AxoMetric

Welcome to AxoMetric: An automatic interval-based quantification platform optimized for axon, mean fluorescence intensity, and cell body quantification. The purpose of this software is to create a tool for researchers to analyze tissue staining within distance intervals in an unbiased, consistent, and accessible manner. This platform will allow users to normalize tissue staining to tissue width based on cellular nuclear staining. This software was developed based on mouse optic and sciatic nerve longitudinal sections, however this may be applicable to other species / types of sections. The platform also includes a tool to measure the number of retinal ganglion cells (RGCs) in retina flatmount images of Rbpms-stained retinas.

The platform is divided into three parts: axon quantification, mean fluorescence intensity (MFI) quantification, and retinal ganglion cell (Rbpms) quantification.

Axon Quantification

Total Number of Axons - Measure the total number of axons in an image at separate intervals from the injury site.

*Developed based on 20x images of mouse optic nerves stained with cholera toxin subunit B (CTB).

Normalized Number of Axons - Measure the total number of axons in an image at separate intervals from the injury site, and then normalize the total number of axons by the nerve width (calculated from nuclear stained image pixel height in inches) at each of the 4 intervals, represented by axons / um of nerve width.

} *Developed based on 20x images of mouse optic nerves stained with cholera toxin subunit B (CTB) & Hoechst.

Multiple File Quantification (Total #) - For multiple image files simultaneously, measure the total number of axons in an image at separate intervals from the injury site.

MFI Quantification

Total MFI - Measure the mean fluorescence intensity (MFI) of an image at separate intervals from the injury site.

*Developed based on 20x images of mouse sciatic nerves stained with Stathmin-2.

Normalized MFI - Measure the MFI in an image at separate intervals from the injury site, and then normalize the MFI by the nerve width (calculated from nuclear stained image and pixel height in inches) at each of the intervals, represented by MFI / um of nerve width.

RGC Quantification

Single File Quantification - Measure the number of Rbpms+ cells in a field-of-view of one image file.

Multiple File Quantification - For multiple image files simultaneously, measure the number of Rbpms+ cells in a field-of-view.

*Developed based on 20x images of mouse retinas stained with Rbpms.

Developer

Matthew Finneran, Giger Laboratory

Neuroscience Graduate Program, University of Michigan Medical School

Department of Cellular and Developmental Biology

University of Michigan Ann Arbor, MI

2025

Acknowledgements

*Figures created with Biorender.com

*Thank you to Craig Johnson, Bioinformatician of the Cellular and Developmental Biology Department at the University of Michigan, for his help with AxoMetric.

*Thank you to Dr. Larry Benowitz for his feedback and support in the development of this tool.

*Thank you to our support from these funding sources: National Institutes of Health (NIH) Ruth L. Kirschstein National Research Service Award (1F31EY036280-01), the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, the Stein Innovation Award for Research to Prevent Blindness, and the Department of Health and Human Services Advanced Research Projects Agency for Health (ARPA-H).

Total Axon Quantification

Image Pre-Processing Instructions

1. Open FIJI Software, select File --> Open --> select image file.

2. To straighten & crop nerves from injury site to the distal nerve, do the following:

a. Select Edit --> Options --> Line Width, set # equal to nerve width (~2500)

b. Right-click line tile --> Select segmented line

c. Click at nerve crush site, and then click in the middle of the nerve until at distal nerve end, then right-click.

d. Search for the straighten plugin and then run.

3. Select Image --> Type --> 8-bit

4. Select Image --> Adjust --> Brightness_Contrast, adjust signal the same for all images in experiment similar to the example image on this page.

5. Select Image --> Scale --> Should be set to 50% (0.5) --> Select OK

6. Select File --> Save as --> Tiff

*Please note that high-quality images are needed for proper quantification (please see example below).

Image Analysis

1. Upload tiff image file and wait for upload to complete.

2. Enter the number of intervals you want to quantify with.

3. Click the submit button to run the image analysis. Please allow a few minutes for image processing.

4. When finished, you may click the reset button to submit another image for analysis.

*Files may generally take ~30 seconds to process.

