Measuring orientation for FRET

[Pablo German]

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Hi,

A question for the FRET experts. According to the literature FRET efficiency
between two fluorophores depends on several parameters including distance
and orientation.

Tipically, orientation is averaged if we can assume that the molecules can
take all possible orientations. But what happens if that cannot be assumed?
Is there a way to measure orientation between two fluorophores? I am working
with tagged receptors so the measurements will be of receptors either in
live cells or purified.

Would really appreciate your comments.

Kind regards,
Pablo

--
Pablo German

Plant and Food Research
Private Bag 92169
Auckland Mail Centre
Auckland 1142
New Zealand
DDI: [phone]
Mobile: [phone]

[Unruh, Jay]

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Pablo,

Unless you have specific data from other techniques besides fluorescence (x ray scattering, modeling, etc), it is difficult to measure orientations.  Nevertheless, if your fluorophores are somewhat mobile or have a dipole shift between absorption and emission, it is possible to calculate the range of fluorophore orientations that will be populated and therefore the "error" in the FRET measurement.  This mobility or dipole shift can be measured using fluorescence anisotropy as described by Van der Meer (2002 Reviews in Molecular Biotechnology vol 82 page 181) and many others that he references.  You can also try to measure "sensitized" anisotropy (the anisotropy of the acceptor upon energy transfer) but this is much more difficult.

Good Luck!
Jay Unruh
Stowers Institute for Medical Research

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Pablo German
Sent: Thursday, August 18, 2011 5:03 PM
To: [hidden email]
Subject: Measuring orientation for FRET

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Hi,

A question for the FRET experts. According to the literature FRET efficiency between two fluorophores depends on several parameters including distance and orientation.

Tipically, orientation is averaged if we can assume that the molecules can take all possible orientations. But what happens if that cannot be assumed?
Is there a way to measure orientation between two fluorophores? I am working with tagged receptors so the measurements will be of receptors either in live cells or purified.

Would really appreciate your comments.

Kind regards,
Pablo

--
Pablo German

Plant and Food Research
Private Bag 92169
Auckland Mail Centre
Auckland 1142
New Zealand
DDI: [phone]
Mobile: [phone]

[Tim Feinstein-2]

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Hi Pablo,  

Our group has some experience with receptor-receptor FRET and designing sensors that report orientation shifts.  Your problem is most likely something that we run across all the time - a FRET interaction that seems like it ought to work but doesn't.  The problem is that distance and orientation are convolved in a manner that (for the most part) is far too mathematically complex to sort out in vivo using fluorescent protein tags.  .  

After anisotropy, as already mentioned, troubleshooting step 1 should be to add a flexible linker between your receptor(s) and the fluorescent tags.  It would also not be a bad idea to use permissive pull-down, e.g. with a reversible cross-linker such as DSP, to verify that your interaction does happen.  It is also possible that your interaction is happening but the signal is swamped by non-interacting background.  In that case I would look into time-resolved FRET using an oscillating LASER-detector combo, which is great for finding signal needles in a background haystack.  

More often we never figure out what went wrong.  FRET experiments, especially biosensor design, have plenty of false negatives (that is why we feel that negative FRET does not constitute definitive proof against an interaction).  In those cases we just move on and try something else.

Good luck,  


TIm Feinstein



On Aug 18, 2011, at 6:02 PM, Pablo German <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> A question for the FRET experts. According to the literature FRET efficiency
> between two fluorophores depends on several parameters including distance
> and orientation.
>
> Tipically, orientation is averaged if we can assume that the molecules can
> take all possible orientations. But what happens if that cannot be assumed?
> Is there a way to measure orientation between two fluorophores? I am working
> with tagged receptors so the measurements will be of receptors either in
> live cells or purified.
>
> Would really appreciate your comments.
>
> Kind regards,
> Pablo
>
> --
> Pablo German
>
> Plant and Food Research
> Private Bag 92169
> Auckland Mail Centre
> Auckland 1142
> New Zealand
> DDI: [phone]
> Mobile: [phone]

[Iain Johnson]

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To join, leave or search the confocal microscopy listserv, go to:
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For the orientation factor to cause a FRET experiment to fail completely is
physically quite improbable.  The orientation factor has possible values
between zero and 4 (with a value of 0.666 representing the orientationally
averaged condition).  To get it to go to zero entails the donor and acceptor
transition dipoles having to be locked at 90 degrees relative to one
another, a condition that is hard to achieve in any sort of molecular
assembly.

