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Using WormSizer to measure Worm Length or Volume

-For Windows, see Zuzana’s protocol for WormSizer. For Mac, follow these instructions.

-Install Leica Application Suite V3.0 from the app store.

-Download Fiji (ImageJ 1.48t) from http://fiji.sc/Downloads

-Download and install WormSizer Plugin by following README.md instructions found at  https://github.com/bradtmoore/wormsizer

-Place worms on unseeded NGM plate for imaging

* wash 20-50 worms 2-3x with M9 in a 1.7ml Eppendorf spinning in benchtop centrifuge

* place 20-50 individuals on unseeded NGM plate

* spread out individuals for better images

-Acquire images using Leica M80 camera and LAS v3.0 for Mac (since Fiji cannot handle acquisition)

*Note which lens you are using: 0.5x or 1.25x. (this protocol uses the 1.25X, but a 0.5X protocol can be developed if necessary)

*File>Preferences, click the “General” tab and add Calibration Settings with the table below

Name

Magnification

Length

Unit

@0.75Xlens1.25

0.463

1.410

mm

@1.0Xlens1.25

0.617

1.058

mm

@1.25Xlens1.25

0.771

0.847

mm

@1.6Xlens1.25

0.987

0.661

mm

@2.0Xlens1.25

1.233

0.529

mm

@2.5Xlens1.25

1.542

0.423

mm

@3.2Xlens1.25

1.973

0.331

mm

@4.0Xlens1.25

2.467

0.265

mm

@5.0Xlens1.25

3.083

0.212

mm

@6.0Xlens1.25

3.700

0.176

mm

*Select the Camera button near the top left of window

*On the “Live Video” section, select the calibration related to your zoom magnification

*On the “Output” section, select Image Size 2048x1536 and Format JPEG

*On the “Output” section, Change “Filename” for each condition, genotype, etc.

*The image #s should automatically increase

*At the bottom left, choose a folder to save all your images. (Note: changing folders between image acquisitions may crash the software)

*Leave all other options on “Auto”  (Note: if you experience image acquisitions in excess of 10 seconds, try unchecking the “Auto” button on the “Live Video” section; average time: 6 seconds)

*Click the “Camera” button at bottom right of the application

**Alternatively, you can use the SD card with an image acquisition time of 2 seconds.

-Batch Convert images to 8-bit grayscale (since Leica cannot acquire 8bitGray, we are told)

*Locate your images and create a new directory for 8-bit grayscale images

*Open Fiji

*Process>Batch>Macro

*Choose your image folder for “Input” and your new, empty directory for “Output”

*”Output Format” JPEG

*In the white box type: run(“8-bit”);

*Click “Process”

*A harmless openImage() error will arise for each .xml file related to the image files.

-Run WormSizer

*Plugins -> WormSizer -> WormSizer GUI

*Play button, then select desired folder of images for both input and output folder options

*If you have separate folders for each condition, check the box “Run on Subdirectories”

*Input microns per pixel (according to the table below)

Lens

Zoom

Meters per Pixel

Microns/pixel

1.25X

0.75

6.84E-06

6.84

1

5.12E-06

5.12

1.25

4.10E-06

4.10

1.6

3.20E-06

3.20

2

2.56E-06

2.56

2.5

2.05E-06

2.05

3.2

1.60E-06

1.60

4

1.28E-06

1.28

5

1.02E-06

1.02

6

8.53E-07

0.85

0.5x

0.75

1.71E-05

17.11

1

1.28E-05

12.80

1.25

1.03E-05

10.26

1.6

8.00E-06

8.00

2

6.40E-06

6.40

2.5

5.12E-06

5.12

3.2

4.00E-06

4.00

4

3.20E-06

3.20

5

2.56E-06

2.56

6

2.13E-06

2.13

*Hit >> and wait (if >1minute per image, and keeps “thinning”, it probably hit a problem)

*Magnifying glass button, then select desired .xml file

*press P(ass) or F(ail) as appropriate

-Find Fiji’s results.csv and open in excel

*select all and filter. Pass = TRUE

*copy PASSed data to second sheet/workbook

*calculate COUNT, AVERAGE, and STDEV, or process in R

*length:meanWidth may be useful as a kind of “BMI” for worms

*Output volume is in picoliters, length & width are in microns

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