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Hi,
with some software you see pixels as squares (imageJ) and with others not (Imaris). I rather like to see them as this is a good warning that the pixel-resolution limit is reached. I had students interpreting interesting triangular patterns created by the software by linking  pixel points, they just did not notice that they zoomed in too much.  This was with a scanning probe microscope. Square pixel also make some good background image for an overlay with higher resolution single molecule tracking data, they give a better idea of the noise in the image. But i think for displaying a most likely version of the object under the microscope i think we we should forget about square pixels. Some flexibility in the image analysis software would be good.

best wishes

Andreas



 

 

 

-----Original Message-----
From: O'Malley, Donald <[log in to unmask]>
To: CONFOCALMICROSCOPY <[log in to unmask]>
Sent: Tue, 17 Apr 2012 16:12
Subject: I FOUND the little SQUARES


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Hi Gang,











Apologies for prolonging this thread (and I do agree with both parties, 





depending on whose email I read last).  I thus thought I might relate the 





finding of little squares in my MRC600 confocal images.  Because my analytical 





abilities were largely limited to what the BioRad confocal software (SOM) could 





do at that time (1994), I could not easily display the individual pixel values: 





all the images we published of nuclear calcium transients and neuronal activity 





in zebrafish (from 1994 to 1996) had a smooth aesthetically pleasing appearance 





(more accurate or less accurate depends I think on the semantic position one 





takes).   I had wanted to see the individual pixels by zooming up (enabled by 





SOM481B), but they were all smoothed out to my annoyance.  Somehow I discovered 





that if one slightly tilted the images, then that knocked out the SOM smoothing 





algorithm and I could now see the individual pixel values.  This enabled me to 





see what was happening on a pixel to pixel basis, in both space and time, in my 





line scans of calcium dynamics.  For anyone wishing to see how these two 





displays of the data vary in a side-by-side, real world sense, you might take a 





look at Fig. 3B and Fig. 3C of my Methods article on Calcium Imaging at the 





Limits of Resolution, O'Malley, et al., 2003, at:





http://www.digital-maze.com/id3.html





[you can actually see the slight tilt of the image in Fig. 3B, which I should 





have adjusted to perfectly register it to the space and time axes, but this was 





the best I could do at that time.].











I get the point that the sharp boundaries we see between adjacent pixels are 





physically impossible (in this case, for sure), but seeing the actual recorded 





data, presented as recorded, was useful at least to me.  Of course, the colors 





in the images are a figment of our artistic imaginations!











Enjoyed all the discussion,





Don











Don O'Malley





Assoc. Professor





Dept. Biology





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