There are two spectroscopic hallmarks of FRET that can be used to confirm
(or disprove) its occurrence without manipulating the donor or acceptor
concentrations.  One is to measure the excitation spectrum of the acceptor
and verify the presence of an extra peak corresponding to the donor
absorption maximum wavelength.  The other is to do a time-resolved
measurement of the acceptor emission and verify the presence of a rise time
corresponding to population of the acceptor excited state via FRET from the
donor.  Of the two, the time-resolved measurement is generally more
practicable in a cellular imaging context.

Iain

Iain Johnson Ph.D
Iain Johnson Consulting
Eugene, OR

On Thu, Aug 18, 2011 at 6:06 PM, Tim Feinstein <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Pablo,
>
> Our group has some experience with receptor-receptor FRET and designing
> sensors that report orientation shifts.  Your problem is most likely
> something that we run across all the time - a FRET interaction that seems
> like it ought to work but doesn't.  The problem is that distance and
> orientation are convolved in a manner that (for the most part) is far too
> mathematically complex to sort out in vivo using fluorescent protein tags.
>  .
>
> After anisotropy, as already mentioned, troubleshooting step 1 should be to
> add a flexible linker between your receptor(s) and the fluorescent tags.  It
> would also not be a bad idea to use permissive pull-down, e.g. with a
> reversible cross-linker such as DSP, to verify that your interaction does
> happen.  It is also possible that your interaction is happening but the
> signal is swamped by non-interacting background.  In that case I would look
> into time-resolved FRET using an oscillating LASER-detector combo, which is
> great for finding signal needles in a background haystack.
>
> More often we never figure out what went wrong.  FRET experiments,
> especially biosensor design, have plenty of false negatives (that is why we
> feel that negative FRET does not constitute definitive proof against an
> interaction).  In those cases we just move on and try something else.
>
> Good luck,
>
>
> TIm Feinstein
>
>
>
> On Aug 18, 2011, at 6:02 PM, Pablo German <[hidden email]>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi,
> >
> > A question for the FRET experts. According to the literature FRET
> efficiency
> > between two fluorophores depends on several parameters including distance
> > and orientation.
> >
> > Tipically, orientation is averaged if we can assume that the molecules
> can
> > take all possible orientations. But what happens if that cannot be
> assumed?
> > Is there a way to measure orientation between two fluorophores? I am
> working
> > with tagged receptors so the measurements will be of receptors either in
> > live cells or purified.
> >
> > Would really appreciate your comments.
> >
> > Kind regards,
> > Pablo
> >
> > --
> > Pablo German
> >
> > Plant and Food Research
> > Private Bag 92169
> > Auckland Mail Centre
> > Auckland 1142
> > New Zealand
> > DDI: [phone]
> > Mobile: [phone]
>

[Zac Arrac Atelaz]

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Some physics I have heard of

Dont get me wrong, love the results and things FRET is helping to show, but, there is a little guy, called the photon, which is the way we use to name a electromagnetic bump in the universe, which behaves both, as something physical (at least in high energies) and as wave, but has not a mass it self, sorry almost pure physics here deeper here:

http://physics.about.com/od/lightoptics/f/photon.htm

http://physics.about.com/od/quantumphysics/a/comptoneffect.htm

The thing is that you can imagine (simplifiying high here) something
like flea-bees flying-jumping at a given wavelength at the speed of light trough this "solar system", and when a big enough group of those get together they create a quantic bee (photon) powerful enough to "show" the interaction of the contact with one planet, as this quantic bee reach and
bounce with planets changes its color when this happens, the trouble that you and any that want to measure light have is that light although would seem macroscopically to bounce back from the wall in your house or a mirror at a given angle is much more complex as the particle-particle interactions are not something easy or direct to measure when it comes to the smaller scale, not as complicated as biological mollecules) as you might imagine you are trying to know the orientation of one small constellation, inside a galaxy, by hiting them with this hordes of bees.

