Temporal Focusing Microscopy

Abstract

Axial localization of multiphoton excitation to a single plane is achieved by temporal focusing of an ultrafast pulsed excitation. We take advantage of geometrical dispersion in an extremely simple experimental setup, where an ultrashort pulse is temporally stretched and hence its peak intensity is lowered outside the focal plane of the microscope. Using this strategy, out-of-focus multiphoton excitation is dramatically reduced, and the achieved axial resolution is comparable to line-scanning multiphoton microscopy for wide-field excitation and to point-scanning multiphoton microscopy for line excitation. In this introduction, we provide a detailed description of the considerations in choosing the experimental parameters, as well as the alignment of a temporal focusing add-on to a multiphoton microscope. We also review current advances and applications for this technique.

Temporal focusing‐based widefield multiphoton microscopy with spatially modulated illumination for biotissue imaging

“…However, the high frame rate needed for dynamic biological observation has motivated the development of MPE microscopy toward highthroughput configurations [8]. Temporal focusingbased multiphoton excitation microscopy (TFMPEM) provides direct widefield TPEF imaging [15][16][17][18][19], in which the diffraction element (e. g., a blazed grating) separates different spectral components of ultrashort laser pulses into different angles according to the diffraction equation and induces spatial dispersion that broadens the laser pulse width. In the so-called 4-f system setup, the diverse spectral components temporally overlap in phase only at the focal plane of the objective lens.…”

Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate

“…We have demonstrated that a moderate increase in repetition rate does not adversely affect photobleaching characteristics, but rather significantly enhances the fluorescence signal. Increasing the repetition rate, in our hands, allowed for the implementation of a setup while solely using off-the-shelf components [34]. Nevertheless, this setup was capable of acquiring clear readouts of neuronal activity from live C. elegans animals under aversive stimulation.…”

HiLo Based Line Scanning Temporal Focusing Microscopy for High-Speed, Deep Tissue Imaging

“…However, the improvement of temporal resolution in LSTPM is at the cost of axial resolution, since the beam is focused only along one spatial dimension [ 12 ]. Thus, line scanning temporal focusing microscopy (LSTFM) was developed, which has the capability of parallel excitation while maintaining tight axial confinement by spatio-temporal focusing [ 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. However, with the increase in penetration depth, the accumulated wavefront distortions would deteriorate axial confinement of the excitation light.…”