FIJI (ImageJ): Extended Depth of Field [Stack Focusing]

[@hamzasaoud7236]

Thanks 👍🏻 😊 This is the best channel to learn Great amazing efforts👏🏻👏🏻👏🏻

[@johanna.m.dela-cruz]

You’re so kind. 😊

[@aisakataiga2522]

Hey, what is the second image from? The image stack of fluorescence? :)

[@johanna.m.dela-cruz]

Hi @Aisaka Taiga. The image stack is from a plant stem stained with DsRed. It was acquired at 5x magnification by one of our clients at the Imaging Facility. (Credit: A. Johnson)

[@aisakataiga2522]

@@johanna.m.dela-cruz Thank you so much :)

[@yashenggao6980]

Thank you so much, Johanna.

[@johanna.m.dela-cruz]

My pleasure, @Yasheng Gao.

[@tritoniadiomedea]

I can not tell if the source images were collected at arbitrary intervals, or if you used a stepper motor to get equal intervals. Are equal intervals necessary, or is the software able to just use the most focused pixels without knowledge of their z-position?

[@johanna.m.dela-cruz]

Hi @tritoniadiomedea. The stacks used in this video were acquired as transmitted light images in a confocal microscope, so step size was constant. However, if images were acquired at various intervals (manual focus adjustment), EDF would still be useful.

[@PumpDisUp]

Hi Johanna, thanks so much for making this. Quick question, is your computer beefy cause everytime I attempt to run the plugin I don't get any progress on the bar. Also using a .czi file.

[@johanna.m.dela-cruz]

Hi @PumpDisUp. How big is your file? Have you tried allocating more memory to your FIJI/ImageJ?

[@beardog6569]

Thank you Johanna

[@johanna.m.dela-cruz]

You're very welcome, @BearDog. :)

[@coolcha]

Is there a way to perform exposure stacking in FIJI?

[@johanna.m.dela-cruz]

Hi there. I’m not sure I understand what you mean by exposure stacking. Are you working with a z stack or a sequence of images with variable exposure times? Have you tried contrast stretching by normalizing or equalizing?

[@coolcha]

@@johanna.m.dela-cruz thanks for responding. Exposure stacking is a technique in photography where you can stack multiple images with shorter exposure times to get a single stacked image with better signal to noise compared to a single long exposure image. Its especially useful for astrophotography, but we have a stain which is very faint for a sample. Was wondering if we can use Fiji to do something like that. Check KZbin for exposure stacking.

[@johanna.m.dela-cruz]

@@coolcha I think EDF can be used in Expert Mode. There are a number of sharpness estimators and other parameters that you can play around with.

[@coolcha]

Sorry I dont know what is EDF. Please could you explain.

[@johanna.m.dela-cruz]

@@coolcha EDF is Extended Depth of Field. You will want to select the one with Expert Mode. I don't know how your images look like, but you can try first complex wavelets for sharpness estimation. Check both items under consistency....you could also use the other default parameters.

[@buuveebayarkhangaibuuveeba7891]

Hi, I really want to try plug-in but link to the plug-in is not working. Is the source removed?

[@johanna.m.dela-cruz]

Hi Buuvee. If you just right click on the Extended_Depth_Field.jar file, you can 'save link as' to download it directly to the plugins folder of ImageJ. Check the plugin on this site: bigwww.epfl.ch/demo/edf/. You will have to restart ImageJ after you have downloaded the plugin. Let me know if this works.