Reference no: EM132574931

Dry Lab: ImageJ software

1. All images to be put into ImageJ programme needs to be in a .tiff format with the same sizes.

-To upload a picture into the programme File→Open, then find a picture from your files where you saved it.

Make sure all the images uploaded into the software correspond to 8-bit (see top line on top the image). To adjust it, go to Image→Type→8-bit. Save your image.

-Make sure that all the images pixel range varies from 0 to 255. To do so, click on the image of interest, go to
Analyze→Histogram. Verify min and max values within 0 and 255.

2. To enhance the quality of your images, click on the image of interest that is black and white, go to Image→Adjust→Brightness/Contrast. Adjust Min and Max so that Min starts where the Histogram begins. You will see the difference.

3. To split channels from a composite image to get a separate image for a corresponding channel: Upload Images with double or triple staining in a .tiff format. From Moodle, choose a picture containing c1-4 in its name. It implies that the picture contains elements derived from three channels. Go to Images→Color→Split channels you will get separate images with Red, Blue and Green annotations on top line of each image. Save each image for lab report.

4. To create a composite image with three channels together in one image from images gotten from different channels:

Upload original (_ORG) black and white pictures in a .tiff format. You have 3 images DAPI_ORG. Alexa 488_ORG, Alexa 555_ORG. Make sure that all the images are of the same size. You can look at the top line of the image. If sizes are different, go to Images→Adjust→Size.
Go to Image→Color→Merge channels. There will be a pop up window where you need to assign C1 Red, choose Alexa 555_ORG; C2 green-Alexa Fluor 488.ORG; C3 blue-DAPI.ORG. Check "Create a Composite" and Check "Keep Source files". Save your composite image. You will need in Step 5 to put text and arrows.

5. To add selection to your images, you have different tools available on your Toolbar including rectangular shape, arrows, freehand selection, text tool (A) etc:
-For text to be present on the image: choose a Text box on the toolbar, and double click on it for formatting options, choose a color, size and font. Type a text, and press Ctrl B to leave the text on the image.

-For arrow to be present: choose an Arrow from the toolbar, or from "More tools". Double click on to launch formatting options. Once arrow is present on the image, click Ctrl B or go to Image→Overlay→Add Selection. Your arrow will stay on the image. In your composite image put DAPI and arrow to indicate the stained cell with the corresponding dye. Repeat the same for Alexa Fluor 488 and 555. Save your image for lab report.

6. To calculate Fluorescence Intensity, put a selection on the image, it can be Rectangular shape, or a freehand selection to choose a region of interest (ROI) for fluorescence. Then, Analyze→Set Measurements→Check Area, St dev, Mean Gray Value (intensity of fluorescence). Press OK button. Then, Analyze→Measure. A separate window will open with Results where you will see the values for your ROI. You can copy and paste those values to Excel spreadsheet to collect your data. Save the screenshot for lab report.

You can also try an option available on the Toolbar, click on the area of interest, go to More tools→Pixel Inspector. You will see a generation of digits. By clicking onto different places on the image, you will see a generation of digits which correspond to pixel intensity. Normally, 0 corresponds to no signal that is black, 255 corresponds to the highest signal, that is white, if your image is initially set to an 8-bit. Save your results for lab report.

7. To setup macros:

This option is useful, if you are doing the same operation all over multiple images, for example if you are going to measure the fluorescence intensity in several images.

Put a selection on the image (freehand or rectangle), then go to Analyze→Set measurements→ Check Area, Mean Gray value. Then go to Plugins→ Macros→Record. Then, Analyze→Measure. Go to the Macros window to click Create and save (File→Save) your Macros in ijm format. Open the next image, go to Plugins→ Macros→Run and on the image click Ctrl M, or Analyze→Measure. Open your Results page and see the values generated. Save your results for lab report.

All screenshots and generated tables should be in your Results part of your report 2.