lifetimes
and strongly double-exponential decay profiles. However, other
publications, such as a protocol in the Molecular Cloning "Bible", do
use fixed samples for FLIM-FRET. Thus, I would welcome any comments or
advice from the community about this matter. Is fixed sample FLIM-FRET
really not recommended, and if it is not true, what would be the best
methodology to use (and pitfalls to avoid). How important would be the
choice of particular fluorescent protein, fixation methods and mounting
media? Obviously, I would also be grateful for links to good reviews and
experimental publications that I might have missed.


Thanks in advance,

Alex

=============================

Alexey Kozlenkov, PhD
Molecular Physiology of Somatic Sensation
Max-Delbruck Centrum for Molecular Medicine
13125 Berlin
Germany
[phone]