Sub-diffraction imaging with total internal reflection fluoresence (TIRF) microscopy by stochastic photobleaching

Abstract

Total internal reflection fluorescence (TIRF) microscopy is widely used in fluorescent imaging. Evanescent wave fields generated by the internal reflection are used to illuminate the sample, and only fluorophores within a thickness of 100 nm thick from the surface are activated, improving the signal-to-noise ratio (SNR) of the image. Sub-diffraction imaging with TIRF microscopy by stochastic photobleaching is studied. Each fluorophore can be localized from the recorded streaming followed by image subtraction. This method can yield contrast-enhanced images with a higher SNR and improve the lateral resolution to approximately 120 nm.