Total Internal Reflection Fluorescence (TIRF) microscopy involves imaging in a limited area adjacent to the glass-water interface (coverslip-sample) with an evanescent wave (generated when light is totally internally reflected at this interface). TIRF involves the excitation light entering the medium at a critical angle using the properties of a high NA lens (TIRF lens) that allows the light to be internally reflected and excite fluorophores within a very thin (100nm) area at the coverslip/sample interface. TIRF also eliminates unwanted background and out of focus fluorescence, providing high signal/noise ratios. This technique is useful in studying molecules close to the cell surface, such as receptors or membrane signaling proteins.
» Watch Video | TIRF imaging of α4-CFP/β1-YFP integrins (expressed in GD25). Movie shows expression only at the spreading cell surface on VCAM-1. Courtesy of Young-min Hyun, University of Rochester Medical Center, USA.
X-Cite® Store