subject: An Introduction To Tirf Microscopy [print this page] Total internal reflection fluorescence microscopy is a highly sensitive technique to perform functional investigations in living cells. Total internal reflection fluorescence is a special technique in fluorescence microscopy developed by Daniel Axelrod at the University of Michigan, Ann Arbor in the early 1980s. This kind of microscopy delivers images with an outstandingly high axial resolution below 100 nm. This allows the observation of membrane-associated processes.
TIRF microscopy allows imaging of fluorescent molecules located close to the glass/water (or glass/specimen) interface. The high signal to noise ratio and a resolution in z direction of usually 70-250 nm above the coverslip/water interface allows to visualize and to analyze vesicles transport and signaling events, as well as kinetic studies and single molecules detection. This is achieved by employing an evanescent wave for excitation of the fluorophores instead of direct illumination via light delivered by an arc lamp, LEDs or lasers.
Physical background is the total reflection of a laser beam at the interface of glass and water and the resulting electromagnetic wave, the so-called evanescent field. The energy of the evanescent field decreases exponentially with the distance of the interface coverslip/water and allows exciting fluorochromes. In biological applications the incident light is usually laser light and the interface the glass of the coverslip and a film of aqueous solution between coverslip and adherent cells.
The energy of the evanescent field decreases exponentially with the distance to the glass/water interface and therefore allows to excitefluorochromes with a high Z-resolution and low background. This allows the creation of images with outstanding signal-to-noise ratio, as fluorophores in the rest of the cell are hardly excited. Additionally, total internal reflection fluorescence delivers images with an outstandingly high axial resolution below 100 nm. This allows the observation of membrane-associated processes like cell adhesion, hormone binding, molecule transport and exocytotic and endocytotic processes
There are two approaches for achieving total internal reflection in optics: one is prism-based and the other objective-based. In the former total internal reflection microscope, a prism is attached to the coverslips surface which directs a focused light beam or laser towards the coverslip or medium interface. With the help of the prism the angle of the penetrating light is adjusted to the critical angle. While the laser in prism-based systems is more or less openly guided into the prism, the laser in objective-based systems is directly coupled into the microscope itself and exits the objective in a much defined manner.