So as quantic it goes, sometimes you get the perfect angle for a great "conjunction" and a beautiful FRET effect happens, but sometimes, as Tim says well the bees and the stars are not aligned that night, and you have to change the telescope for, maybe some gold deposition or other nanocontrast.

Quantum luck

Gabriel OH

> Date: Thu, 18 Aug 2011 21:06:34 -0400
> From: [hidden email]
> Subject: Re: Measuring orientation for FRET
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Pablo,  
>
> Our group has some experience with receptor-receptor FRET and designing sensors that report orientation shifts.  Your problem is most likely something that we run across all the time - a FRET interaction that seems like it ought to work but doesn't.  The problem is that distance and orientation are convolved in a manner that (for the most part) is far too mathematically complex to sort out in vivo using fluorescent protein tags.  .  
>
> After anisotropy, as already mentioned, troubleshooting step 1 should be to add a flexible linker between your receptor(s) and the fluorescent tags.  It would also not be a bad idea to use permissive pull-down, e.g. with a reversible cross-linker such as DSP, to verify that your interaction does happen.  It is also possible that your interaction is happening but the signal is swamped by non-interacting background.  In that case I would look into time-resolved FRET using an oscillating LASER-detector combo, which is great for finding signal needles in a background haystack.  
>
> More often we never figure out what went wrong.  FRET experiments, especially biosensor design, have plenty of false negatives (that is why we feel that negative FRET does not constitute definitive proof against an interaction).  In those cases we just move on and try something else.
>
> Good luck,  
>
>
> TIm Feinstein
>
>
>
> On Aug 18, 2011, at 6:02 PM, Pablo German <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi,
> >
> > A question for the FRET experts. According to the literature FRET efficiency
> > between two fluorophores depends on several parameters including distance
> > and orientation.
> >
> > Tipically, orientation is averaged if we can assume that the molecules can
> > take all possible orientations. But what happens if that cannot be assumed?
> > Is there a way to measure orientation between two fluorophores? I am working
> > with tagged receptors so the measurements will be of receptors either in
> > live cells or purified.
> >
> > Would really appreciate your comments.
> >
> > Kind regards,
> > Pablo
> >
> > --
> > Pablo German
> >
> > Plant and Food Research
> > Private Bag 92169
> > Auckland Mail Centre
> > Auckland 1142
> > New Zealand
> > DDI: [phone]
> > Mobile: [phone]

     

[Deepak nair]

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On 19 Aug 2011 08:07, "Zac Arrac Atelaz" <[hidden email]> wrote:

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin...
Some physics I have heard of

Dont get me wrong, love the results and things FRET is helping to show, but,
there is a little guy, called the photon, which is the way we use to name a
electromagnetic bump in the universe, which behaves both, as something
physical (at least in high energies) and as wave, but has not a mass it
self, sorry almost pure physics here deeper here:

http://physics.about.com/od/lightoptics/f/photon.htm

http://physics.about.com/od/quantumphysics/a/comptoneffect.htm

The thing is that you can imagine (simplifiying high here) something
like flea-bees flying-jumping at a given wavelength at the speed of light
trough this "solar system", and when a big enough group of those get
together they create a quantic bee (photon) powerful enough to "show" the
interaction of the contact with one planet, as this quantic bee reach and
bounce with planets changes its color when this happens, the trouble that
you and any that want to measure light have is that light although would
seem macroscopically to bounce back from the wall in your house or a mirror
at a given angle is much more complex as the particle-particle interactions
are not something easy or direct to measure when it comes to the smaller
scale, not as complicated as biological mollecules) as you might imagine you
are trying to know the orientation of one small constellation, inside a
galaxy, by hiting them with this hordes of bees.

