Speed-dependent resolution analysis of ultrafast laser-scanning fluorescence microscopy

The image resolution of an aberration-corrected laser-scanning fluorescence microscopy (LSFM) system, like all other classical optical imaging modalities, is ultimately governed by diffraction limit and can be, in practice, influenced by the noise. However, consideration of only these two parameters is not adequate for LSFM with ultrafast laser-scanning, in which the dwell time of each resolvable image point becomes comparable with the fluorescence lifetime. In view of the continuing demand for faster LSFM, we here revisit the theoretical framework of LSFM and investigate the impact of the scanning speed on the resolution. In particular, we identify there are different speed regimes and excitation conditions in which the resolution is primarily limited by diffraction limit, fluorescence lifetime, or intrinsic noise. Our model also suggests that the speed of the current laser-scanning technologies is still at least an order of magnitude below the limit (∼sub-MHz to MHz), at which the diffraction-limited resolution can be preserved. We thus anticipate that the present study can provide new insight for practical designs and implementation of ultrafast LSFM, based on emerging laser-scanning techniques, e.g., ultrafast wavelength-swept sources, or optical time-stretch.