Scattering reduction by structured light illumination in line-scanning temporal focusing microscopy

Abstract

Line-scanning temporal focusing microscopy (LineTFM) is capable of imaging biological samples more than 10 times faster than two-photon laser point-scanning microscopy (TPLSM), while achieving nearly the same lateral and axial spatial resolution. However, the image contrast taken by LineTFM is lower than that by TPLSM because LineTFM is severely influenced by biological tissue scattering. To reject the scattered photons, we implemented LineTFM using both structured illumination and uniform illumination combined with the HiLo post-processing algorithm, called HiLL microscopy (HiLo-Line-scanning temporal focusing microscopy). HiLL microscopy significantly reduces tissue scattering and improves image contrast. We demonstrate HiLL microscopy with in vivo brain imaging. This approach could potentially find applications in monitoring fast dynamic events and in mapping high resolution structures over a large volume.

Scanless volumetric imaging by selective access multifocal multiphoton microscopy

“…Accurately locating several hundreds of submicrometer excitation spots on the targeted positions from the 3D structure "map" requires highly precise registration between the TF system and the saMMM. Thus, we integrated the auxiliary line-scan TF multiphoton imaging system within the saMMM setup [37], while spatial resolution of the line-scan TF is similar to standard PS two-photon microscopy ( Fig. S1 of Supplement 1) due to spectral and spatial filling of the objective back aperture [20][21][22]37].…”

“…Thus, we integrated the auxiliary line-scan TF multiphoton imaging system within the saMMM setup [37], while spatial resolution of the line-scan TF is similar to standard PS two-photon microscopy ( Fig. S1 of Supplement 1) due to spectral and spatial filling of the objective back aperture [20][21][22]37]. After structural imaging with the line-scan TF, we selected spots along multiple dendrites of the same cell.…”

Multiline Orthogonal Scanning Temporal Focusing (mosTF) microscopy for reducing scattering in high-speed in vivo brain imaging

“…2b-d . When compared to the speed of TPLSM [3][4][5] , which takes about 30 s to collect sufficient emissions from one plane in our in vivo preparation, mosTF shows an eight-fold improvement in imaging speed. Noticed that time comparison between different systems are based on collecting similar number of emission photons.…”

“…As an alternative to TPLSM, line-scanning temporal focusing microscopy (lineTF) has been used for fast two-photon imaging in mouse brain [5][6] . lineTF focuses light spatially along one axis and modulates pulse dispersion along the other axis using the principle of "temporal focusing" 7-9 . Instead of point-scanning, line scanning improved imaging speed by over an order of magnitude 5 . Even though lineTF has the same excitation point spread function as TPLSM, the theoretical resolution has never been realized in vivo.…”

Temporal focusing multiphoton microscopy with optimized parallel multiline scanning for fast biotissue imaging

“…[46][47][48][49] The TFMPM image contrast can be further improved by eliminating the background noise. The HiLo technique [50][51][52] and Hilbert transform 53 can retrieve the in-focus image, which is modulated by the applied structural pattern, while rejecting the out-of-focus background. The optical pattern in the TFMPM changes the filling ratio on the back-focal plane of the objective and has been shown to affect the system AEC as well.…”