So as quantic it goes, sometimes you get the perfect angle for a great
"conjunction" and a beautiful FRET effect happens, but sometimes, as Tim
says well the bees and the stars are not aligned that night, and you have to
change the telescope for, maybe some gold deposition or other nanocontrast.

Quantum luck

Gabriel OH




> Date: Thu, 18 Aug 2011 21:06:34 -0400
> From: [hidden email]
> Subject: Re: Measuring orientation for FRET
> To: [hidden email]

>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.ed...

[Mark Cannell]

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Quite apart from the fact that anchored proteins are not free to tumble through all possible angles, let's no forget the dielectric constant too...

Mark

On 19/08/2011, at 6:58 AM, Iain Johnson wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> For the orientation factor to cause a FRET experiment to fail completely is
> physically quite improbable.  The orientation factor has possible values
> between zero and 4 (with a value of 0.666 representing the orientationally
> averaged condition).  To get it to go to zero entails the donor and acceptor
> transition dipoles having to be locked at 90 degrees relative to one
> another, a condition that is hard to achieve in any sort of molecular
> assembly.
>
> There are two spectroscopic hallmarks of FRET that can be used to confirm
> (or disprove) its occurrence without manipulating the donor or acceptor
> concentrations.  One is to measure the excitation spectrum of the acceptor
> and verify the presence of an extra peak corresponding to the donor
> absorption maximum wavelength.  The other is to do a time-resolved
> measurement of the acceptor emission and verify the presence of a rise time
> corresponding to population of the acceptor excited state via FRET from the
> donor.  Of the two, the time-resolved measurement is generally more
> practicable in a cellular imaging context.
>
> Iain
>
> Iain Johnson Ph.D
> Iain Johnson Consulting
> Eugene, OR
>
> On Thu, Aug 18, 2011 at 6:06 PM, Tim Feinstein <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Pablo,
>>
>> Our group has some experience with receptor-receptor FRET and designing
>> sensors that report orientation shifts.  Your problem is most likely
>> something that we run across all the time - a FRET interaction that seems
>> like it ought to work but doesn't.  The problem is that distance and
>> orientation are convolved in a manner that (for the most part) is far too
>> mathematically complex to sort out in vivo using fluorescent protein tags.
>> .
>>
>> After anisotropy, as already mentioned, troubleshooting step 1 should be to
>> add a flexible linker between your receptor(s) and the fluorescent tags.  It
>> would also not be a bad idea to use permissive pull-down, e.g. with a
>> reversible cross-linker such as DSP, to verify that your interaction does
>> happen.  It is also possible that your interaction is happening but the
>> signal is swamped by non-interacting background.  In that case I would look
>> into time-resolved FRET using an oscillating LASER-detector combo, which is
>> great for finding signal needles in a background haystack.
>>
>> More often we never figure out what went wrong.  FRET experiments,
>> especially biosensor design, have plenty of false negatives (that is why we
>> feel that negative FRET does not constitute definitive proof against an
>> interaction).  In those cases we just move on and try something else.
>>
>> Good luck,
>>
>>
>> TIm Feinstein
>>
>>
>>
>> On Aug 18, 2011, at 6:02 PM, Pablo German <[hidden email]>
>> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi,
>>>
>>> A question for the FRET experts. According to the literature FRET
>> efficiency
>>> between two fluorophores depends on several parameters including distance
>>> and orientation.
>>>
>>> Tipically, orientation is averaged if we can assume that the molecules
>> can
>>> take all possible orientations. But what happens if that cannot be
>> assumed?
>>> Is there a way to measure orientation between two fluorophores? I am
>> working
>>> with tagged receptors so the measurements will be of receptors either in
>>> live cells or purified.
>>>
>>> Would really appreciate your comments.
>>>
>>> Kind regards,
>>> Pablo
>>>
>>> --
>>> Pablo German
>>>
>>> Plant and Food Research
>>> Private Bag 92169
>>> Auckland Mail Centre
>>> Auckland 1142
>>> New Zealand
>>> DDI: [phone]
>>> Mobile: [phone]
>>

