Setting "placement.uniform.num_spots" for different submasks

Bayesian analysis of blinking and bleaching

Setting "placement.uniform.num_spots" for different submasks

Postby hooying » Sat Mar 29, 2014 1:08 am

Hi,

When I run the 3B calculation on a 128-by-128-pixel image with 10-by-10-pixel submasks with a 5-pixel overlap, the reconstructed image after I piece together all submasks tends to lack contrast, or has more or less the same brightness for all features across the image. I wonder if by modifying the initial guess of the number of spots for each submask based on its averaged intensity level we can improve the overall image contrast. If so, how can we execute it since concurrent executions call a single file multispot5.cfg for initial conditions?

Thanks,
Ying
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Re: Setting "placement.uniform.num_spots" for different subm

Postby edrosten » Wed Apr 02, 2014 8:48 pm

hooying wrote:When I run the 3B calculation on a 128-by-128-pixel image with 10-by-10-pixel submasks with a 5-pixel overlap,


Just a note: 10x10 is pretty small for sub masks. I usually use 15x15 if possible. It's also quite a high level of overlap, you can probably get away with 3 pixels, if you want it to run faster.

the reconstructed image after I piece together all submasks tends to lack contrast, or has more or less the same brightness for all features across the image.


I'm not 100% sure I follow. In this algorithm, the intensity is a probability density of a fluorophore being present, and is not directly related to the intensity. 3B can give bad results if there is insufficient blinking in the data or there is no real underlying structure. Can you provide more details of the imaging conditions and the structure that you're looking at?

I wonder if by modifying the initial guess of the number of spots for each submask based on its averaged intensity level we can improve the overall image contrast.


The initial number of spots shouldn't affect the final contrast: if the initial number of spots is far off, the algorithm will take a while to converge to the correct number of spots and then run from there.

If so, how can we execute it since concurrent executions call a single file multispot5.cfg for initial conditions?


The program also accepts commandline arguments. The commandline arguments will override any of the variables in the config file. The syntax is --VAR VALUE, so you could add:

Code: Select all
--placement.uniform.num_spots 15


to the commandline to specify a different value.
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Re: Setting "placement.uniform.num_spots" for different subm

Postby hooying » Sun Apr 06, 2014 4:36 am

Thank you for the clarification.
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Re: Setting "placement.uniform.num_spots" for different subm

Postby desheikh » Sat Feb 14, 2015 5:19 am

Can you provide more details of the imaging conditions and the structure that you're looking at?
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Re: Setting "placement.uniform.num_spots" for different subm

Postby Brunswick » Mon Nov 27, 2017 8:59 am

Makes a lot of sense now. Thanks for the info edro.
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