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Dual-Color Auto-Calibration Scanning-Angle Evanescent Field Microscope

Ames Laboratory researchers have developed a new microscope that can be used for live cell imaging as well as for examining single molecule dynamics. Total internal reflection fluorescence microscopy (TIRFM) is a mode of fluorescence microscopy that has been widely used for live-cell imaging at the interface between a biological sample and a cover slip or tissue culture well. TIRFM is based on the induction of an evanescent wave in the liquid adjacent to the interface, which is created when reflected light penetrates the interface, propagates parallel to the surface of the plane of incidence, and decays exponentially with distance.
There are two basic TIRFM systems: prism-based and objective based. Prism-based systems are preferable since they have lower costs, wider range of incident angles, less excitation light scattering, and higher accuracy in the incident angle determination. However, the prism-based method has geographical constraints on sample manipulation–it is difficult to recalibrate the system manually for all incident angles–and because image reconstruction can be difficult. To overcome these drawbacks, Ames Laboratory researchers have developed an innovative dual-color auto-calibration scanning-angle evanescent field microscope that is easier to operate and more reproducible than existing approaches. This microscope has utility for live-cell imaging to examine cellular organization and dynamic processes that occur in the cell/ substrate contact regions. A computer-controlled automatic high-precision calibration procedure is used to find the incident angles in the full range, and this microscope is able to achieve better axial resolution than currently available systems.
1. Permits high axial resolution (5-10 nm); 2. Provides quick and automatic creation of an evanescent field for any incident angle in the full range; 3. Enables dual-color auto-calibration and scanning capability; 4. Quickly scans through a set of samples; 5. Allows rapid re-calibration of new samples; 6. Enables fine adjustment of the optical trapping forces created by the evanescent field.
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