Total Internal Reflection (TIRFM)
Total Internal Reflection Fluorescence Microscopy is set up on our Olympus 'Xcellence' microscope and on the Zeiss Elyra PS1 system.
Principles of the technique
TIRF microscopy exploits the formation of evanescent waves, at the interface between two medias of significantly different refractive index, to create a thin field of fluorescence excitation with an axial extent which doesn't exceed about 200nm and provides intrinsic optical sectioning and high contrast fluorescent imaging.
In general, rays of light with almost normal incidence (perpendicular to the surface) will mostly refract ('go through') at the interface between two surfaces of different refractive indices. However, a small portion of light will reflect backwards. For example, between glass and air, back reflections typically consist of about 4% of the incident beam, with visible light.
TIRF consists in adjusting the incident angle up to a critical angle, above which reflection will be dominant. With the right combination of refractive indices, this full reflection will be accompanied by evanescent waves propagating along the surface/interface. These waves typically extend beyond the interface and their axial extent is tunable typically between 100 and 200nm. Only high Numerical Aperture Objective lenses (NA > 1.45) can achieve critical angle.
Features / Specifications
1) Mounted on our Motorized Widefield Fluorescence Microscope Olympus ScanR/CellR, TIRF is available with two laser lines: 488nm and 561nm, and with special objective lenses for TIRF available: 100x /1.45 oil, 150x / 1.45 oil.
To use this instrument, please contact Anna Lladó and Julien Colombelli: microscopy (at) irbbarcelona.org
1) Mounted on our Motorized Elyra PS1 super resolution system, TIRF is available with three laser lines: 488nm 561nm and 640nm, and with special objective lenses for TIRF available: more info to come.
Microtubule ends (labelled with EB3-YFP) in cultured cells, imaged with TIRF.