Abstract
Due to diffraction of light, the conventional fluorescent microscope is a system with a band-limited information spatial channel. Every super-resolved fluorescent microscope circumvents this limitation by avoiding the simultaneous signaling of adjacent (< diffraction limit distance) fluorophores recording them sequentially in time. Notably, the confocal microscope also agrees to this perspective. Thus, the current super-resolved systems encode extra spatial information in the temporal channel.Here we discuss if other channels (dimensions) can be used to further improve the resolution of a fluorescence microscope. In particular, we describe time-gated STED microscopy and image-scanning microscopy (ISM) as examples of exploring extra dimensions, respectively the photon arrival time and the spatial image plane dimensions.Confocal scanning microscopy (CSM) can theoretically surpass the diffraction limit by a factor of √2. Practically, this improvement is sacrificed to obtain a good signal-to-noise ratio (SNR). Image scanning microscopy (ISM) solved this limitation by substituting the single point detector with a 2D array of detector. We showed that ISM can be straightforwardly implemented by using a quadrant detector. This implementation offers resolution close to the CSM theoretical value, improves the SNR by factor of 1.5 with respect to the CSM counterpart, and may be implemented without losing the optical sectioning capability and the system versatility.Time-gated detection increases the spatial resolution of a gated continuous-wave-(CW-) STED microscope, with the drawback of reducing the SNR. Thus, in sub-optimal conditions, such as a low-photons budget regime, the SNR reduction can cancel-out the expected gain in resolution. We developed a method which does not discard photons, but instead sorts all the photons in different time-gates according to their arrival time and recombines them through a multi-image deconvolution. Our results show that the SNR of the restored image improves, thereby improving the effective resolution.
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