*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****
Hi all,
just for completeness, I did use the fixed 1/7 threshold for the
BIOP-FRC and one block only within squirrel. So I expected (near)
identical results when comparing the same image pair.
I can easily imagine that image pairs taken at different areas of the
same sample lead to different results even with the same algorithm and
we indeed found that when looking at beads. Different structures may hit
the edge, or whatever. That's not my point.
But the very same image pair put into different tools with the same
parameters (1 block, threshold 1/7), I had expected more similar
results. When both tools do use the same engine, as you say, Ricardo,
Kyle probably has a point with different pretreatments. I don't read
Java, so I can't check, but it would explain the outcome.
Another thing I still have to wrap my brain around is the fact that for
a 'non-confocal' (open pinhole) image pair of 26 nm beads excited with
645 nm we are getting an FRC resolution of 198/200 nm (BIOP/squirrel,
respectively) while according to 0.5*lambda/NA and lambda=645 nm, NA=1.4
the resolution limit is supposed to be at 230 nm. So it seems we are
getting a resolution that is better than theoretically possible. Not
usually a good sign.
Steffen
Am 31.01.2018 um 13:09 schrieb Ricardo Henriques:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Steffen,
>
> Both BIOP and SQUIRREL use the same engine for FRC (Alex Herbert's
> original code).
> SQUIRREL does default to the 1/7 threshold while BIOP allows you to both
> select different ones + plus gives detailed information on the frequency
> profile of the correlation. I think the differences are more about the fact
> that SQUIRREL breaks the image into blocks instead of using a pre-defined
> ROI.
>
>> So i am wondering why that is. And what it means for practical purposes,
> i.e. how reliable is an FRC measurement on real life data.
>
> I would always suggest taking FRC measurements with a grain of salt. There are
> many ways the readout can be biased (e.g.: constant patterning/artefacts
> across both images). Unfortunately I don't believe we have an unbiased way
> to calculate resolution (yet), at the end of the day most of us resort to
> looking at line profiles across 2 structures + FRC and use these as a rough
> suggestion for resolution.
>
> I would actually welcome the feedback of the community for other
> resolution measurement
> alternatives.
>
> All the best,
> -Ricardo
>
>
--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging
Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany
http://www.bioimaging.bmc.med.uni-muenchen.de
|
