Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Yang and Musser (Methods 39: 316-328) figure 4A 
vs 4B tells the story: widefield epi-illumination 
fluorescence has a high background due to glare, 
scattered light, etc. Placing matched pinholes on 
the illumination and detection sides reduces 
glare, etc., even more than it decreases 
"signal". At the specimen plane, the smallest 
illuminated(and detected) area they show is 
approximately 5 um diameter (for a 200 um 
diameter apertures). Using a back-illuminated EMCCD (~90% QE) helps.

The "~15 nm resolution for a stationary particle" 
is the localization precision based on a Gaussian 
fit to the shape of each particle. This is NOT 
the size of the particle. For a 100x/1.45 NA oil 
immersion lens, resolution is either Rayliegh 
limited or pixel size limited, depending on intermediate optics used.

Several chapters in the Pawley3 book point out 
that shrinking the epi-field aperture has effects 
on the PSF that could (would) affect the shape of 
the PSF in ways that could be bad for 
deconvolution. It would be interesting to see
1. the PSF shape under the Yang and Musser paired 
pinhole condition (at different locations in the area viewed), and
2. whether any of the current deconvolution 
algorithms improve the spatial (lateral) resolution
3. whether the deconvolution improves the 
localization precision (would need to be able to 
move the stage in nm's to test this).


George

At 01:11 AM 9/22/2006, you wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>I was happy to read this article in the latest 
>issue of the journal "Methods" :
>
>Visualizing single molecules interacting with 
>nuclear pore complexes by narrow-field epifluorescence microscopy
>Weidong Yanga and Siegfried M. Musse
>doi:10.1016/j.ymeth.2006.06.002
>
>I think I remember having read on this list that 
>using an open pinhole with a confocal microscope 
>will lead to a result worse than with a 
>conventionnal epifluorescence setup. In this 
>paper, the authors claim that using big pinholes 
>( some kind of very small field stop, ~400 µm) 
>in the excitation path increases the S/N 
>compared to regular epifluorescence. The S/N is 
>further incereased by placing a 300 µm pinhole 
>in the emission path. I wonder what are the 
>theoretical basis of these observations ?
>
>Christophe Leterrier
>
>Ionic Channels and Neuronal Polarity Team :: Bénédicte Dargent
>INSERM UMR 641
>Neurobiology of Ionic Channels Lab :: Mike Seagar
>Institut Jean Roche :: Université de la Méditerranée
>51, boulevard Pierre Dramard :: 13919 Marseille Cedex 20
>Telephone 04 91 69 89 30 :: Fax 04 91 09 05 06
>Website : http://ifrjr.nord.univ-mrs.fr
>Email : [log in to unmask]





George McNamara, Ph.D.
Glendale, CA
818-547-6909
[log in to unmask]