Temporal‐Focusing Multiphoton Excitation Single‐Molecule Localization Microscopy Using Spontaneously Blinking Fluorophores

Abstract Single‐molecule localization microscopy (SMLM) based on temporal‐focusing multiphoton excitation (TFMPE) and single‐wavelength excitation is used to visualize the three‐dimensional (3D) distribution of spontaneously blinking fluorophore‐labeled subcellular structures in a thick specimen with a nanoscale‐level spatial resolution. To eliminate the photobleaching effect of unlocalized molecules in out‐of‐focus regions for improving the utilization rate of the photon budget in 3D SMLM imaging, SMLM with single‐wavelength TFMPE achieves wide‐field and axially confined two‐photon excitation (TPE) of spontaneously blinking fluorophores. TPE spectral measurement of blinking fluorophores is then conducted through TFMPE imaging at a tunable excitation wavelength, yielding the optimal TPE wavelength for increasing the number of detected photons from a single blinking event during SMLM. Subsequently, the TPE fluorescence of blinking fluorophores is recorded to obtain a two‐dimensional TFMPE‐SMLM image of the microtubules in cancer cells with a localization precision of 18±6 nm and an overall imaging resolution of approximately 51 nm, which is estimated based on the contribution of Nyquist resolution and localization precision. Combined with astigmatic imaging, the system is capable of 3D TFMPE‐SMLM imaging of brain tissue section of a 5XFAD transgenic mouse with the pathological features of Alzheimer's disease, revealing the distribution of neurotoxic amyloid‐beta peptide deposits.