ImageJ Custom Macros Plugin - 02 Filename-based Calibration Tutorial

● 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.

00:56 - ● To customize the sample image, press ‘shift-V’. ● Let’s create a sample image with the following parameters: ● 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. ● 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.

02:46 - ▪ 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 unit² (typically μm²) per pixel calibration is calculated based on the technical specifications of your camera’s sensor chip. ▪ This sensor value is the ‘base calibration’.

04:00 - ○ 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 μm² ÷ 20 = 0.3225 μm²/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.

05:12 - ● 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. ● 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.

06:51 - ○ 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. ○ Holding control closes only images. ○ Holding space closes only the top-most image. .