ABSTRACTTotal internal reflection fluorescence (TIRF) microscopy represents a method of exciting and visualizing fluorophores present in the near‐membrane region of live or fixed cells grown on coverslips. TIRF microscopy is based on the total internal reflection phenomenon that occurs when light passes from a high‐refractive medium (e.g., glass) into a low‐refractive medium (e.g., cell, water). The evanescent field produced by total internally reflected light excites the fluorescent molecules at the cell‐substrate interface and is accompanied by minimal exposure of the remaining cell volume. This technique provides high‐contrast fluorescence images, with very low background and virtually no out‐of‐focus light, ideal for visualization and spectroscopy of single‐molecule fluorescence near a surface. This unit presents, in a concise manner, the principle of operation, instrument diversity, and TIRF microscopy applications for the study of biological samples.
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