We propose HiLo based line-scanning temporal focusing microscopy to enhance contrast and axial confinement in deep imaging, and demonstrate its superiority by volumetric imaging of microglia and neurons in mouse brains in vivo.
To stimulate neural ensemble selectively at high resolution, we propose non-convex optimization with spherical aberration compensation algorithm for two-photon optogenetics, which achieves high excitation efficiency and precise axial positioning.
Wide-field fluorescence microscopy (WFFM) is widely adopted in biomedical studies. However, axial resolution in most WFFM is poor due to the absence of optical-sectioning capability. To achieve wide-field optical-sectioning, several methods have been proposed, most of which need at least two images to reconstruct one optical sectioning image. So, the frame rate of current wide-field optical sectioning microscopy is no more than half of that of conventional WFFM, which may not meet the speed requirement of fast biodynamic studies. We introduce a novel high-speed, wide-field optical sectioning method based on local contrast weighting function and two-dimensional Hilbert-Huang transform, in which only one structured image is required to reconstruct an optical sectioning image. In this way, the loss of temporal resolution in conventional wide-field optical sectioning microscopy is compensated. We validated this method with the imaging of mouse brain slices.
To reveal fast biological dynamics in deep tissue, we combine two wavefront engineering methods that were developed in our laboratory, namely optical phase-locked ultrasound lens (OPLUL) based volumetric imaging and iterative multiphoton adaptive compensation technique (IMPACT). OPLUL is used to generate oscillating defocusing wavefront for fast axial scanning, and IMPACT is used to compensate the wavefront distortions for deep tissue imaging. We show its promising applications in neuroscience and immunology.
In vivo flow cytometry has found numerous applications in biology and pharmacology. However, conventional cytometry does not provide the detailed morphological information that is needed to fully determine the phenotype of individual circulating cells. Imaging cytometry, capable of visualizing the morphology and dynamics of the circulating cells at high spatiotemporal resolution, is highly desired. Current wide-field based image cytometers are limited in the imaging depth and provide only two-dimensional resolution. For deep tissue imaging, laser scanning two-photon fluorescence microscopy (TPM) is widely adopted. However, for applications in flow cytometry, the axial scanning speed of current TPMs is inadequate to provide high-speed cross-sectional imaging of vasculature. We have integrated an optical phase-locked ultrasound lens into a standard TPM and achieved microsecond-scale axial scanning. With a galvo scanner for transverse scanning, we achieved kHz cross-sectional frame rate. Here we report its applications for in vivo deformability cytometry and in vivo imaging flow cytometry, and demonstrate the capability of imaging dynamical morphologies of flowing cells, distinguishing cells and cellular clusters, and simultaneously quantifying different cell populations based on their fluorescent labels.
We demonstrate a fiber-based two-color source of picosecond pulses for coherent Raman scattering (CRS) microscopy.
An Yb-doped fiber laser combined with a divided-pulse amplifier produce up to 3 W of power tunable from 1030 nm to
1040 nm. A normal dispersion photonic crystal fiber is used to blue-shift the pulses through seeded four-wave mixing.
Pulses with up to 150 mW of average power are produced, tunable between 770 nm and 800 nm. Imaging of animal
tissue and cells is demonstrated.
Nonlinear microscopies based on coherent anti-Stokes Raman scattering (CARS) and stimulated Raman
scattering (SRS) have demonstrated important capabilities for
non-invasive label-free imaging of biological
samples [1, 2]. In contrast to other nonlinear microscopies, Raman imaging is best performed with
excitation pulses in the 2-7 ps range, which overlap spectrally with the desired Raman resonances. There is
interest in performing CARS and SRS imaging with femtosecond pulses or picosecond/femtosecond
combinations, or with chirped femtosecond pulses. This article will outline the challenges associated with
the demonstration of a picosecond fiber source for Raman microscopies, and assess approaches to such a
source.
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