Why is HILO microscopy useful?
The beam of light is both highly inclined and also incredibly thin. This has the effect of increasing the image intensity, while at the same time reducing the background noise. Overall, the signal to background noise ratio can be up to eight times greater than standard epifluorescence illumination.
Crucially, the technique is not limited to imaging the surface of samples as is the case for TIRF. In TIRF microscopy, the evanescent wave is limited to 200 nm from the glass surface so where deeper sample penetration is required, HILO becomes a very desirable mode. Often this is needed when imaging tissues, nuclei or other cell material that sit beyond the membrane.
This advantage, coupled to reduced background noise, facilitates the quantification of molecular dynamics, kinetics and interactions across biological systems.