What Is TIRF Microscopy?

Introduction

Total internal reflection (TIR) can occur when light is moving from one medium to a different medium e.g. air to glass, glass to water, water to oil. At certain angles, rather than being refracted into the new medium, the light is reflected back into the medium it came from. TIR only occurs at certain angles, as demonstrated in Figure 1.

**Figure 1:** The concept of TIR. A-C shows a beam of light coming from one medium (glass) into a different medium (air) at three different angles. A) Small angle, the light is refracted into the air. B) The critical angle, where the light is refracted along the boundary between the two media. C) TIR, where the light is reflected back into the glass. D) TIR can occur within a glass tube, this is how fiber optics work. E) TIR also works if the fiber optic cable is curved. F) The concept of total internal reflection fluorescence (TIRF) microscopy, where the light is reflected at the point of a sample (image from www.pages.drexel.edu/~bio/cores/cic/tirf-microscopy.html, images A-E from www.bbc.co.uk/bitesize/guides/zctmh39/revision/1).

In the mid-1950s, E.J. Ambrose had the idea to use TIR to image cells that were adhered to a surface (Ambrose 1956), an idea that was expanded into total internal reflection fluorescence (TIRF) microscopy by Daniel Axelrod in 1981 (Axelrod, 1981).

Epifluorescence vs TIRF Microscopy

Traditional widefield fluorescent microscopy involves illuminating and exciting the whole sample, with light penetrating more than 1 mm into the sample. However, because the whole sample is illuminated at once emission light is received from the regions of interest and all other regions, meaning that out‑of-focus light emission interferes with the signal and introduces substantial background fluorescence and a poor quality image. The quality of widefield images compared to TIRF can be seen in Figure 2.

**Figure 2:** Difference in illumination from widefield (left) and TIRF (right). Widefield illumination moves in a line through the sample and can excite fluorophores >1 mm in depth. Evanescent waves generated by TIRF decay quickly and can only penetrate up to ~100 nm into the sample, exciting a far more select group of molecules.

Thanks to the small optical section that is illuminated at the slide interface, there is far less out‑of‑focus light, a better signal, lower light intensity and higher resolution compared to widefield fluorescence, a comparison seen in Figure 3. TIRF is particularly favorable for live cell imaging as low light levels mean the cells are not at risk of phototoxic damage, and for studying areas of the sample close to the surface such as the surface of cell membranes.

**Figure 3:** Comparison between widefield (left) and TIRF (right) illumination of HeLa cells. The images are taken at the same focal point. *Left* Widefield illumination produces more out of focus light shown as diffuse signal in the cytoplasm. *Right* TIRF illumination produces a higher signal-to-noise by producing an optical section only within a superficial 100 nm. Scale bar: 10 µm. Adapted from Mattheyses *et.al.* (2010).

References

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