Calibration

Calibration can be done at any time. However, quantitative measures that involve x and y components in their calculation (i.e., flat area and volume) will not be accurate until the series is calibrated. If a series is calibrated after traces have been made, the contours will be resized proportionately with the underlying images.

Instruction	Image(s)
Go to the calibration grid section (typically section 0).
2. Name two palette squares calibrate_x{1} and calibrate_y{1}. Note: Using curly braces in trace palette names allow you to easily change the number associated with each trace by clicking the plus button to the right of the palette. For example, "calibrate_x1" becomes "calibrate_x2" after incrementing with the + button.
3. Zoom in until you see about 12 boxes across your screen. That is how close you should be in order to accurately trace across 10 boxes from corner to corner. Avoid extreme edges and far corners, as this is where most distortion occurs.
4. With the calibrate_x{1) palette selected, use the "open trace" tool, and trace across 10 boxes from corner to corner in the x-direction. (link to open trace section here)
5. Select the calibrate_y{1} palette, and repeat #4 by tracing across 10 boxes in the y-direction.
6. Move to another region on the calibration grid. Use the "+" button to the right of the palette to automatically increase your numbers in curly braces by +1, and lay down your second -x and -y calibration traces by repeating steps 4 and 5 above.
7. Repeat step 6 until you have a total of 5 calibration traces in x and y.
8. When your traces are complete, select all your traces (Ctrl+A).
9. From the series dropdown menu, select "Calibrate pixel size."
10. A dialogue box will appear asking for the length of your calibration trace in microns. If you are in the Harris lab, enter 4.63 and select ok. This dialogue box will pop up for every calibration trace you have selected. Continue to enter 4.63, and select ok. Once you have done this for all of your traces, PyReconstruct will automatically change the series magnification by averaging your calibration traces (a progress bar will appear informing you that this is occurring). *IMPORTANT NOTE* This number will depend on the calibration grid used. For the grids commonly used in the Harris lab, each box is 0.463 microns. Therefore, the length of 10 boxes (your calibration trace) is 4.63 microns.
11. After series calibration all of your existing traces, including your calibration traces, will be scaled proportionately with the image magnification. You can check to see if a series has been calibrated by opening the trace list, and checking the length of your calibration traces. They should be close to 4.63 microns.