Michael Hooker Microscopy Facility (MHMF.ORG)

Scale Bars / Calibration - General Information

In order to create a scale bar of know dimension enough information to calculate the um/pixel size for the particular microscope, objective & camera is required.  Given this information one can calculate how long (in pixels) the calibration bar needs to be in order to represent a desired um length. The specific information required is listed below.  Note that labeling photomicrographs with a calibration bar is the desired method of displaying the calibration of an image.  Labeling an image such as "taken at 20x" is uncalibrated and obscure given that the image could be blown up or reduced by unspecified amounts in the steps taken in laying out, printing and preparation the image for publication. 

In order to add a calibrated scale bar to an acquired image basic information about the image acquisition is required:

• Which microscope?
• Objective magnification? Also numerical aperture (NA) helps to uniquely identify an objective.
• Intermediate zoom factor? e.g. Optivar magnification: typically 1x, 1.5x, 1.6x, etc
• Which camera was used? e.g. On the Leica inverted widefield scope MicroPublisher (color) versus Photometrics HQ2 (b/w)
• Acquisition binning factor? Usually this is 1, but could be 2 (half size), 3 (one third size), etc.

With the above information one piece of data is required to make a scale bar of known dimension:

• An image of a calibration slide,  e.g. stage micrometer, taken at the same settings, as specified above. 

Note: For the widefield scopes and the spinning disk confocal in the MHMF stock images of a calibration slide are stored in the D:\_calibration directory (or \\computername\users2\_calibration share\directory over the network) of each respective computer workstation associated with the microscope.  These images are of a 1 to 2 mm stage micrometer with 10 um small divisions.

Given the above information it is possible to calculate the um/pixel size of the acquired image.  Scale bars of desired length can be created by one of two general ways:

• pixels[#] = length [um] / microns per pixel [um/#]      (# is number of pixels)
• Draw a line over the calibration micrometer image of desired length. e.g. smallest divisions is 10 um.  Draw or copy line of same length (pixel length) on your sample image.

Important: Scale bars should be added to the image before it is resized in, for example, Photoshop.  Otherwise resizing amount needs to be factored into the microns per pixel calibration.

Notes for Laser Scanning Confocal Microscopes:

• For the laser scanning confocal scopes calibration meta information is stored for each image in the image data base.  There is no need to scan a calibration slide!
• The on line and off line software for the Zeiss and Leica confocals allow the insertion of a calibration bar (to at least primary scans, and possibly derived images, such as overlays).

		Copyright 2001-2014  Dr. M. Chua, Program in Molecular Biology & Biotechnology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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