[Tim Feinstein-2]

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

Hi Iain,

Yes, in theory transfer will happen as long as the dipoles are not locked at a ninety degree angle.  In my experience signal-to-noise, bleed-through/cross-talk and background signal constraints usually render energy transfers well above zero more or less undetectable.  Approaches that work reliably with a stirred cuvette in a spectrofluorimeter get messy when working with live cells.  Of course my experience should be salted with the usual caveat that I am statistically certain to be doing at least one thing wrong.  

cheers,


TF

Timothy Feinstein, PhD
Postdoctoral Fellow
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

On Aug 19, 2011, at 1:58 AM, Iain Johnson wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> For the orientation factor to cause a FRET experiment to fail completely is
> physically quite improbable.  The orientation factor has possible values
> between zero and 4 (with a value of 0.666 representing the orientationally
> averaged condition).  To get it to go to zero entails the donor and acceptor
> transition dipoles having to be locked at 90 degrees relative to one
> another, a condition that is hard to achieve in any sort of molecular
> assembly.
>
> There are two spectroscopic hallmarks of FRET that can be used to confirm
> (or disprove) its occurrence without manipulating the donor or acceptor
> concentrations.  One is to measure the excitation spectrum of the acceptor
> and verify the presence of an extra peak corresponding to the donor
> absorption maximum wavelength.  The other is to do a time-resolved
> measurement of the acceptor emission and verify the presence of a rise time
> corresponding to population of the acceptor excited state via FRET from the
> donor.  Of the two, the time-resolved measurement is generally more
> practicable in a cellular imaging context.
>
> Iain
>
> Iain Johnson Ph.D
> Iain Johnson Consulting
> Eugene, OR
>
> On Thu, Aug 18, 2011 at 6:06 PM, Tim Feinstein <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Pablo,
>>
>> Our group has some experience with receptor-receptor FRET and designing
>> sensors that report orientation shifts.  Your problem is most likely
>> something that we run across all the time - a FRET interaction that seems
>> like it ought to work but doesn't.  The problem is that distance and
>> orientation are convolved in a manner that (for the most part) is far too
>> mathematically complex to sort out in vivo using fluorescent protein tags.
>> .
>>
>> After anisotropy, as already mentioned, troubleshooting step 1 should be to
>> add a flexible linker between your receptor(s) and the fluorescent tags.  It
>> would also not be a bad idea to use permissive pull-down, e.g. with a
>> reversible cross-linker such as DSP, to verify that your interaction does
>> happen.  It is also possible that your interaction is happening but the
>> signal is swamped by non-interacting background.  In that case I would look
>> into time-resolved FRET using an oscillating LASER-detector combo, which is
>> great for finding signal needles in a background haystack.
>>
>> More often we never figure out what went wrong.  FRET experiments,
>> especially biosensor design, have plenty of false negatives (that is why we
>> feel that negative FRET does not constitute definitive proof against an
>> interaction).  In those cases we just move on and try something else.
>>
>> Good luck,
>>
>>
>> TIm Feinstein
>>
>>
>>
>> On Aug 18, 2011, at 6:02 PM, Pablo German <[hidden email]>
>> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi,
>>>
>>> A question for the FRET experts. According to the literature FRET
>> efficiency
>>> between two fluorophores depends on several parameters including distance
>>> and orientation.
>>>
>>> Tipically, orientation is averaged if we can assume that the molecules
>> can
>>> take all possible orientations. But what happens if that cannot be
>> assumed?
>>> Is there a way to measure orientation between two fluorophores? I am
>> working
>>> with tagged receptors so the measurements will be of receptors either in
>>> live cells or purified.
>>>
>>> Would really appreciate your comments.
>>>
>>> Kind regards,
>>> Pablo
>>>
>>> --
>>> Pablo German
>>>
>>> Plant and Food Research
>>> Private Bag 92169
>>> Auckland Mail Centre
>>> Auckland 1142
>>> New Zealand
>>> DDI: [phone]
>>> Mobile: [phone]
>>

