Setting scale: This may need to be done depending on the metadata contained in your image file. Most raw images directly from microscopes have scale data embedded. If this is not the case for your image and you know the resolution of your image, set the scale by going to Analyze > Set Scale and input the number of pixels per micron.
Measuring length: To obtain measurements of specimen length.
- Click on the line tool and measure desired object.
- Choose Analyze tab and select “Measure”. A new window will open. Scroll to the right to see “Length”
Measuring single area: To obtain area measurements of a single region of interest.
- Click on the appropriate area selection tool for your object and outline your ROI (region of interest).
- Choose the Analyze tab and select “Measure”. A new window will open. Scroll to the right to see “Area”.
Measuring the area of complex objects or multiple regions: To obtain area measurements for many ROIs or a complex object within an image.This method can also be used to count objects.
- Convert your image to grayscale by selecting Image > Type > 8-bit.
- Threshold the image by selecting Process > Binary > Make Binary. This creates a two-color (black and white) image based on a threshold of the intensities of pixels in the original image.
- If this mask does not look correct for your image, manually threshold your image by selecting Image > Adjust > Threshold. A histogram will open and masks will appear on your image. Adjust the slider below the histogram to change the pixel value cutoff until the mask selects all your pixels of interest. Select “Apply” and close the window.
- To count your objects, select Analyze > Analyze Particles. Once the Analyze Particles window opens, define your minimum and maximum size of your object (pixels), select “Show Outlines” and check “Display Results”, “Summarize” and OK.
- Additional windows will open with all data for each object, as well as summary data for all of the objects. This can all be copied and pasted into a spreadsheet.