Large-scale two-photon calcium imaging in freely moving mice.

We developed a miniaturized two-photon microscope (MINI2P) for fast, high-resolution, multiplane calcium imaging of over 1,000 neurons at a time in freely moving mice. With a microscope weight below 3 g and a highly flexible connection cable, MINI2P allowed stable imaging with no impediment of behavior in a variety of assays compared to untethered, unimplanted animals. The improved cell yield was achieved through a optical system design featuring an enlarged field of view (FOV) and a microtunable lens with increased z-scanning range and speed that allows fast and stable imaging of multiple interleaved planes, as well as 3D functional imaging. Successive imaging across multiple, adjacent FOVs enabled recordings from more than 10,000 neurons in the same animal. Large-scale proof-of-principle data were obtained from cell populations in visual cortex, medial entorhinal cortex, and hippocampus, revealing spatial tuning of cells in all areas.

Functional network topography of the medial entorhinal cortex.

The medial entorhinal cortex (MEC) creates a map of local space, based on the firing patterns of grid, head-direction (HD), border, and object-vector (OV) cells. How these cell types are organized anatomically is debated. In-depth analysis of this question requires collection of precise anatomical and activity data across large populations of neurons during unrestrained behavior, which neither electrophysiological nor previous imaging methods fully afford. Here, we examined the topographic arrangement of spatially modulated neurons in the superficial layers of MEC and adjacent parasubiculum using miniaturized, portable two-photon microscopes, which allow mice to roam freely in open fields. Grid cells exhibited low levels of co-occurrence with OV cells and clustered anatomically, while border, HD, and OV cells tended to intermingle. These data suggest that grid cell networks might be largely distinct from those of border, HD, and OV cells and that grid cells exhibit strong coupling among themselves but weaker links to other cell types.

Miniature two-photon microscopy for enlarged field-of-view, multi-plane and long-term brain imaging.

We have developed a miniature two-photon microscope equipped with an axial scanning mechanism and a long-working-distance miniature objective to enable multi-plane imaging over a volume of 420 × 420 × 180 µm3 at a lateral resolution of ~1 µm. Together with the detachable design that permits long-term recurring imaging, our miniature two-photon microscope can help decipher neuronal mechanisms in freely behaving animals.

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 microscope's robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.

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.

Core-pumped femtosecond Nd:fiber laser at 910 and 935 nm.

We report a core-pumped all-normal dispersion mode-locked Nd-doped fiber laser at 910 and 935 nm. The pulse is compressed to 198 fs, and the pulse energy is 1.3 nJ. The slope efficiency is more than 14%. This laser is tested as the optical source for the two-photon fluorescence imaging of pollen.

In vivo Imaging of Calcium Activities from Pancreatic ß-cells in Zebrafish Embryos Using Spinning-disc Confocal and Two-photon Light-sheet Microscopy.

Visualizing the function of pancreatic ß-cells in vivo has been a long-sought goal for ß-cell researchers. Unlike imaging of ß-cells in mammalian species with conventional positron emission tomography and single-photon emission computed tomography, which only provides limited spatial-temporal resolution, transparent zebrafish embryos are a unique model that allows high-resolution fluorescent imaging of ß-cells in their native physiological microenvironment in vivo. Here, we detail a protocol for real-time visualization of individual ß-cell function in vivo in a non-invasive manner, through combination of a novel transgenic zebrafish reporter line Tg (ins:Rcamp1.07) with both a commercial spinning-disc confocal microscope and an in-house developed super-resolution microscope (2P3A-DSLM). The protocol described here allows for the longitudinal monitoring of dynamic calcium activities from heterogeneous ß-cells in early developing zebrafish embryos and is readily adaptable for use in imaging other important processes in islet biology, as well as screening new compounds that can promote ß-cell function or maturation using a living whole organism system.

37.4 fs pulse generation in an Er:fiber laser at a 225 MHz repetition rate.

We report 37.4 fs pulse generation from a ring-cavity Er:fiber laser at a repetition rate of 225 MHz. The spectral bandwidth of the pulse is 135 nm. The single-pulse energy is 0.31 nJ.

New solution for fast axial scanning in fluorescence microscopy.

A novel technique based on the remote-focusing concept, using a galvanometer scanner combined with a self-fabricated "step mirror" or "tilted mirror" to transform fast lateral scanning into axial scanning, was reported as a new solution for fast, subcellular, 3D fluorescence imaging.

