I made several measurements of area and length in CellProfiler. In which units of measure are the data presented?
Take a look at the little box called “Pixel size” at the bottom center of the main window of CellProfiler.
What is the pixel size?
The pixel size is the number of micrometers per pixel. This number is used to convert measurements to micrometers instead of pixels, if you would like the size measurements to be scaled for your images. By default, the pixel size is set to “1” which means that all distance measurements will be in units of pixel lengths.
You can let CellProfiler convert pixel lengths to absolute units of measure (microns (micrometers)) for you by changing the pixel size or you can do the conversion yourself later. The default pixel size can be set in File > Set preferences. Upon startup, the default preferences are loaded or you can load preferences using File > Load Preferences. Either way, the preference for pixel size will be shown in the main window of CellProfiler. You can change the pixel size for the current session by typing it into the main window of CellProfiler. This value is stored along with any pipelines you save, so you can check what pixel size was used in an old experiment by loading the pipeline from a pipeline file or output file.
How do you know what value to use for the pixel size?
The pixel size depends on the resolution and binning of the camera and the magnification of the objective lens of the microscope, in addition to the physical setup of the microscope itself. You have two options: (1) check with the microscope manufacturer or service person and ask them for a table of pixel sizes for each possible combination of resolution/binning/objectives for your scope, or (2) get a ‘stage micrometer’ (a glass slide with precise markings of distances) and take pictures of it at all possible combinations of resolution/binning/objectives for your scope. Once the pictures are acquired, open them in CellProfiler, zoom in on them and take a look at the markings on the slide relative to a single pixel in the image. Make a table for yourself of the pixel size at each microscope/camera setting for future reference.
Warning: some CellProfiler modules might currently ignore the pixel size and produce data in pixel length units no matter what pixel size is set. We are working to fix this.