Practice question

Total internal reflection fluorescence excels for single-molecule sensitivity near plasma membrane. A laser totally reflects at glass-water interface, generating an evanescent field decaying within 100-200 nanometers above coverslip, selectively illuminating fluorophores in basal membrane while excluding bulk cytoplasm fluorescence. This extreme background suppression dramatically improves signal-to-noise, enabling detection of individual GFP-tagged receptors, vesicle fusion events and motor protein stepping. Confocal excites deeper volumes causing out-of-focus noise, while DIC and phase contrast lack molecular specificity, making TIRF preferred for membrane dynamics and single-molecule biophysics.