FILENAME-BASED CALIBRATION TUTORIAL TRANSCRIPT
 
• In this tutorial, we’ll go through sample image generation,  filename-based calibration, and the Close button.

• Run ImageJ with Custom Macros installed.
• Remember, if you need help with procedures or a quick list of shortcuts, press the ‘Help button’.
• Login by pressing the ‘Start button’.
• Since we’re simply exploring functionality rather than running an experimental analysis, login in as default.
• Press keyboard shortcut ‘v’ to create the standard sample image.
• To customize the sample image, press ‘shift-V’.
• Let’s create a sample image with the following parameters:
	° // 300 x 300, red, 20x.
• Pressing ‘v’ automatically creates the customized sample image.
• The standard ImageJ ‘close all’ menu function prompts users to save images. It also only closes images, not text or function windows.
• Instead press the Custom Macros’ ‘close button’ to close all windows.
• Lets now explore Custom Macros’ filename based calibration system.
• Press ‘shift-V’ to create the following sample image.
	° // 300 x300, gray, 20x.
• Note that we set the calibration multiplier to 20x.
• Press keyboard shortcut 'F11' to open the Info Window.
	° The calibration multiplier is a 1-4 digit number preceded by a space and followed by an x and then another space or period.
		° For example, the calibration multiplier for the file:
		° Archaeopteryx_19 T-maze 20x SNc section_12.tif is 20.
		° Just as it is in:
		° Archaeopteryx_19 T-maze SNc section_12 20x.tif.
	° The calibration multiplier is intended to correspond to the magnification at which an image was taken. The resulting unit˛  (typically µm˛) per pixel calibration is calculated based on the technical specifications of your camera’s sensor chip.
		° This sensor value is the ‘base calibration’.
	° Base calibration can be easily adjusted to match your microscope’s camera by editing a text file called Calibration.ijm in the Custom Macros folder – please see documentation for details.
	° Scale is set by dividing the base calibration by the calibration multiplier:
	° Base calibration ÷ calibration multiplier  =     scale
	°    6.45 µm˛      ÷          20             =  0.3225 µm˛/px
• Finally, note that calibration multipliers can be very useful independent of actual camera settings as a way of scaling images as-needed. See documentation on the Scaling Tool (keyboard shortcut ‘k’) for details.
• Let’s see what filename based calibration accomplishes.
• Press ‘shift-V’ to create the same sample image, but with a 10x magnification.
• Select the 20x image and measure it using the ImageJ menu command.
	° // Analyze -->  Measure.
• Now do the same for the 10x image.
	° Note that while the mean, mode, area fraction, and other distribution based measurements remain constant regardless of calibration,
	° The area and integrated density are four times as large.
• Press ‘shift-V’ one last time to create an image with a 0x calibration multiplier.
	° // Analyze -->  Measure.
	° Note that measurements are no longer scaled but simply the raw pixel values.
• Finally, the function of the close button can be modified depending on whether an additional key is pressed:
	° Holding shift while pressing the button closes all windows except images.
		° // Analyze -->  Measure.
	° Holding control closes only images.
		° // Keyboard shortcut ‘v’ (twice).
	° Holding space closes only the top-most image.