CalibrationTutorial copy

CalibrationTutorial copy - Calibrating a microscope using a...

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Unformatted text preview: Calibrating a microscope using a calibration target Hernan G. Garcia March 30, 2009 1 Introduction When we take an image using a CCD all distances are given in pixels, the fundamental spatial unit of a digital camera. Of course, we can guess the size of what we’re looking at from the supposed magnification of the scope (the magnification of the objective that was chosen). This tutorial will walk you through calibrating a microscope such that you can include a scale bar on all your images. Unfortunately, even though widespread, this is not common practice in all biology. You’ll often find images on text books without any scale bars. 2 Taking a good image You will take an image of your calibration target using Micro-Manager (refer to the Micro-Manager manual and the TAs!). The calibration target is a cross with lines spaced every 10 μ m. Notice that the targets we have already come with a coverslip on top of the slide. We will always image through the coverslip. For 100x imaging we’ll have to put oil on it. When you’re done imaging make sure you wipe the oil off using a cotton swab and isopropanol. You can find both of these cleaning supplies in the drawers underneath the microscopes. Fig. 2.1 shows two representative images of the same calibration target. Both images are good enough for the purposes of calibration. On the one hand, there’s a clear aesthetic difference between the two of them. On the other hand, the one that looks “nicer” is a little bit crooked. The image on the left presents some “bubbles” which could be due to the coverslip being dirty....
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