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Optical imaging through the intact mouse skull is challenging because of skull-induced aberrations and scattering. We found that three-photon excitation provided improved optical sectioning compared with that obtained with two-photon excitation, even when we used the same excitation wavelength and imaging system. Here we demonstrate three-photon imaging of vasculature through the adult mouse skull at >500-μm depth, as well as GCaMP6s calcium imaging over weeks in cortical layers 2/3 and 4 in awake mice, with 8.5 frames per second and a field of view spanning hundreds of micrometers.
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http://dx.doi.org/10.1038/s41592-018-0115-y | DOI Listing |
J Biophotonics
August 2025
State Key Laboratory of Extreme Photonics and Instrumentation, Centre for Optical and Electromagnetic Research, College of Optical Science and Engineering, International Research Center for Advanced Photonics, Zhejiang University, Hangzhou, China.
Three-photon fluorescence microscopy (3PFM) enables high-resolution volumetric imaging in deep tissues but is often hindered by motion artifacts in dynamic physiological environments. Existing solutions, including surgical fixation and conventional image registration algorithms, frequently fail under intense and nonuniform motions, particularly in low-texture or highly deformed regions. To overcome these problems, we propose StabiFormer, a transformer-based optical flow learning network designed for robust motion correction.
View Article and Find Full Text PDFLight Sci Appl
August 2025
Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Label-free detection of biological events at single-cell resolution in the brain can non-invasively capture brain status for medical diagnosis and basic neuroscience research. NADH is an universal coenzyme that not only plays a central role in cellular metabolism but may also be used as a biomarker to capture metabolic processes in brain cells and structures. We have developed a new label-free, multiphoton photoacoustic microscope (LF-MP-PAM) with a near-infrared femtosecond laser to observe endogenous NAD(P)H in living cells.
View Article and Find Full Text PDFChem Sci
August 2025
Division of Physics and Applied Physics, School of Physical and Mathematical Sciences, Nanyang Technological University 21 Nanyang Link Singapore 637371 Singapore
Single- and multi-photon absorption cross-sections quantify the likelihood that a material will absorb one or more photons at a given wavelength. This critical parameter is fundamental to understanding light-matter interactions that underpin key applications in spectroscopy, photochemistry and advanced imaging techniques like multi-photon microscopy and deep tissue imaging. Conventional methods for measuring absorption cross-sections are often limited by sensitivity to sample morphology, type, concentration, and high excitation intensities - factors that can compromise reliability, increase experimental complexity, and risk sample damage.
View Article and Find Full Text PDFPhotochem Photobiol Sci
July 2025
Institute of Photonics and Photon-Technology, Northwest University, #1 Xuefu Avenue, Guodu Education and Industry Zone, Chang'an, Xi'an, 710127, Shaanxi, China.
Nonlinear optical imaging (NLOI) provided detailed morphological information about biological systems, whereas confocal Raman micro-spectral imaging (CRMI) identified the biochemical properties of tissue samples. In this work, we proposed an integrated microscopy system by combining NLOI and CRMI together. An Er⁺-doped femtosecond fiber laser at 1560 nm serves as the excitation source for NLOI modalities, and a semiconductor laser at 830 nm was used for spectra excitation during CRMI investigations.
View Article and Find Full Text PDFNeurophotonics
April 2025
Beijing Municipal Education Commission, Beijing Laboratory of Biomedical Imaging, Beijing, China.
Significance: Miniature multiphoton microscopy has revolutionized neuronal imaging in freely behaving animals. However, its shallow depth of field-a result of high axial resolution-combined with a limited field of view (FOV), makes it challenging for researchers to identify regions of interest in three-dimensional space across multimillimeter cranial windows, thereby reducing the system's ease of use.
Aim: We aimed to develop a multimodal imaging platform with enhanced guidance and a standardized workflow tailored for efficient imaging of freely behaving animals.