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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Eric,

Without more information, this sounds like an artifact problem. On 
what basis have you determined that the object of interest is not 
moving? There is always the trivial  issue of estimating the exact 
position of an object related to noise in the measurement but I think 
you are referring to something that's giving larger apparent 
displacements?

Sub-resolution fluorescent lipid vesicles electrostatically absorbed 
onto slides/coverslips, when imaged at a high frame rate, can appear 
to move about owing to thermal or Brownian motion but on average stay 
at one x-y position. Objects can move owing to displacement of fluid 
because the medium is drying out and there is actual hydrostatic 
induced displacement of the object related to asymmetry in fluid 
movement. Mechanical vibrations, and other artifacts can cause 
apparent positional displacements.

Can you be more specific about what you mean by sampling frequency 
(confocal dwell time? or is it W.F. frame rate?), live cell or fixed 
cell imaging, and what distances are you referring to relative to 
optical resolution?

Mario

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>I am curious how people have dealt with the issue of variance in 
>positional measurement ( using the measuring tool in ImageJ, for 
>example) and its contribution to particle speed measurements.  The 
>positional variance produces paradoxical "speed" for non moving 
>particles - if particle displacement is determined between 
>successive images. This effect directly increases with sampling 
>frequency.
>
>I have looked at a number of papers on cell migration and not found 
>a correction or mention of this effect - which can be large for high 
>sampling frequencies.
>One idea would be to define a minimum detectable displacement based 
>on the standard deviation of the measurement.  Every value less than 
>that minimum would be set to zero???
>
>Thanks,
>Eric
>
>
>Eric C. Olson, PhD
>Assistant Professor
>Department of Neuroscience and Physiology
>SUNY Upstate Medical
>3295 Weiskotten Hall
>766 Irving St.
>Syracuse, NY 13210
>
>office: 315-464-7776
>lab    : 315-464-8157


-- 
________________________________________________________________________________
Mario M. Moronne, Ph.D.
ph (510) 528-2400
Fax (510) 528-8076
cell (510) 367-8497

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