HILO microscopy is a mode of imaging where the laser is directed at a sharp angle through the sample. This affords an imaging depth of up to 10 µm, at a signal to background noise ratio (SNR) only 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.
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 like TIRF. In TIRF microscopy, the effervescent wave is limited to 200nm 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 with the clearing up of background noise means it becomes possible to quantify molecular dynamics, kinetics and interactions in biological systems.
The Nanoimager microscope allows full control over the illumination angle so three imaging modes - EPI, TIRF and HILO - are all possible. Each of these types of illumination can be used to acquire super-resolution, smFRET or tracking data. A full field of view takes only milliseconds to record and reduced frames can be imaged at up to 5 kHz frame rate.
It is possible to change the illumination angle in 0.5 degree increments. Seeing the SNR changes in real time, in practice, 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.
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