CoccoBiom - extra notes on getting started
Downloading
A problem with downloading text files is that extra carriage returns are often inserted by the download program. For a computer program this can be disastrous, causing numerous bugs. To stop this happening adjust the preferences in your download program or use the zipped archive file (this contains all the macros).
Calibrating magnification
For measurements the image magnification needs to set, it is then stored in the tiff header of the image. The basic procedure is
1. Obtain a stage micrometer (slide with engraved scale).
2. Take an image of the micrometer.
3. Use the line tool in Image to measure the pixel length of a segment of the micrometer.
4. Use the set scale dialog (analyze menu) to set the scale for this image and note the scale it calculates, in pixels per micron.
5. From this calculate a microscope calibration. microscope_calib = scale/(objective magnification). (e.g. If with an x40 objective the scale of the captured image is 5pixels per micron then the microcope calibration is 5/40 = 0.125)
6. Open the macro file (using the open file command in Image), substitute this value of microscope_calib for the value in the file (in procinit). Save the file.
7. The magnification can now be set using macros without further calibration.
N.B. Microscope lenses are normally very accurately calibrated, so once the microscope calibration has been worked out for one lens the image scale can be calculated for any lens. In the macros this principle is applied, a dialog box asks for the objective lens used and the program then calcultates the image scale.
Calibrating pixel aspect ratio
Not all systems produce accurately square pixels - i.e. the captured image may be slightly distorted. Our approach is to undistort images as they are incorporated into the mosaics. To do this the pixel aspect ratio of the captured images needs to be known - this can be determined by the same process as the scale setting (see also the NIH-Image manual). This pixel aspect ratio then needs to be substituted into the macro file. Typical process;
1. Capture an image of a horizontally oriented object and measure length.
2. Rotate stage through 90 degrees, image specimen again and measure length in this orientation.
3. Calculate camera pixel aspect ratio (camerapar); camerapar= (length when horizontal) / (length when vertical). Typically values are in the range 1.00 to 1.05.
4. Open the macro file (using the open file command in Image), substitute this value of camerapar for the value in the file (in procinit). Save the file.
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