07 Aug 2000 : TILL-TIRF Total Internal Reflection Fluorescence Microscopy

TILL-TIRF Total Internal Reflection Fluorescence Microscopy
Variable Angle TIRF through the objective

TIRF microscopy is a powerful method to observe and measure membrane associated processes in living cells, biological or chemical events at liquid-solid interfaces and even single molecule dynamics.

TIRF microscopy is based on the excitation of fluorochromes by the evanescent field of totally internally reflected light. The evanescent field propagates several 10 to several 100 nm from the solid-liquid interface and thereby provides z-super resolution without exciting background fluorescence from fluorophores further away. The penetration depth is determined by the refractive index difference, the wavelength and the angle of the incident light.

TILL-Photonics is proud to present a newly designed TIRF setup, which is based on TILL's well known high-speed Imaging System and the Olympus Apo 100x/1.65 Objective. The new optical design of the TILL-TIRF system and the extreme numerical aperture of the objective allow varying the incident angle by almost 108. The fluorescence signal is collected through the full numerical aperture (no central annulus). By this design all advantages of an objective setup - full access to the specimen, easy handling and adjustment - are combined with the advantages of a classical prism setup - variable angle, high signal collection efficiency. The TILL-TIRF is fully integrated into the TILL-Imaging System. Within milliseconds TIRF, wide field fluorescence and transmitted illumination can be alternated and fast time-lapse experiments can be performed.

	Wide field fluorescence image of YFP-tubulin in a fibroblast. The entire tubulin network in the cell can be recognized.
	TIRF-image of the same cell. Only the contact sites of the tubulin network to the surface-attached cell membrane are visible.