Function: TIRF

Function: TIRF

The TIRF function allows increasing the signal-to-noise ratio for super-resolution imaging. By default, the illumination on the Nanoimager is the same as epifluorescence wide-field microscopy. This corresponds to a degree of 0, in which light is coming out straight from the objective to illuminate the sample. To activate TIRF illumination, set the illumination angle to 50~55 degrees depending on the system.

Instructions

  1. Connect to the microscope and place a bead slide with fresh oil.
  2. Set the focus reference and enable the Z lock.
  3. Change the contrast method to Photon Count and turn on the green laser such that the beads are visible in at least one channel.
  4. Starting from 45 degrees up to 57 degrees, gradually increase the illumination angle at an interval of 0.5 degrees to find the angle that provides the best signal-to-noise ratio. At this angle, the bead signal (foreground) should be at its brightness and the noise signal (background) should be minimized:

Discussions

  1. The Nanoimager integrates the objective-based TIRF technique.
  2. The ONI TIRF angle does not match the theoretical critical angle due to an offset, and therefore this angle can slightly vary from one Nanoimager to another.
  3. The TIRF angle is wavelength-dependent rather than sample-dependent, it is recommended to use the green laser to determine the best angle for imaging.
If at any point you are having issues with the TIRF function, please do not hesitate to contact the CX team.


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