Introduction to Fiji (ImgeJ)
This is basic training on how to use Fiji which is a version of ImageJ with specialist plugins for Biological application.
Last updated
This is basic training on how to use Fiji which is a version of ImageJ with specialist plugins for Biological application.
Last updated
This is a pilot introductory course to Fiji which is a version of ImageJ that is packaged with a number of plugins useful for image analysis. It should take less than half a day to complete the whole course. The course will provide an overview of Fiji along with a set of exercises which cover the common operations that researchers may like to apply to their images. This includes opening an image, adding a scale bar, adjusting contrast and brightness, setting the pixel size, applying a Fourier Transform and saving an image. The course will use images taken by cryo-electron microscopy and light microscopy.
This course is aimed at research PhD students and researchers working with experimental imaging equipment.
The data used in this course is very small so you should be able to follow all but the last exercise of the course on most desktops or laptops. If you use larger data (after you have finished this course) you may find that you need more memory or disk space to store the data.
This course was designed in collaboration with researchers and academics that run imaging systems at the University of Leeds. The images have been provided by Matt Iadanza a researcher at the University of Leeds who uses cryo-em. He also provided suggestions for the exercises as these are common tasks for researchers who use imaging facilities. He choice of exercises was endorsed by Sally Boxall and Zabeda Aslam who manage imaging facilities at the University of Leeds. While the exercises are designed for cryo-em data most can also be applied to other types of image data.
The exercises build on each other so explanations of the tasks are given in more details at the beginning. That detail is not repeated in later exercises.
Go to the fiji download web page in your browser. The web address is currently https://imagej.net/Fiji/Downloads or you can do a web search to find the download site.
I have a Windows 10 systems so I select the latest version for 64 bit Windows systems at the top of the web page. The process is similar for Linux and macOS systems so download the latest version that matches the system you are using.
Then click on the link for the download which is a zip file and in my case is namedfiji-win64.zip
Go to the directory where the file downloaded to.
Right click on the file in the file browser and select the option toExtract All. Extract to the download folder.
Once the files are extracted go into the folder that you created when you extracted the files, it is called Fiji.app.
Find the file ImageJ-win64.exe and then right click on it. Select the optionSend toand select the option Desktop (Create shortcut).
Now when you look at the desktop there is an ImageJ icon and shortcut. Double left click on it and if need be select the option to run it.
https://www.youtube.com/watch?v=-MwyhFKuOHA&feature=youtu.be
negative_stain_001.dm3 - A negative stain image of human antibodies, in Gatan’s proprietary dm3 format.
From the File menu select the Open option. Notice that as you move your mouse over the menus and other options information about them appears at the bottom of the Fiji menuing window.
In the file browser navigate to the file and select it to open it. A new window will open to display the image.
From the Analyze menu select Tools and the Scale Bar option. A new window will open so you can set the scale bar settings. You will also see a scale bar appear over the image in the bottom right corner.
In the text box named Width in nm change the value to 100 and change the Height in pixels to32. Look at the scale bar at the bottom of the image and if it looks good click on the OK button at the bottom of the Scale Bar window.
From the File menu select the Save As option and from the drop down menu select the Jpeg option. A file browser will appear so use that to pick the directory where you wish to save the image.
Click on the X at the top right of the window that is displaying the image to close it.
https://www.youtube.com/embed/uksbngcSYPg
cryoEM_001.mrc. A cryoEM image of icosahedral viruses, in mrc format.
Open the image.
From the Image menu select Adjust and from the drop down menu Brightness/Contrast option. A new window will open so you can set the options for the brightness and contrast of the image. Notice the image histogram at the top of the window.
Click on the button with the word Auto on it in the bottom left of the window. Notice the image looks brighter and clearer and the line on the histogram has changed.
Click on the button with the word Apply on it in the bottom right of the window.
Save the image as a jpeg and close the image display window.
negative_stain_002.tif - A negative stain image of a helical virus, in 16-bit tif format.
Open the image. You will be able to see the image in a display window and at the top left it will have the dimensions of the image in microns.
From the Image menu select the Properties option.
In the text box labelled Unit of Length delete the word micron and replace it with nm. Look again at the size of the image in the top left of the image display window, it should now be in nm rather than microns. Now close the B&C window by clicking on the OK button at the bottom of the window.
From the Process menu select the FFT option and then from the drop down menu select the FFT option. A new window appears showing the results of the FFT operation.
Select the Strainght Line tool from the tool bar below the menus which we can use as a ruler.
Click on and hold down the left mouse button on the centre of the FFT image. Drag thisout to the first halo on the image, this is the first layer line. Read the length from the information bar under at the bottom of the Fiji control panel. Do this in 3 directions from the centre and take the average. What is the length in nm of the repeating feature?
Close the image display windows.
cryoEM_003.mrc - A cryoEM image of a small membrane protein with crystalline ice contamination, in mrc format.
Open the image. You will be able to see the image in a display window and at the top left it will have bit depth of the image and in this case will read 32-bit.
Save this file as a jpeg, put “-32” at the end of the filename before the .jpg file extension.
From the Image menu select the Type option and from the pull down menu you will see there is a tick by the 32-bit option. Change that by selecting the 16-bit option.
Save this image with a new file name.
Now change the bit depth to 8-bit.
Save this image with a new file name.
Compare the 3 images and the file sizes for the files you have saved.
Close the image display windows.
cryoEM_003.mrc - A cryoEM image of a small membrane protein with crystalline ice contamination, in mrc format.
Open the image.
From the Image menu select the Properties option to open a window to alter the settings.
Change the Unit of Length from pixel to nm. In each of the text boxes Pixel width, Pixel height and Voxel depth change the value 1.0000to0.107.
Perform a FFT on the altered image.
Use the ruler tool to measure from the centre to the outer bright ring on the FFT in 3 places. Take the average to find the resolution that the ice is diffracting at.
cryoEM_004.mrc - A cryoEM image of a 2-dimensional protein crystal, in mrc format.
Open the image.
Open the Image Properties window. Change the Unit of Length from cm to nm. In each of the text boxes Pixel width and Pixel height change the value 5000.0000 to 0.083 and Voxel depth from 10000000.0000 to 0.083.
From the Image menu select the Transform option and then the Bin option. An Image Shrink window will appear.
In the text boxes X shrink factor and Y shrink factor change the value from 2 to 4. It is a very large image. Then click on the OK box at the bottom of the window.
Open the image properties window and check the pixel width and height have changed so that they have increased by a factor of 4.
Produce a FFT of this image.
From the Image menu select Adjust and then the Brightness and Contrast option. Increase the contrast almost so you can just see the regular grid of white dots and the illumination ring around them. Then adjust the brightness down slightly until the image noise is reduced.
Use the ruler tool to measure the distance of the centre to the furthest points in the grid in 3 places. This is the resolution to which this crystal shows spots?
cryoEM_stack_001.mrc - An image stack from a dose fractionated cryoEM image, in mrc stack form.
Warning: This exercise requires good computational resources. If you do not have enough memory or compute power your system may be slow or even hang so that you are not able to complete it.
Open the image stack.
From the Image menu select the Stack option and then select the Z-Projection option. Select OK in the pop up window.
When a new display window opens it will be very dark. Open the Brightness and Contrast window. Select the Auto option and then Apply.