How to measure distance from cellular particles to nucleus?

Image Analysis
#1

Hello everyone,

Is there anyone I could have quick online support while learning how to use Fiji software?

I’m trying to measure the distribution of lysosomes after I knocked down some gene in the cells. So what I need to quantify is the distance from each lysosome to the nucleus. I stained lysosome with LysoTracker and nucleus with DAPI, and the images were taken use Zeiss 710 confocal.

I know how to identify lysosomes by the size in Fiji. And I have read some paper which has measured lysosome distance to the nucleus, but I can’t find any guideline of how to do the measurement.

I would really appreciate the help, and I can also talk on whatssap/ messanger if it’s too complicated to explain.

Best,
Yuanyuan

#2

Hi @YQin,

Welcome to the forum. If you took some time to get a look around the forum, you might have noticed the common reply to questions like yours, without an initial image: if you could share an image with us that depicts your particular needs, people on the list are much better to help you.
Will it be 2D, 3D? What did you try and where did you get stuck?

#3

@eljonco
Thank you for the suggestions. It’s 2D images as I attached below. I don’t know how to measure the distance of a cellular particle to the nucleus, and I can’t find any information online.

Lys NTC 10.1
Lys NTC 10.1512×512 27.9 KB

#4

Hi
@YQin

Your picture seems to be a jpg
If you have a tif image, please look at this document for more informations

https://alfresco.uclouvain.be/alfresco/service/guest/streamDownload/workspace/SpacesStore/b35713b6-fa0e-4d3f-a256-42aa4c4b2d09/ImageJ%20-%20distance%20lysosomes-nucleus.pdf?guest=true

I hope it will help

#5

@Mathew
Here is the image in tif format.Lysotracker HepG2 control.tif (515.6 KB)
Thank you Mathew! for the information. It is very helpful. I can definitely use it for the calculation of lysosome-nucleus distance.

But what I’m actually looking for is cumulative intensity distribution of lysosomes (the example pic is attached)

example
example734×540 160 KB
: averaged percentage distribution at 0-5 um from nuclear rim, 5-15 um from nuclear rim, and total signal 15 um to cell boarder.

Based on the paper which did the lysosome distribution using Fiji, “Individual cell ROI’s were outlined using a cytosolic protein marker and whole cell lysosome signal fluorescence were measured. ROI was then decreased by 10% and lysosome signal fluorescence was measured at each decreased. To generate a lysosome distribution curve, the signal intensity of each fraction was divided by the total cell signal.”

I guess my question right now is, how to decrease ROI by 10% each time? And do I need to manually decrease 10% for 10 times? Is there possibly a macro for it?

#6

You can use Edit>Selection>Enlarge… and enter a number of pixels to inset a smaller ROI and a positive number to outset the ROI. That is a quick and dirty way :sunglasses: . Keeping them in the ROI Manager allows to quickly obtain the cumulative values.

Given the varying distances of two consecutive contours in your sample line image, in/out setting a number of pixels rather than a percentage of distance might even be a better measure. Or do you expect/want distant objects to be measured in an ‘expanding universe’, ie. the further a lysosome is away from the nucleus, the relatively further it has to move to reach the next ridge?

BTW. is it the distance to the outline of the nucleus (the nuclear envelope) you want, or to its centroid?

#7

You need to first generate a mask from of the nucleus.
Process > Filters > Median Filter…
Median to preserve the edges of the nucleus.
The radius should be no larger than the nucleus and large enough to smooth out the noise in the image.

Image > Adjust > Auto Threshold…
You can adjust the mask by binary methods: https://imagej.nih.gov/ij/docs/menus/process.html#binary

Then invert the mask:
Edit > Invert
Now you can create an euclidean distance map:
Process > Binary > Distance Map

This will output the distance of any white (foreground) pixel to the next black (background) pixel. The measurement is expressed in pixels. Since one inverted the nucleus mask this means you can measure the euclidean distance from the nucleus.

You can then do different things.
Detect or segment the individual lysosomes and measure their distance.
Or you could threshold the euclidean distance map (EDM) and measure the lysosome intensity at different distances.

With your images at the moment you just need to be aware that you measure the shortest distance from any nucleus. This does not 100% reflect the distance from the nucleus of a single cell. In order to do so you would need to have a different marker to delineate the cell it self and create the EDM only in this cell.

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