TIRF | TIRF
Key Words: time-lapse imaging, Fluorescence, widefield, GFP, fluorophore, N.A., oil immersion, SRIC, white-light TIRF, resolution, signal-to-noise-ratio
Definition:TIRF, or evanescent field microscopy, is a form of fluorescence microscopy where excitation light is confined to a small area at the interface of the specimen and the glass coverslip or culture vessel. TIRF allows nanometre-scale imaging of single molecules.
When light travelling from a high refractive index medium (e.g. glass) to a medium of lower refractive index (e.g. water / cells) strikes the interface of these media at a small enough angle of incidence, the light is prevented from propagating into the second material and is reflected back into the first. Light penetrates the second medium (cells) only to a depth of a few hundred nanometres and decays exponentially with distance. Excitation of fluorophores, therefore, occurs in an area typically less than 100 nm thick. By confining excitation energy to this thin section, a high signal-to-noise-ratio is obtained, making it possible to observe single molecule fluorescence.
TIRF is suitable only for aqueous samples (i.e. not fixed, mounted specimens) and cell adhesion to the glass surface is a pre-requisite for TIRF microscopy. TIRF can be used to image minute structures or single molecules. It has been used to observe; vesicles undergoing endocytosis or exocytosis, single protein trafficking in cells, the absorption of blood proteins on biomaterials, cell adhesion to various surfaces, and dynamics of neurotransmitter release. It has also been used to characterize nanoparticle arrays, electron transport in mitochondrial and photosynthetic membranes, and the forces exerted on a surface during cell movement. TIRF can be combined with FRAP and FRET to obtain information on molecular dynamics and molecular interactions.
TIRF can be carried out on almost any research-level upright or inverted microscope by modification of epi-illumination used in widefield techniques. TIRF is easier to configure on an inverted microscope although upright microscopes can also be used. Imaging requires high N.A. TIRF objectives.
Nikon's Eclipse laser TIRF-2 system with Ti (Ti-E, Ti-U, Ti-Perfect Focus System) inverted microscope (with epi-fluorescence attachment) and Evanescent Wave Illumination Unit: Laser: blue (488), green (543), red (633) together with Nikon's dedicated TIRF Plan Apo 60x NA 1.45 oil and TIRF Plan Apo 100x 1.45 oil immersion objectives. The TIRF Plan Apo 60x NA 1.45 oil objective corrects for spherical aberration induced by temperature changes in the refractive index of immersion oil.
The modular design of the Ti allows TIRF to be used in combination with the C1 confocal system to provide extremely high signal-to-noise image at the cell membrane and 3D images of the whole cell. SRIC (Surface Reflective Interference Contrast) method can confirm focal contacts prior to switching to TIRF.