ImageJ 'Custom Macros' Plugin - 02 Filename-based Calibration Tutorial
Channel: ImageJ Custom Macros Plugin
Date: 17/01/2020

https://ijmacros.com

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 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’.
 ○ 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.
● 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.