Identified Objects with Analysis Intervals

Number of Axons

Normalized Axon Quantification

Image Pre-Processing Instructions

1. Open FIJI Software, select File --> Open --> select image file.

2. To straighten & crop nerves from injury site to the distal nerve, do the following:

a. Select Edit --> Options --> Line Width, set # equal to nerve width (~2500)

b. Right-click line tile --> Select segmented line

c. Click at nerve crush site, and then click in the middle of the nerve until at distal nerve end, then right-click.

d. Search for the straighten plugin and then run.

3. Select Image --> Type --> 8-bit

4. Select Image --> Adjust --> Brightness_Contrast, adjust signal the same for all images in experiment similar to the example image on this page.

5. Select Image --> Scale --> Should be set to 50% (0.5) --> Select OK

6. Select File --> Save as --> Tiff

7. Record the pixel height in inches: Select Image --> Properties

8. Repeat steps 1-5 with an image file of nuclear staining (i.e., DAPI).

*Please note no nuclear staining can be on the edges of the cropped image or the code may fail to acquire the upper/lower limit of the nerve width.

*Please note that high-quality images are needed for proper quantification (please see example below).

CTB Example

Hoechst Example

Image Analysis

1. Upload tiff image file and wait for upload to complete.

2. Upload nuclear stained tiff image file and wait for upload to complete.

3. Enter the number of intervals you want to quantify with.

4. Input the height per pixel in inches for the image.

5. Click the submit button to run the image analysis. Please allow a few minutes for image processing.

6. When finished, you may click the reset button to submit another image for analysis.

*Files may generally need ~2 minutes of processing time.

Number of Axons

Axon Quantification for Multiple Files

Image Pre-Processing Instructions (to do for each file)

1. Open FIJI Software, select File --> Open --> select image file.

2. To straighten & crop nerves from injury site to the distal nerve, do the following:

a. Select Edit --> Options --> Line Width, set # equal to nerve width (~2500)

b. Right-click line tile --> Select segmented line

c. Click at nerve crush site, and then click in the middle of the nerve until at distal nerve end, then right-click.

d. Search for the straighten plugin and then run.

3. Select Image --> Type --> 8-bit

4. Select Image --> Adjust --> Brightness_Contrast, adjust signal the same for all images in experiment similar to the example image on this page.

5. Select Image --> Scale --> Should be set to 50% (0.5) --> Select OK

6. Select File --> Save as --> Tiff

*Please note that high-quality images are needed for proper quantification (please see example below).

Image Analysis

1. Upload your tiff image files and wait for upload to complete. Please upload image files (select multiple files at once, we recommend no more than 10 at a time) instead of a folder of files.

2. Enter the number of intervals you want to quantify with.

3. Click the submit button to run the image analysis. Please allow a few minutes for image processing.

*One file may generally take 1-2 minutes to process, so please be patient for the program to finish all files.

Number of Axons

*If submitting more than one job for batch processing, please upload the next batch of images to process once the original batch finishes and hit submit again.

Total Mean Fluorescence Intensity (MFI) Quantification

Image Pre-Processing Instructions

1. Open FIJI Software, select File --> Open --> select image file.

2. To straighten & crop nerves from injury site to the distal nerve, do the following:

a. Select Edit --> Options --> Line Width, set # equal to nerve width (~2500)

*For MFI quantification, it is important that this number is optimized to fit the nerve and reduce background that may dilute the overall MFI quantification. If this is a concern, please use the normalization tab to disregard background MFI.

b. Right-click line tile --> Select segmented line

c. Click at nerve crush site, and then click in the middle of the nerve until at distal nerve end, then right-click.

d. Search for the straighten plugin and then run.

*Make sure empty space is not present in the cropped image, or this will interfere with the MFI calculation.

3. Select Image --> Type --> 8-bit

4. Select Image --> Scale --> Should be set to 50% (0.5) --> Select OK

5. Select File --> Save as --> Tiff

*Please note that high-quality images are needed for proper quantification (please see example below).

Image Analysis

1. Upload tiff image file and wait for upload to complete.

2. Enter the number of intervals you want to quantify with.

3. Click the submit button to run the image analysis. Please allow a few minutes for image processing.

4. When finished, you may click the reset button to submit another image for analysis.

*Files may generally need ~1 minute of processing time.

Mean MFI

Normalized Mean Fluorescence Intensity (MFI) Quantification

Image Pre-Processing Instructions

1. Open FIJI Software, select File --> Open --> select image file.

2. To straighten & crop nerves from injury site to the distal nerve, do the following:

a. Select Edit --> Options --> Line Width, set # equal to nerve width (~2500)

b. Right-click line tile --> Select segmented line

c. Click at nerve crush site, and then click in the middle of the nerve until at distal nerve end, then right-click.

d. Search for the straighten plugin and then run.

