TIRF (Total Internal Reflection Fluorescence) Imaging
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.
Traditionally, TIRF was performed using multiple lasers, but this set-up can be rather expensive and laser excitation can contribute to significant photobleaching of the fluorophores, skewing the acquired data and affecting normal cell physiology.
Studies have shown that effective TIRF illumination can be achieved with a broadband light source such as the X-Cite® 120Q or X-Cite XYLIS. This could also be extended to the X-Cite 200DC, or the X-Cite exacte for longer studies requiring stable intensity. If using the X-Cite XLED1 or X-Cite TURBO, the light source can
be switched accurately and quickly.
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.