[Periasamy, Ammasi (ap3t)]

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

Ideal method to measure the orientation of the molecule is time-resolved anisotropy. If you do not have the system you can also use steady state anisotropy. The question also arise.... is it important to measure the orientation of the molecule to estimate the energy transfer efficiency or distance between donor and acceptor molecules. Since the sixth root is involved in the estimation of the distance one can use 2/3 for orientation in the calculation and the error involved is about 10-20% depending on the structure of the molecules, shape, viscosity, biology, etc.
If you would like to read more about the anisotropy you can look at the Prof. Joseph Lakowicz book on Principles of Fluorescence Spectroscopy. Also, there is a good basic explanation about anisotropy connected to biology a book chapter written by Dr. Steven Vogel in my book on FLIM Microscopy in Biology and Medicine, CRC Press 2010.
If you have the set up, yes you can measure the orientation and verify the difference in E%. We are demonstrating or the participants can physically measure the orientation under FRET and non FRET conditions in our upcoming workshop on FRET microscopy.
http://www.kcci.virginia.edu/workshop/workshop2012/index.php 
Hope this helps.

Dr. Ammasi Periasamy
Professor & Center Director
Keck Center for Cellular Imaging (KCCI)
Biology, Gilmer Hall (064), 485 McCormick Rd
University of Virginia
Charlottesville, VA 22904
(Campus Mail - P.O. Box 400328)
Voice: 434-243-7602 (Office); 982-4869 (lab)
Fax:[phone]; Email:[hidden email]
http://www.kcci.virginia.edu/Contact/peri.php 
************************
11th Annual Workshop on FRET Microscopy, March 5-10, 2012
http://www.kcci.virginia.edu/workshop/workshop2012/index.php 
*************************


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Pablo German
Sent: Thursday, August 18, 2011 6:03 PM
To: [hidden email]
Subject: Measuring orientation for FRET

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

Hi,

A question for the FRET experts. According to the literature FRET efficiency
between two fluorophores depends on several parameters including distance
and orientation.

Tipically, orientation is averaged if we can assume that the molecules can
take all possible orientations. But what happens if that cannot be assumed?
Is there a way to measure orientation between two fluorophores? I am working
with tagged receptors so the measurements will be of receptors either in
live cells or purified.

Would really appreciate your comments.

Kind regards,
Pablo

--
Pablo German

Plant and Food Research
Private Bag 92169
Auckland Mail Centre
Auckland 1142
New Zealand
DDI: [phone]
Mobile: [phone]

[Michelle Digman]

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Dear Colleagues,

Announcing the 6th LFD Workshop in Advanced Fluorescence Imaging and Dynamics

October 24–28, 2011

For full details on course content, registration procedures, and much more
information, please see the attached flyer and consult our website at

http://www.lfd.uci.edu/workshop/

Please note that there remain a limited number of laboratory training
section spaces available.  We assure our applicants that the computer
training covers the exact same course material, and we also maintain a
laboratory training wait list.

Features lectures and hands on training:
Introduction to fluorescence correlation spectroscopy (FCS)
The photon counting histogram (PCH)
Raster image correlation spectroscopy (RICS)
The number and brightness method (N&B)
The pair correlation approach (pCF)
FLIM: Biosensors and computational tools
2D and 3D Particle tracking

Thank you for forwarding this announcement to colleagues you think may be
interested in our workshop!

Kind regards,
Michelle

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Michelle Digman, Ph.D.
University of California, Irvine
Laboratory for Fluorescence Dynamics
3204 Natural Sciences II
Office:[phone]
Mobile: [phone]
http://www.lfd.uci.edu/
http://digmanlab.webstarts.com/services.html