principle of TIRF

Total Internal Reflection Fluorescence microscopy (TIRF) or evanescent wave microscopy is very appropriate for studies of the localization and dynamics of plasma membrane molecules.

When the excitation beam passes from a medium of refractive index n1 (glass plate n1 =1.51) to a medium of lower refractive index n2 (for water n2 =1.33), there is a critical incident angle, above which the laser beam is completely reflected. In TIRF microscopy the illumination is done with an angle giving a total reflection which can be obtained in 2 ways :

1) the beam and the detection are separated. The excitation beam passes through a prism and excites the sample in total reflection and then the fluorescence is recovered by the objective.
2) the excitation beam passes through a large numerical aperture objective with an angle greater than the critical angle. The fluorescence is then recovered by the same objective.

The advantage of TIRF microscopy is the selective excitation of the sample, because only a zone of a few tens of nanometers is excited above the slide (interface of the 2 media). The TIRF excitation field decreases exponentially with the distance from the slide, where the cells are grown. Fluorescence from the other levels is then eliminated (no excitation above ≈100nm) and the signal-to-noise ratio is high for near-surface markings.

Video Tutorial on ibiology.org.

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