Increasing signal-to-noise ratio by use of highly inclined thin illumination beam

Signal-to-noise ratio (SNR) is a crucial parameter in imaging. In order to improve it researchers use different tricks, optimizing the labels and reducing the background.

Here is one interesting imaging trick called Highly Inclined and Laminated Optical sheet (HILO) microscopy. Using highly inclined thin imaging beam it is possible to increase SNR dramatically.

In a sense this approach is similar to TIRF, where illuminating light is approaching the sample at a critical angle, so that only molecules very close to the coverslip surface are illuminated by the evanescent wave. The problem with TIRF is exactly that - only molecules very close to the surface can be imaged, which is fine for working with membrane proteins, but not so for the cytosolic ones.

HILO can do just that: instead of illuminating only the molecules in close proximity to the coverslide (as TIRF does) it illuminates a thin slice cutting through the cell at an angle.

References:

Tokunaga et al. Nature Methods 2008, 5 (2) pp. 159-161 PIMD 18176568