EDOF

EDOF imaging and deconvolution

EDOFSpheroid.mov

Contents

• Overview
• Beads in a gel
• Spheroid measurement
• Deconvolution example

Overview

Here we include MATLAB codes and data for the EDOF quantitative analysis performed on beads suspeneded in a gel. In addition, we provide Zenodo links to raw measurements of a spheroid with the standard PSF (unmodified objective lens) and an EDOF PSF. Finally, we provide a demo example of applying a few iterations of Lucy-Richardson deconvolution to improve the resolution of the EDOF measurements.

Beads in a gel

The folder Beads should contain two data subfolders: EDOF and ST corresponding to z-stacks (saved as individual tif images) of the same FOV one acquired with the standard PSF (ST) and the other with an EDOF PSF. These zstacks are inside the compressed folder EDOF_Beads_low_density.zip which can be downloaded from Zenodo:

https://zenodo.org/records/10928122/files/EDOF_Beads_low_density.zip?download=1

Thunderstorm was run on each zstack to quickly find the beads positions. These are saved in excel files ThunStormMAX*.csv. In addition Thunderstorm proximity filter was applied to avoid beads that are too close. Any group of proximal beads is registered in excel files *close_locs.csv, specifying beads that will be excluded from analysis. To reproduce the analysis from the paper, you need to run the scripts Analyze_multiple_beads.m and Display_DOF_Multiple_beads.m:

• Analyze_multiple_beads.m - This code crops the useful beads, and performs 2D gaussian fit per frame around the focus of each bead (takes a long time to run). The generated results are Crops_*.mat (fast) and fitted*.mat (slow), where * is either ST or EDOF.
• Display_DOF_Multiple_beads.m - This code displays the results from fitted*.mat and performs 1D gaussian fitting to the recovered PSF widths (also quite slow). The results are saved as multi_bead_fits_results.mat.

Expected results of running these scripts are included inside Beads. We also performed a similar analysis for samples with a higher density of beads. The respective code and data are inside the compressed folder EDOF_Beads_high_density.zip which can be downloaded from Zenodo:

https://zenodo.org/records/10928122/files/EDOF_Beads_high_density.zip?download=1

Spheroid measurement

The raw data of a spheroid measured with both the standard PSF (unmodified objective lens) and the EDOF PSF are inside the compressed folder EDOF_Spheroid.zip which can be downloaded from Zenodo:

https://zenodo.org/records/10928122/files/EDOF_Spheroid.zip?download=1

Deconvolution example

The folder Deconvolution includes a demo of Lucy-Richardson deconvolution applied to the measured spheroid with the Standard/EDOF PSF to improve the lateral resolution. Note that to run this demo you are required to download the EDOF data from the url in the previous section and locate the desired frame to be deconvolved inside the folder input. A single example frame with the Standard/EDOF PSF are provided in the input folder. The demo script deconvolution_demo.m runs both deconvolution functions deconvolve_ST.m and deconvolve_EDOF.m to demonstrate their use. The respective expected output *_deconvolved_image.tif where * is either ST or EDOF, are also provided in the folder output.