Total Internal Reflection Fluorescence (TIRF) microscopy employs a special method of illumination which will only excite fluorophores within a 100-200 nm distance of a coverslip-aqueous medium interface.  Using TIRF microscopy to image cells selectively visualises events occurring at or near the plasma membrane adjacent to the coverslip surface. Examples of studies which have employed this methodology include binding of molecules at membrane receptors, near-membrane calcium transients, cell adhesion, cytoskeletal and trafficking events, secretion, single molecule interactions and protein translocation.

Highly Inclined and Laminated Optical (HILO) sheet) illumination microscopy is a widefield fluorescence imaging technique that improves contrast and signal-to-noise when imaging near the coverslip.  HILO works by directing laser excitation light into the sample at a steeply inclined angle to creates a thin, tilted light sheet that penetrates only a few microns into the specimen.  This results in reduced background fluorescence (compared with standard epifluorescence microscopy), but has a greater penetration depth compared to TIRF.

The Imaging Facility’s Nikon SIM/STORM microscope system is equipped with a motorised TIRF illuminator and multiple excitation wavelengths to image blue, green, red and far-red fluorescence using TIRF or HILO.

Our System: