# Unguided example of widefield particle extinction image development and analysis using Extinction Suite v4.1.0+

This example dataset provides two possible analysis paths, and accordingly two zipped folders, (1) containing the raw experimentally acquired data, and (2) the developed extinction images from (1). The sample used in the experiment contained nominally spherical 30nm and 50nm diameter gold nanospheres in oil. Weak dielectric debris might be present in very small amounts.
1. Use RawData.zip in order to test knowledge of both (a) extinction image development and (b) particle analysis in extinction images.
• It may be relevant to note the unzipped data size is 1.06 GB.
• For this specific dataset, developing extinction images will temporarily occupy anywhere from an additional 144MB to an additional 576MB and will result in a final additional data storage cost of 125.8 MB.
2. Use Data.zip in order to test knowledge of only (b) particle analysis in extinction images.
• The unzipped size is 113.2 MB.
• For this specific dataset, analyzing the data will result in a final additional data storage cost of ~23 MB.
Unzip (1) or (2) as needed.
• "RawData.zip" contains 1 folder: "ExpData", which should be treated as the base folder for this analysis.
• "ExpData" contains 1 folder: "Raw".
• "Raw" contains three signal image stacks "bright_450.tif", "bright_550.tif", "bright _600.tif" and three associated background (dark sensor) image stacks in the format "BG_xxx.tif".
• "Data.zip" contains 1 folder: "Data"
• "Data" contains 1 folder: "Processed Images".
• "ProcessedImages" contains 18 developed extinction images: "Extinction_xxx_yyy.tif", where xxx indicates the center wavelength of the color filter ranges with $$xxx\in\{450,\,550,\,600\}\,$$nm and yyy indicates the polariser angle with $$yyy\in\{000,\,030,\,060,\,090,\,120,\,150\}^\circ$$.
The data was taken in the synchronously triggered stage-camera format using a single recording over all polariser angles, but separate recordings for each color channel. Hence, there are three saved signal files, and three saved background (dark sensor) files, one of each per channel. The shifted reference method was chosen for this experiment. The recording settings are
• 128 images per position (signal and reference).
• (2, 2, 8) complete shift repetitions (P1->P2 + P2->P1) associated with the (450, 550, 600) nm color channels, respectively. (This information is needed for pathway 1).
•  (1024, 1024, 4096) total brightfield acquisitions per polariser angle in the three respective color channels. (This information is needed for pathway 2)
• Note for reference that the camera was set to average 128 acquisitions into a single saved frame, hence this means in practice (8, 8, 32) images were saved per polariser angle for the three the respective color channels.
• Note for reference, each background file contains as many images as that for a single polariser angle in the case of each color channel (i.e. 8, 8, or 32 images), since the dark sensor offset depends only on the exposure time (which is different for the different color channels), but is independent of polariser angle.
• The NA of the microscope objective used was 0.95NA.
• The total magnification was 60x.
• Measurements were taken for linear polarisation along angles 0 degrees to 150 degrees in 30 degree steps.
• The number of photoelectrons detected be assumed to be 30000.

### 1. Run ExtinctionSuite v4.1.0+

• Plugins $$\rightarrow$$ ExtinctionSuite v4
• Make sure no other images are open in ImageJ/Fiji, and then close the welcome prompt.

### 2. Mode selection

• Choose Polarized.
• Choose Run Extinction Suite.

### 3. Choose modules to run

• This example provides the raw data so that the user can test their knowledge of the Extinction Image Development module.
• If the user/examiner wishes to test this, make sure the appropriate file has been unzipped (see above).
• If the user/examiner wishes to test this, make sure to tick the Extinction Image Development in the module selection dialogue.
• Tick the Particle Analysis module in the module selection dialogue.
• If user/examiner will only test knowledge of the Particle Analysis module, make sure the appropriate file has been unzipped (see above).

### 3. Perform analysis

• Perform the analysis using the above settings and check against the known results (request access once you have attempted the example by yourself).

Created by Lukas Payne, 24/01/2021, edited 27/01/2021 WL.