Calculate TIRF

Author: Sebastian Rhode (Sebastian.Rhode at
History: 2008/04/01: First version
2008/04/16: Version 1.1
2008/05/09: Version 1.2
Installation: Download Calc_TIRF.class to the plugins folder and restart ImageJ.
Description: This plugin calculates the penetration depth of the evanescent field for imaging application using TIRF microscopy. Additionally it displays the critical angle (where Total Internal reflection starts) and the maximum working angle of the used objective and the evanescent field intensity for a desired penetration depth.

The required input parameters are:

  • Wavelength of laser - right now 3 lines are implemented; in [nm]
  • Refractive index n1 - this is the index of the immersion oil; usually 1.518
  • Refractive Index n2 - this is the index of the medium; usually 1.330 (some people use 1.360 for the cytosol)
  • Numerical aperture - numerical aperture of the used TIRF objective
  • Desired penetration depth - this value will be used to calculate the required incident angles
If the calculated incident angle = NaN -> the desired penetration depth is not possible for the respective wavelength and the given parameters. If the value for the angle is bigger than maxAngle, the penetration depth is theoretically possible, but can not be achieved with the given parameters --> NA, n1 & n2. [Plot]

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