- HOW TO MEASURE SCRATCH ASSAY WITH FIJI IMAGEJ SOFTWARE HOW TO
- HOW TO MEASURE SCRATCH ASSAY WITH FIJI IMAGEJ SOFTWARE INSTALL
- HOW TO MEASURE SCRATCH ASSAY WITH FIJI IMAGEJ SOFTWARE SOFTWARE
To do this, go to Image > Color > Split Channels. To quantify signal in green and red channels, we have to split them and treat separately. It will merge all the slices of the image to a single one with pixels containing the maximum value from all images in the stack at the given location.įrom this point, file processing will be identical for. In the Z-Project options window, choose all your stacks and Max Intensity as a Projection type. lsm file, go to Image > Stacks > Z-project. To process the image, we need to have it in the right format: single channel, 8-bit images. 3: Image Sequence Options Image processing
If you want to import everything, just leave the values as default. Next, you will need to choose Sequence Options by selecting the number of stacks to import. If you took a Z-stack image of your sample and exported the file as a series of separate images, you can import it to Fiji by selecting File > Import > Image Sequence… and opening the folder with the Z-stacks. You can cycle through channels and z-stacks using sliders below the image preview.
However, the open file will be a composite image with three different channels in a Z-stack that you can cycle through. lsm file (a common format for ZEISS confocal microscopes), you can open it using the same method. For single files like this, simply go to File > Open and select your file. tif image exported from the microscope software. There are instructions for modifications included directly in the macro file.
HOW TO MEASURE SCRATCH ASSAY WITH FIJI IMAGEJ SOFTWARE INSTALL
You can follow the steps below for the manual quantification, but you can also download our Fiji macro and install it by going to Plugins > Macros > Install… and then use it by selecting it from Plugins > Macros > “live dead quantification.” You might need to modify specific settings of the macro to suit your needs by going to Plugins > Macros > Edit and selecting our macro file. Green (calcein) stains for live cells, red (EthD) for dead cells. Microscope image of Live/Dead assay of neural stem cells. To follow along, you can download an example Live/Dead image here. It is available for Mac, Windows, and Linux platforms.
HOW TO MEASURE SCRATCH ASSAY WITH FIJI IMAGEJ SOFTWARE SOFTWARE
While this approach can be used for 3D cell culture, the results are more reliable if the sample is relatively thin and there are not many cells overlapping in different focal planes.įor this analysis, we will be using free image processing software Fiji, an extended version of ImageJ. This workflow is suitable for images of 2D and thin 3D controls captured using a fluorescent or confocal microscope for samples stained with calceinAM/ethidium homodimer (EthD).
HOW TO MEASURE SCRATCH ASSAY WITH FIJI IMAGEJ SOFTWARE HOW TO
This guide will walk you through a step-by-step guide on how to perform a Live/Dead quantification using Fiji. You can read more about different types of viability assays on our assays for 3D culture page. Dead cells are labeled with the ethidium homodimer dye (EthD) which binds to their DNA and fluoresces red. Live cells are stained with calcein and generate green fluorescence upon the excitation of their cytoplasm. Live/Dead assay is a very common cell staining procedure.
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