Dear All:
I want to quantify the fluorescence intensity of images using fluorescence microscope.
I would really appreciate it if someone could tell me how to use image j correctly.
Hadiseh Amani Beni
Open ImageJ: Download and install ImageJ (or FIJI, an enhanced version of ImageJ) if you haven't already. Open the Fluorescence Image: Go to File > Open and select your fluorescence image (typically in formats like TIFF, PNG, or JPEG). Convert to Grayscale (if necessary): If your image is in color, convert it to grayscale. This step is important because intensity measurements are typically based on grayscale values. Go to Image > Type > 8-bit to convert the image to grayscale. Set the Scale (optional, if you need physical units): If the image includes scale information, go to Analyze > Set Scale. Input the scale information (e.g., pixels per micron), if available. Subtract Background: To improve the accuracy of the intensity measurement, subtract any background fluorescence. Go to Process > Subtract Background and set an appropriate rolling ball radius (e.g., 50-100 depending on your image). Select the Region of Interest (ROI): Use the Rectangle, Oval, or Polygon selection tool to outline the area of interest (the region where you want to quantify fluorescence). You can skip the selection for the entire image. Measure Fluorescence Intensity: Once the ROI is selected, go to Analyze > Measure or simply press M. A results window will appear showing several metrics, including the Mean Gray Value (this is the average intensity of the selected area). You can repeat this for different regions of the image if needed. Quantify Intensity for Multiple ROIs (if required): You can measure fluorescence intensity in multiple regions by selecting new areas and repeating the measurement process. Alternatively, use Analyze > Analyze Particles to automatically select multiple regions and measure their intensities. Export and Analyze Data: The results (including fluorescence intensity) can be copied from the results window or saved as a .csv file by going to File > Save As. Optional: Intensity Calibration: If needed, perform intensity calibration to convert pixel intensities to actual concentration units. This requires creating a standard curve from known concentrations of fluorescent molecules.
To quantify fluorescence intensity from DCFH-DA staining (or any fluorescence-based assay) using ImageJ, follow these steps:
Step-by-Step Guide:
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