Improvement of axial resolution and contrast in temporally focused widefield two-photon microscopy with structured light illumination

Abstract

Although temporally focused wide-field two-photon microscopy (TFM) can perform depth resolved wide field imaging, it cannot avoid the image degradation due to scattering of excitation and emission photons when imaging in a turbid medium. Further, its axial resolution is inferior to standard point-scanning two-photon microscopy. We implemented a structured light illumination for TFM and have shown that it can effectively reject the out-of-focus scattered emission photons improving image contrast. Further, the depth resolution of the improved system is dictated by the spatial frequency of the structure light with the potential of attaining depth resolution better than point-scanning two-photon microscopy.

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

“…Integrated with patterned illumination, the system could further be applied for functional excitation [17,28,29,31], 3D fabrication [30], and structured illumination purposes [39][40][41][42]. In this paper, we have demonstrated that the AEC would vary with different patterns at the same single spatial frequency and different orientations, and could reach nearly 29 % difference, e. g., from À11 % to + 18 % with 1.09 mm À1 spatial frequency patterns at the 908 and 08 orientations.…”

High throughput second harmonic imaging for label-free biological applications

“…In the imaging system of Fig. 2, the illuminating section has a larger depth of field compared to scanning nonlinear microscopes and spatiotemporal focusing microscopes [54][55][56][57][58]. It is possible to limit the illumination depth by e.g.…”

“…It is possible to limit the illumination depth by e.g. using a 5 micron high sample cell [65] or by producing axial crosssections using structured illumination [58]. The latter is possible but has not been implemented in the present microscope.…”

“…The second one is to parallelize the imaging process. Recent advancements [46] include wide-field counter propagating SHG geometries [10,11], lens-less imaging approaches [49], harmonic holography [50,51], multi-confocal imaging [52,53], and spatiotemporal wide-field illumination [54][55][56][57][58].…”

Accumulating advantages, reducing limitations: Multimodal nonlinear imaging in biomedical sciences – The synergy of multiple contrast mechanisms

“…The scattering problem can be overcome by projecting illumination patterns into the specimen, i.e., by structured illumination [98]. Another approach is based on temporal focusing in combination with structured illumination, which allows to visualize larger areas without mechanical scanning and prospectively a further substantial increase in frame rate [99,100].…”