Biological microscopes are often used to measure artifacts, cells, cellular components (i.e. nuclear diameter) and other features as necessary using an eyepiece reticle. Typically, the objectives used on even a very high end research microscope do not exhibit exact magnification(s). The actual magnification values may vary as much as 2-3% so that a 10X objective, for example, may in reality be as low as 9.7x or as high as 10.3x. Clearly this will affect the accuracy of any measurements that are made and the variation may be significant depending on the ultimate use of the values collected. This potential source of error is easily overcome by calibrating the objective/eyepiece reticle set using a stage micrometer. Stage micrometers are quite accurate and can be acquired with a calibration certificate traceable to NIST, JIS, DIN or other recognized national standards organizations. Each objective must be individually calibrated, but once done, the values remain valid as long as the microscope set-up remains intact and unchanged. The procedure and mathematics are quite simple and step by step instructions are available either online or in printed hardcopy. In essence, the graduations in the eyepiece reticle are compared to those on the stage micrometer and a correction factor is calculated. Once the correction factor is determined for each objective a spreadsheet can be easily created to correct the “raw” readings. NIKON and UNICO are two of the many microscope manufacturers that offer stage micrometers for use in quantitative microscopy.
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