Weijian Zong

Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice

Developments in miniaturized microscopes have enabled visualization of brain activities and structural dynamics in animals engaging in self-determined behaviors. However, it remains a challenge to resolve activity at single dendritic spines in freely behaving animals. Here, we report the design and application of a fast high-resolution, miniaturized two-photon microscope (FHIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a hollow-core photonic crystal fiber delivering 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors (GFP and GCaMP6) at high spatiotemporal resolution (0.64 μm laterally and 3.35 μm axially, 40 Hz at 256 × 256 pixels for raster scanning and 10,000 Hz for free-line scanning). We demonstrate the microscopes robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.

Large-field high-resolution two-photon digital scanned light-sheet microscopy.

Recent advent of light-sheet fluorescent microscopy (LSFM) has revolutionized three-dimensional biological imaging with high temporal resolution and minimal photodamage, enabling long-term fluorescence imaging of tissues and small organisms [1-2]. By combining two-photon fluorescence excitation with LSFM, Truong et al. [3] have created a two-photon digital scanned light-sheet microscope (2P-DSLM), allowing for deep-tissue imaging of highly scattering Drosophila embryos and fast beating hearts of zebrafish. Similar to classical LSFM configurations, a 2P-DSLM uses a low numerical aperture (NA < 0.1) to achieve a long and homogenous illumination. This leads to a thick light sheet and thus reduces axial resolution and image contrast. On the other hand, Betzig and colleagues have used a Bessel beam to generate thin single-photon light sheet that yields superb axial resolution [4]. However, the field of view and the penetration depth are limited in such system. To achieve high axial resolution (thin light sheet) and large field of view simultaneously, here we have developed a novel two-photon three-axis digital scanned light-sheet microscope (2P3A-DSLM) based on ultrafast axial scanning of illumination focal spot with a tunable acoustic gradient (TAG) index device. This new technique provides a sub-micron axial resolution, a large field of view of 170 × 170 μm 2 in deep tissues, and a high temporal resolution limited only by the detection camera. In vivo imaging with 2P3A-DSLM resolved subcellular structures and dynamic processes in small organisms. A schematic illustration and configuration of the 2P3A-DSLM is shown in Figure 1A. A TAG lens uses acoustic wave to radially excite a fluid-filled cylindrical cavity and produce continuous changes in refrac-tive power that enables rapid change of its axial focal plane within 10 μs [5-6]. Here we used the TAG lens to rapidly translate the focal spot of a femtosecond laser (140 fs, repetition rate, 80 MHz, Chameleon Version II, Coherent) along the optics axis (x-direction) in front of the illumination objective (40× NA 0.8, Nikon). A gal-vo scanning mirror (GSM) was used to scan the laser beam vertically to the axis of the illumination objective (y-direction). By driving the TAG lens to refocus at ~450 kHz and the GSM at 1 kHz, we were able to create an ultra-thin scanned 2P light sheet within one millisecond. The size of the scanned light sheet can be modulated by adjusting the modulation intensity of the TAG (x-direction) and scanning amplitude of the galvo mirrors (y-direction) (Figure 1B-1D). During the fast …

Diacylglycerol Guides the Hopping of Clathrin-Coated Pits along Microtubules for Exo-Endocytosis Coupling

Many receptor-mediated endocytic processes are mediated by constitutive budding of clathrin-coated pits (CCPs) at spatially randomized sites before slowly pinching off from the plasma membrane (60-100 s). In contrast, clathrin-mediated endocytosis (CME) coupled with regulated exocytosis in excitable cells occurs at peri-exocytic sites shortly after vesicle fusion (∼10 s). The molecular mechanism underlying this spatiotemporal coupling remains elusive. We show that coupled endocytosis makes use of pre-formed CCPs, which hop to nascent fusion sites nearby following vesicle exocytosis. A dynamic cortical microtubular network, anchored at the cell surface by the cytoplasmic linker-associated protein on microtubules and the LL5β/ELKS complex on the plasma membrane, provides the track for CCP hopping. Local diacylglycerol gradients generated upon exocytosis guide the direction of hopping. Overall, the CCP-cytoskeleton-lipid interaction demonstrated here mediates exocytosis-coupled fast recycling of both plasma membrane and vesicular proteins, and it is required for the sustained exocytosis during repetitive stimulations.

Shadowless-illuminated variable-angle TIRF (siva-TIRF) microscopy for the observation of spatial-temporal dynamics in live cells

Total-internal-reflection fluorescence (TIRF) microscopy provides high optical-sectioning capability and a good signal-contrast ratio for structures near the surfaces of cells. In recent years, several improvements have been developed, such as variable-angle TIRF (VA-TIRF) and spinning TIRF (sp-TIRF), which permit quantitative image analysis and address non-uniform scattering fringes, respectively. Here, we present a dual-color DMD-based shadowless-illuminated variable-angle TIRF (siva-TIRF) system that provides a uniform illumination field. By adjusting the incidence angle of the illuminating laser on the back focal plane (BFP) of the objective, we can rapidly illuminate biological samples in layers of various thicknesses in TIRF or hollow-cone epi-fluorescence mode. Compared with other methods of accomplishing VA-TIRF/sp-TIRF illumination, our system is simple to build and cost-effective, and it provides optimal multi-plane dual-color images. By showing spatiotemporal correlated movement of clathrin-coated structures with microtubule filaments from various layers of live cells, we demonstrate that cortical microtubules are important spatial regulators of clathrin-coated structures. Moreover, our system can be used to prove superb axial information of three-dimensional movement of structures near the plasma membrane within live cells.

910nm femtosecond Nd-doped fiber laser for in vivo two-photon microscopic imaging.

Pre-chirp technique was used in an Nd-doped fiber amplifier to optimize high-quality 910 nm pulses with the pulses width of 114 fs and pulse energy of 4.4 nJ. The in vivo zebrafish imaging results from our totally home-made microscopy proves our femtosecond Nd fiber laser an ideal source in two-photon microscopic imaging.

High power 780 nm femtosecond fiber laser

We demonstrate an 880 mW 780 nm femtosecond laser based on a high-order dispersion compensated Er-doped fiber laser and frequency doubling. It has been used on a two-photon three-axis digital scanned light-sheet microscopy.

In vivo Imaging β-cell Function Reveals Two Waves of β-cell Maturation

The insulin-secreting cells generated from stem cells in vitro are less glucose responsive than primary β-cells. To search for the missing ingredients that are needed for β-cell maturation, we have longitudinally monitored function of every β-cell in Tg (ins:Rcamp1.07) zebrafish embryos with a newly-invented two-photon light-sheet microscope. We have shown that β-cell maturation begins from the islet mantle and propagates to the islet core during the hatching period, coordinated by the islet vascularization. Lower concentration of glucose is optimal to initiate β-cell maturation, while increased glucose delivery to every cell through microcirculation is required for functional boosting of the β-cells. Both the initiation and the boosting of β-cell maturation demands activation of calcineurin/NFAT by glucose. Calcineurin activator combined with glucose promotes mouse neonatal β-cells cultured in vitro to mature to a functional state similar to adult β-cells, suggesting a new strategy for improving stem cell-derived β-like cell function in vitro.

Imaging functional maturation of pancreatic beta-cells and neuronal circuit activity in Zebrafish in vivo using two-photon three-axis digital scanned lightsheet microscopy

Here we have developed a novel large-field high-resolution two-photon digital scanned light-sheet microscopy (2P3A-DSLM) based on ultrafast axial scanning of illumination focal spot with a tunable acoustic gradient (TAG) index device. This new technique provides a sub-micron axial resolution with a large field of view in deep tissues. In vivo imaging with 2P3A-DSLM resolves functional maturation of pancreatic beta cells and neuron circuit activities in live Zebrafish embryos.

4.4 nJ, 114 fs Nd-doped fiber laser pulses at 920nm for in vivo two-photon microscopic imaging

We optimized pre-chirp in an Nd-doped fiber amplifier to achieve high-quality femtosecond 920 nm pulses. The in vivo zebrafish imaging proves the laser amplifier an ideal source in two-photon microscopic imaging.

Two-photon three-axis digital scanned light-sheet microscopy (2P3A-DSLM)

We demonstrate a new two-photon scanned light-sheet microscopy with diffraction-limited thickness and tailorable illumination area from 50×50 μm² to 500×500 μm², capable of multi-scale live imaging in one setup.