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Hi Michelle,

Thanks! Jay Unruh's http://research.stowers.org/imagejplugins/   PCH 
page  ( http://research.stowers.org/imagejplugins/pch_plugins.html ) 
indicates the PCH analysis includes N&B:

        A quick glance at our page on PCH
        <http://research.stowers-institute.org/microscopy/external/Technology/PCH/index.html>
        reveals that there are three ways to obtain molecular brightness
        information: 1.) from the amplitude of the FCS curve itself, 2.)
        from "moment analysis" also known as N&B analysis, and 3.) from
        PCH analysis. So which method should be used to obtain the
        molecular brightness? The answer depends on the application. If
        the data has been obtained from a single point measurement, the
        answer is almost always to determine the brightness from a fit
        of the FCS curve. I recommend this because this method allows
        for visual inspection of the quality of the fit given the
        expected diffusion profile of the sample under study. In
        addition, this allows for rejection or detrending of curves
        which show bleaching or slow dynamics like cell motion. Of
        course, one can always divide the curve up into many small
        sections and perform N&B analysis, but it can be difficult to
        decide how large to make these sections. These issues are also
        prevalent in PCH analysis. Once bleaching is involved, the PCH
        curve becomes a convolution of all possible average
        concentration values present throughout the aquisition. In
        addition, both N&B and PCH contain complex corrections for
        detector afterpulsing and triplet blinking that are typically
        not present in fitting software (including mine).

        So when should N&B analysis be used? There are several
        circumstances under which FCS cannot be used. These include
        circumstances under which samples are not obtained in a
        continuous stream (e.g. images). Here the diffusion time is much
        smaller than the time between frames, but much larger than the
        pixel dwell time. Here FCS will not work, but N&B will. Note
        that it may be useful to try RICS analysis for this circumstance
        as well. In addition, the simplicity of the N&B analysis makes
        it useful for simplistic analysis of many samples when automated
        fitting is not trivial.

        Finally, when should PCH analysis be used? The major advantage
        and disadvantage of the PCH method is its ability to represent
        complexity in the sample. One must be careful not to "overfit"
        the PCH curve to represent more complexity than is justified in
        a noisy curve. Nevertheless, with high quality data, one can
        obtain information about multiple species in a sample. In
        addition, it can be much more straightforward to assess the
        error limits on parameters from PCH data as opposed to N&B
        analysis (typically requires the use of factorial cumulants). In
        addition, multispecies N&B models for multicolor analysis can
        become quite complex while 2D PCH retains a simplicity that
        allows for straightforward model testing.


        http://research.stowers-institute.org/microscopy/external/Technology/PCH/index.html#NBBright



I will check it out.

Sincerely,

George

On 7/15/2011 3:01 PM, [log in to unmask] wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear George,
> I don't know if anyone has an image J plugin for the N&B analysis. However
> Jay Unruh from Stowers has a collection of image J plugins for FCS,
> CCRICS, RICS and PCH. Here is a link to those plugins:
> http://research.stowers.org/imagejplugins/zipped_plugins.html
>
> The SIMFCS software can be used for N&B analysis. If you are interested
> you can download the software from: http://www.lfd.uci.edu/globals/
>
> We can send you tutorials on how to use the program for the N&B analysis.
> Kind regards,
> Michelle
>
> *******************************
> Michelle Digman, Ph.D.
> University of California, Irvine
> Laboratory for Fluorescence Dynamics
> 3204 Natural Sciences II
> (949) 824-2992
> http://www.lfd.uci.edu/
>
>
>
>
>    
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Confocal listserv,
>>
>> If anyone is aware of an ImageJ plugin for Number and Molecular
>> brightness (N&B) Method, could you please post the link here?
>>
>> An explanation of N&B can be found in
>> http://www.lfd.uci.edu/workshop/2010/files/LFDWorkshop2010-Lecture05.pdf
>> as well as various papers by Digman, Gratton et al.  (page 33 of the pdf
>> file).
>>
>> If there is one, could someone please write one in time for me to use
>> for a CW-STED demo Aug 22-Sept 6?
>>
>> If you are feeling ambitious, Cross N&B would be great to have, too. The
>> pdf is not explicit, but usually 100 images are acquired. Figure on
>> calculating from a stack in ImageJ (two stacks for cross). I'll be happy
>> to send you nice data to use in your publication announcing your plugin.
>> You are welcome to visit Miami during the demo to acquire however you
>> think is best - however, this is the rainy season (and I have no travel
>> budget). I anticipate using the demo instrument's HyD detector in photon
>> counting mode for most of this.
>>
>>
>> thanks,
>>
>> George
>>
>>
>> --
>>
>>
>> George McNamara, PhD
>> Analytical Imaging Core Facility
>> University of Miami
>>
>>
>>      
>    


-- 


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami