Leilei Peng

Understanding how neurons integrate synaptic inputs requires imaging techniques capable of capturing rapid, three-dimensional dendritic events. These processes occur on millisecond timescales and submicron spatial scales, exceeding the speed of conventional two-photon microscopy (2PM). We developed dual-view Bessel two-photon projection microscopy (dv-B2PM), a high-speed volumetric imaging approach that achieves 100 Hz whole-volume acquisition with synaptic-level resolution. dv-B2PM simultaneously records two orthogonal projections of the same 3D volume, preserving spatial information while minimizing ambiguity from structural overlap. Combining dv-B2PM with two-photon glutamate uncaging, we visualized 3D Ca²⁺ dynamics in neurons following localized stimulation. Multi-timescale analysis revealed dendrite-to-soma Ca²⁺ signal propagation, back propagated Ca²⁺ signal from the soma, and multi-frequency (5–40 Hz) Ca²⁺ transients activated along apical dendrites at speeds from ten of microns per second to millimeters per seconds. These findings demonstrate dv-B2PM as a powerful tool for direct visualization of 3D calcium dynamics associated with dendritic integration across extended neuronal structures, bridging the gap between optical imaging and the dynamic biophysics of neuronal integration.

Two-photon light-sheet fluorescence microscopy enables high-resolution imaging of neural activity in brain tissue at a high frame rate. Traditionally, light-sheet microscopy builds up a 3D stack by multiple depth scans with uniform spatial intervals, which substantially limits the volumetric imaging speed. Here, we introduce the depth random-access light-sheet microscopy, allowing rapid switching scanning depth for light-sheet imaging. With a low-cost electrically tunable lens and minimum modification of an existing two-photon light-sheet imaging instrument, we demonstrated fast random depth hopping light-sheet imaging at 100 frames per second in the live brain slice. Through depth random-access, calcium activities for an astrocyte were recorded on four user-selected detection planes at a refreshing rate of 25 Hz.

Light-sheet fluorescence microscopy serves as a fast high-resolution imaging method for neural imaging. However, its 3D imaging ability is often limited by the speed of scanning the detection focal plane in the z-direction. Herein, we develop a rapid random z-access two-photon light-sheet microscope, which incorporates a two-photon Bessel beam light-sheet microscope with a dynamically driven electrical tunable lens (ETL). With a precise ETL calibration process and a novel rapid random z-access method, our system can selectively scan any desired z-section in the 3D imaging volume at the speed of 100 frame-per-second, and allows neural activates monitoring on the living brain tissue at video rate.

SPIE Digital Library Proceedings

SPIE Digital Library Proceedings

Fluorescence lifetime imaging microscopy (FLIM) is a widely used functional imaging method in bioscience. Fourier multiplexed FLIM (FmFLIM), a frequency-domain lifetime measurement method, explores the principle of Fourier (frequency) multiplexing to achieve parallel lifetime detection on multiple fluorescence labels. Combining FmFLIM with a confocal scanning microscope allows multiplexed 3D lifetime imaging of cells and tissues. FmFLIM can also be integrated with the scanning laser tomography imaging method to perform 3D multiplex lifetime imaging of whole embryos and thick tissues.

Structured illumination microscopy (SIM) is a well‐established method for optical sectioning and super‐resolution. The core of structured illumination is using a periodic pattern to excite image signals. This work reports a method for estimating minor pattern distortions from the raw image data and correcting these distortions during SIM image processing. The method was tested with both simulated and experimental image data from two‐photon Bessel light‐sheet SIM. The results proves the method is effective in challenging situations, where strong scattering background exists, signal‐to‐noise ratio (SNR) is low and the sample structure is sparse. Experimental results demonstrate restoring synaptic structures in deep brain tissue, despite the presence of strong light scattering and tissue‐induced SIM pattern distortion.

Multiplexed imaging is a powerful tool for studying complex interactions inside biological systems. Spectral imaging methods that capture multiple fluorescent markers synchronously without sacrificing the imaging speed or resolution are most suitable for live imaging. We describe spectral-encoded structured illumination (spectral-SIM) light-sheet microscopy, which enables parallel multi-excitation-channel imaging in 3D. Spectral-SIM encodes the excitation wavelength as the phase of the illumination pattern, and allows synchronous image capture over multiple excitation channels at the same speed and spatial resolution as mono-channel structured light-sheet imaging. The technique retains structured light-sheet microscopy’s ability in removing out-of-focus and scattered emission background, and generates clear 3D multiplexed images in thick tissue. The capability of this technique was demonstrated by the imaging of live triple-labeled transgenic zebrafish to over 300 μm deep with 0.5μm-by-2μm (lateral-by-axial) resolution.

Fourier multiplexed FLIM (FmFLIM) tomography enables multiplexed 3D lifetime imaging of whole embryos. In our previous FmFLIM system, the spatial resolution was limited to 25 μm because of the trade-off between the spatial resolution and the imaging depth. In order to achieve cellular resolution imaging of thick specimens, we built a tomography system with dual-color Bessel beam. In combination with FmFLIM, the Bessel FmFLIM tomography system can perform parallel 3D lifetime imaging on multiple excitation-emission channels at a cellular resolution of 2.8 μm. The image capability of the Bessel FmFLIM tomography system was demonstrated by 3D lifetime imaging of dual-labeled transgenic zebrafish embryos.

Current deep tissue microscopy techniques are mostly restricted to intensity mapping of fluorophores, which significantly limit their applications in investigating biochemical processes in vivo. We present a deep tissue multiplexed functional imaging method that probes multiple Förster resonant energy transfer (FRET) sensors in live embryos with high spatial resolution. The method simultaneously images fluorescence lifetimes in 3D with multiple excitation lasers. Through quantitative analysis of triple-channel intensity and lifetime images, we demonstrated that Ca²⁺ and cAMP levels of live embryos expressing dual FRET sensors can be monitored simultaneously at microscopic resolution. The method is compatible with a broad range of FRET sensors currently available for probing various cellular biochemical functions. It opens the door to imaging complex cellular circuitries in whole live organisms.