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Using Imagej: Kameron June 2020

This document provides instructions for processing immunofluorescence and FISH images in ImageJ. It describes how to [1] open multi-channel image stacks, [2] split channels, [3] adjust brightness and contrast, [4] assign color lookup tables, [5] merge channels, and [6] merge multiple z-slices to create a single maximum intensity projection image. The steps allow researchers to analyze fluorescence signals from multiple proteins or chromosomes in cell images.

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Stephanie Williams
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39 views

Using Imagej: Kameron June 2020

This document provides instructions for processing immunofluorescence and FISH images in ImageJ. It describes how to [1] open multi-channel image stacks, [2] split channels, [3] adjust brightness and contrast, [4] assign color lookup tables, [5] merge channels, and [6] merge multiple z-slices to create a single maximum intensity projection image. The steps allow researchers to analyze fluorescence signals from multiple proteins or chromosomes in cell images.

Uploaded by

Stephanie Williams
Copyright
© © All Rights Reserved
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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Kameron June 2020

Using ImageJ

Where to download/install ImageJ: https://imagej.nih.gov/ij/download.html

For Immunofluorescence:

We have an automated multi-channel acquisition setup for


immunofluorescence, using Micromanager. Michael Cammer has trained us,
and Jerome has worked with it extensively. When images are acquired, it is
saved as a multi-channel stack, with each channel a 16-bit tiff file.

You’ll notice when you open a 16-bit image, it is completely black


(compared to the picture you saw when imaging). Because it is in 16-bit, the
pixels have not yet been assigned color information – this will colorized in
ImageJ using an assigned look-up table (LUT). When you open the file in
ImageJ, it will by default assign the grays LUT to the 16-bit image(s). It is
not until you convert it to an RGB image (as described below) that it is
assigned real color and you can open it in Preview/Photoshop/Powerpoint.

I will now walk you through how I process images. In this example, I have
imaged early-passage WI38 fibroblasts with DAPI, FITC (anti-ACA), and
TRITC (anti-TRF1) channels on our microscope. My process is by no means
the most streamlined way you can do this – you are welcome to write your
own macros to make things more efficient. This is the way I (someone who
is not an expert in ImageJ) have done it.

1. First, drag your file into ImageJ.


Kameron June 2020

2. The image will open as a 3-channel stack. You can switch between
the 3 channels using the scrollbar.

3. Split the channels up by going to Image  Color  Split channels.


Each individual channel will now be its own window
Kameron June 2020

4. To adjust contrast, go to Image  Adjust  Brightness/Contrast. You


can adjust by using the Minimum, Maximum, Brightness, and
Contrast scrollbars, or use the ‘Set’ button to enter specific
Minimum/Maximum values.

5. The LUTs used for FITC and TRITC are already in ImageJ. To assign
color for the FITC and TRITC channels, have the image (needs to be
in 16-bit format, you cannot change the LUT for an RGB image)
selected, then go to Image  Color  Channels Tool.
Kameron June 2020

6. The Channels Tool will pop up. Go to More  Red or Green or


whatever LUT you’re looking to color the channel.

7. For the DAPI channel, ImageJ does not have a built-in LUT that
resembles the color we used in Openlab. I have extracted the DAPI
LUT from the Openlab software – this is found on the server in the
‘06 – SMITH LAB’ folder. To apply this to the DAPI channel, make
sure the channel is selected, then go to Image  Color  Edit LUT.
Kameron June 2020

8. The LUT editor will open. Click ‘Open’ then find and open the DAPI
LUT. Your image should now be colored with DAPI.

9. If you are not merging your channels, then for each of them go to
Image  Type  RGB Color. Then, go to File  Save as  tiff.
These saved images can be taken out of ImageJ and processed further.

10.For merging the 3 channels, go to Image  Color  Merge


Channels.
Kameron June 2020

11.The ‘Merge Channels’ pop-up will open. Disregard what the channel
labels on the left say, i.e. ‘C1 (red)’ or ‘C3 (blue)’ – as long as you
have ‘Create Composite’ checked, it will retain the LUTs you applied.
I also ‘Keep Source Images’ in case something messes up. Press
‘OK,’ and you will get a 16-bit merged image. Convert it to an RGB
image and save as a tiff, as described in Step 9.
Kameron June 2020

For chromosome-specific FISH:

Because we need to acquire multiple images through z for one channel (to
get every signal) when doing FISH, we don’t have automation and instead
take individual images using Ocular. Each image is saved as a 16-bit tiff file.

The important thing to know when processing FISH is how to merge


multiple layers of one channel. The way I’ve done it is by using the ‘Images
to Stack,’ then ‘Z-project’ with ‘Max Intensity’ tools. This produces a
merged 16-bit FITC image that I can work with. I will break down each step
below:

1. First, have the FITC layers you need to merge open and go to Image
 Stacks  Images to Stack.

2. The ‘Images to Stack’ window should pop up. Here, name your
merged stack (here I named it ‘FITC merge’) and indicate what files
should be included in the stack with ‘Title Contains’ (here I put
‘FITC’ to merge all of my open FITC images). press ‘OK.’
Kameron June 2020

3. This now produces a stack with all of the FITC images. To get the
merged 16-bit image, go to Image  Stacks  Z Project.

4. In the Z Projection window, make sure the projection is including


every image slice in the stack (my stack has 2 slices, so it’s going
from 1 to 2); this normally is set correctly. Then for ‘Projection type’
select ‘Max Intensity.’
Kameron June 2020

5. You now have a 16-bit merged image for FITC, that you can contrast,
color (using the Channels Tool) and merge with other channels as
described above.

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