What is HILO microscopy?
In HILO microscopy, the laser is directed at a sharp angle through the sample. This allows an imaging depth of up to 10µm, at a signal to background noise ratio (SNR) slightly lower than that of TIRF.
HILO stands for highly inclined and laminated optical sheet but is affectionately referred to as “Dirty TIRF” due to the slightly lower SNR in comparison to TIRF imaging.
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.
Can I do HILO microscopy on the Nanoimager?
The Nanoimager microscope allows full control over the illumination angle, supporting the three imaging modes of epifluorescence, HILO and TIRF. Each of these types of illumination can be used to acquire super-resolution images, such as PALM microscopy or dSTORM microscopy, smFRET or data for single-particle tracking.
A full field of view takes milliseconds to record and reduced frames can be imaged at up to 5kHz frame rate. It is possible to change the illumination angle in 0.5 degree increments. Seeing the signal to background noise ratio changes in real time, allows the user to scroll through all illumination modes on the same sample. It is therefore possible to quickly adapt to the specific sample requirements without having to select the correct mode up front, allowing you to achieve the best imaging for your sample.