Avalanche photodiode detection with object scanning and image restoration provides 2–4 fold resolution increase in two‐photon fluorescence microscopy

High-quantum-efficiency photodetection, millisecond pixel dwell time stage scanning and image restoration by maximum-likelihood estimation are synergetically combined and shown to improve the resolution of two-photon excitation microscopy 2–4 fold in all directions. Measurements of the two-photon excitation point-spread function (PSF) of a 1.4 aperture oil immersion lens are carried out by imaging fluorescence beads with a diameter of one seventh of the excitation wavelength (830 nm) and subsequent deconvolution with the bead object function. The proposed method of resolution increase is applied to beads as well as to rhodamine labelled actin fibres in mouse fibroblast cells. As the resolution improvement is not based on the non-linear effect of two-photon excitation, the results imply a comparable resolution increase in single-photon excitation confocal microscopy. In the fibroblasts, we established a three-fold improvement in axial resolution, namely from 840 nm before, to 280 nm after restoration (full-width at half-maximum). Actin fibres with axial distances of 850 nm, otherwise difficult to discern, are fully separated. In the lateral direction, images of fluorescence beads of about 110 nm diameter are restored to the real dimensions of the beads with an accuracy of better than one pixel (41 nm).