3. Select Image --> Type --> 8-bit

4. Select Image --> Scale --> Should be set to 50% (0.5) --> Select OK

5. Select File --> Save as --> Tiff

6. Record the pixel height in inches: Select Image --> Properties

7. Repeat steps 1-5 with an image file of nuclear staining (i.e., DAPI).

*Please note no nuclear staining can be on the edges of the cropped image or the code may fail to acquire the upper/lower limit of the nerve width.

*Please note that high-quality images are needed for proper quantification (please see example below).

SCG10 Example

Hoechst Example

Image Analysis

1. Upload tiff image file and wait for upload to complete.

2. Upload nuclear stained tiff image file and wait for upload to complete.

3. Enter the number of intervals you want to quantify with.

4. Input the height of pixels in inches for the image.

5. Click the submit button to run the image analysis. Please allow a few minutes for image processing.

6. When finished, you may click the reset button to submit another image for analysis.

*Files may generally need ~8 minutes of processing time.

Mean MFI

Mean Fluorescence Intensity (MFI) Quantification for Multiple Files

Image Pre-Processing Instructions (to do for each file)

1. Open FIJI Software, select File --> Open --> select image file.

2. To straighten & crop nerves from injury site to the distal nerve, do the following:

a. Select Edit --> Options --> Line Width, set # equal to nerve width (~2500)

*For MFI quantification, it is important that this number is optimized to fit the nerve and reduce background that may dilute the overall MFI quantification. If this is a concern, please use the normalization tab to disregard background MFI.

b. Right-click line tile --> Select segmented line

c. Click at nerve crush site, and then click in the middle of the nerve until at distal nerve end, then right-click.

d. Search for the straighten plugin and then run.

*Make sure background is not present in the cropped image, or this will interfere with the MFI calculation.

3. Select Image --> Type --> 8-bit

4. Select Image --> Scale --> Should be set to 50% (0.5) --> Select OK

5. Select File --> Save as --> Tiff

*Please note that high-quality images are needed for proper quantification (please see example below).

Image Analysis

1. Upload your tiff image files (select multiple files at once, we recommend no more than 10 images at once) and wait for upload to complete. Do not upload a folder but just the files.

2. Enter the number of intervals you want to quantify with.

3. Click the submit button to run the image analysis. Please allow a few minutes for image processing.

*One file may generally take 1-2 minutes to process, so please be patient for all files to finish.

*Please reset browser after quantifying set of images if doing more multiple file quantification.

Mean MFI

*If submitting more than one job for batch processing, please upload the next batch of images to process once the original batch finishes and hit submit again.

RGC Quantification

Image Pre-Processing Instructions

1. Open FIJI Software, select File --> Open --> select image file.

2. Select Image --> Type --> 8-bit

3. Select Image --> Adjust --> Brightness_Contrast, adjust to have signal similar to the example image on this page.

4. Select Image --> Scale --> Should be set to 50% (0.5) --> Select OK

5. Select File --> Save as --> Tiff

*Please note that high-quality images are needed for proper quantification (please see example below).

Image Analysis

1. Upload tiff image file and wait for upload to complete.

2. Click the submit button to run the image analysis. Please allow a few minutes for image processing.

3. When finished, you may click the reset button to submit another image for analysis.

Number of RGCs

RGC Quantification for Multiple Files

Image Pre-Processing Instructions (to do for each file)

1. Open FIJI Software, select File --> Open --> select image file.

2. Select Image --> Type --> 8-bit

3. Select Image --> Adjust --> Brightness_Contrast, adjust to have signal similar to the example image on this page.

4. Select Image --> Scale --> Should be set to 50% (0.5) --> Select OK

5. Select File --> Save as --> Tiff

*Please note that high-quality images are needed for proper quantification (please see example below).

Image Analysis

1. Upload tiff image files (select multiple files at once) and wait for upload to complete.

2. Click the submit button to run the image analysis. Please allow a few minutes for image processing.

3. When finished, you may click the reset button to submit another image for analysis.

Number of RGCs

*If submitting more than one job for batch processing, please upload the next batch of images to process once the original batch finishes and hit submit again.