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The signal-to-noise ratio (SNR) and three-dimensional localization precision of a double helix point spread function (DH-PSF) can be significantly improved by applying variable-angle illumination epifluorescence microscopy (VAI, also commonly known as “pseudo-TIRF” or “quasi-TIRF”). Here, we performed a quantitative analysis of the dependence of SNR and localization precision on the number of measured photons and the incident angle for static particles under both low (at a planar index-matched interface) and high (within a porous silica matrix) fluorescent background conditions. We found that under noisier imaging conditions, the SNR and localization precision obtained using VAI are up to fivefold and threefold greater, respectively, than those obtained using epi-illumination. Moreover, we demonstrate that the combination of DH-PSF and VAI can significantly improve the accuracy of the measured diffusion coefficient for mobile particles, even at a relatively large distance (50 μm) from the boundary of the ...

applied physics letters

Enhanced information content for three-dimensional localization and tracking using the double-helix point spread function with variable-angle illumination epifluorescence microscopy