## Setting "placement.uniform.num_spots" for different submasks

Bayesian analysis of blinking and bleaching

### Setting "placement.uniform.num_spots" for different submasks

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
hooying

Posts: 6
Joined: Tue May 14, 2013 10:03 pm

### Re: Setting "placement.uniform.num_spots" for different subm

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.
edrosten

Posts: 42
Joined: Wed Jul 18, 2012 2:54 pm

### Re: Setting "placement.uniform.num_spots" for different subm

Thank you for the clarification.
hooying

Posts: 6
Joined: Tue May 14, 2013 10:03 pm

### Re: Setting "placement.uniform.num_spots" for different subm

Can you provide more details of the imaging conditions and the structure that you're looking at?
desheikh

Posts: 1
Joined: Sat Feb 14, 2015 4:54 am

### Re: Setting "placement.uniform.num_spots" for different subm

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

Posts: 1
Joined: Sat Nov 18, 2017 9:15 am