Two-photon fluorescence imaging of mitochondria viscosity with water-soluble pyridinium inner salts

Rational design of mitochondrial viscosity probes with water-soluble two-photon (TP) fluorescence probe still remains a challenge in practical biological applications. Herein, three novel water-soluble TP active sulfosalt derivatives (Mito-Z, Mito-H,...

In vivo volumetric imaging of calcium and glutamate activity at synapses with high spatiotemporal resolution

Studying neuronal activity at synapses requires high spatiotemporal resolution. For high spatial resolution in vivo imaging at depth, adaptive optics (AO) is required to correct sample-induced aberrations. To improve temporal resolution, Bessel focus has been combined with two-photon fluorescence microscopy (2PFM) for fast volumetric imaging at subcellular lateral resolution. To achieve both high-spatial and high-temporal resolution at depth, we develop an efficient AO method that corrects the distorted wavefront of Bessel focus at the objective focal plane and recovers diffraction-limited imaging performance. Applying AO Bessel focus scanning 2PFM to volumetric imaging of zebrafish larval and mouse brains down to 500 µm depth, we demonstrate substantial improvements in the sensitivity and resolution of structural and functional measurements of synapses in vivo. This enables volumetric measurements of synaptic calcium and glutamate activity at high accuracy, including the simultaneous recording of glutamate activity of apical and basal dendritic spines in the mouse cortex.

Non-Diffracting Light Wave: Fundamentals and Biomedical Applications

The light propagation in the medium normally experiences diffraction, dispersion, and scattering. Studying the light propagation is a century-old problem as the photons may attenuate and wander. We start from the fundamental concepts of the non-diffracting beams, and examples of the non-diffracting beams include but are not limited to the Bessel beam, Airy beam, and Mathieu beam. Then, we discuss the biomedical applications of the non-diffracting beams, focusing on linear and nonlinear imaging, e.g., light-sheet fluorescence microscopy and two-photon fluorescence microscopy. The non-diffracting photons may provide scattering resilient imaging and fast speed in the volumetric two-photon fluorescence microscopy. The non-diffracting Bessel beam and the Airy beam have been successfully used in volumetric imaging applications with faster speed since a single 2D scan provides information in the whole volume that adopted 3D scan in traditional scanning microscopy. This is a significant advancement in imaging applications with sparse sample structures, especially in neuron imaging. Moreover, the fine axial resolution is enabled by the self-accelerating Airy beams combined with deep learning algorithms. These additional features to the existing microscopy directly realize a great advantage over the field, especially for recording the ultrafast neuronal activities, including the calcium voltage signal recording. Nonetheless, with the illumination of dual Bessel beams at non-identical orders, the transverse resolution can also be improved by the concept of image subtraction, which would provide clearer images in neuronal imaging.

Two Photon Fluorescence Microscopy of the Unstained Human Cochlea Reveals Organ of Corti Cytoarchitecture

Sensorineural hearing loss (SNHL) is the most common sensory deficit worldwide, and it typically originates from the cochlea. Methods to visualize intracochlear cells in living people are currently lacking, limiting not only diagnostics but also therapies for SNHL. Two-photon fluorescence microscopy (TPFM) is a high-resolution optical imaging technique. Here we demonstrate that TPFM enables visualization of sensory cells and auditory nerve fibers in an unstained, non-decalcified adult human cochlea.