Light-sheet fluorescence imaging charts the gastrula origin of vascular endothelial cells in early zebrafish embryos.

It remains challenging to construct a complete cell lineage map of the origin of vascular endothelial cells in any vertebrate embryo. Here, we report the application of in toto light-sheet fluorescence imaging of embryos to trace the origin of vascular endothelial cells (ECs) at single-cell resolution in zebrafish. We first adapted a previously reported method to embryo mounting and light-sheet imaging, created an alignment, fusion, and extraction all-in-one software (AFEIO) for processing big data, and performed quantitative analysis of cell lineage relationships using commercially available Imaris software. Our data revealed that vascular ECs originated from broad regions of the gastrula along the dorsal-ventral and anterior-posterior axes, of which the dorsal-anterior cells contributed to cerebral ECs, the dorsal-lateral cells to anterior trunk ECs, and the ventral-lateral cells to posterior trunk and tail ECs. Therefore, this work, to our knowledge, charts the first comprehensive map of the gastrula origin of vascular ECs in zebrafish, and has potential applications for studying the origin of any embryonic organs in zebrafish and other model organisms.

Long-term, in toto live imaging of cardiomyocyte behaviour during mouse ventricle chamber formation at single-cell resolution.

Mapping of the holistic cell behaviours sculpting the four-chambered mammalian heart has been a goal or previous studies, but so far only success in transparent invertebrates and lower vertebrates with two-chambered hearts has been achieved. Using a live-imaging system comprising a customized vertical light-sheet microscope equipped with a mouse embryo culture module, a heartbeat-gated imaging strategy and a digital image processing framework, we realized volumetric imaging of developing mouse hearts at single-cell resolution and with uninterrupted cell lineages for up to 1.5 d. Four-dimensional landscapes of Nppa+ cardiomyocyte cell behaviours revealed a blueprint for ventricle chamber formation by which biased outward migration of the outermost cardiomyocytes is coupled with cell intercalation and horizontal division. The inner-muscle architecture of trabeculae was developed through dual mechanisms: early fate segregation and transmural cell arrangement involving both oriented cell division and directional migration. Thus, live-imaging reconstruction of uninterrupted cell lineages affords a transformative means for deciphering mammalian organogenesis.

In vivo imaging of ß-cell function reveals glucose-mediated heterogeneity of ß-cell functional development.

How pancreatic ß-cells acquire function in vivo is a long-standing mystery due to the lack of technology to visualize ß-cell function in living animals. Here, we applied a high-resolution two-photon light-sheet microscope for the first in vivo imaging of Ca2+activity of every ß-cell in Tg (ins:Rcamp1.07) zebrafish. We reveal that the heterogeneity of ß-cell functional development in vivo occurred as two waves propagating from the islet mantle to the core, coordinated by islet vascularization. Increasing amounts of glucose induced functional acquisition and enhancement of ß-cells via activating calcineurin/nuclear factor of activated T-cells (NFAT) signaling. Conserved in mammalians, calcineurin/NFAT prompted high-glucose-stimulated insulin secretion of neonatal mouse islets cultured in vitro. However, the reduction in low-glucose-stimulated insulin secretion was dependent on optimal glucose but independent of calcineurin/NFAT. Thus, combination of optimal glucose and calcineurin activation represents a previously unexplored strategy for promoting functional maturation of stem cell-derived ß-like cells in vitro.

A frequency domain SIM reconstruction algorithm using reduced number of images.

Conventional two-dimensional structured illumination microscopy (SIM) requires 9 raw images to reconstruct a super-resolved image. In order to increase the frame rate of 2DSIM, attempts are being made to reduce the number of raw SIM images. However, all the proposed SIM reconstruction algorithms (SIM-RA) capable of reconstructing super-resolution (SR) image with a reduced number of raw SIM images operate in the spatial domain. Here, we present a frequency domain SIM-RA based on ordinary least squares technique, which enables reconstruction of SR image using 4 raw SIM images. Unlike the spatial domain RA, which produces the SR image through iterative convergence, the presented RA provides a single step solution. It also reveals the fundamental limitation of least number of raw images required for resolution doubling in SIM.

Two-photon light-sheet nanoscopy by fluorescence fluctuation correlation analysis.

Advances in light-sheet microscopy have enabled the fast three-dimensional (3D) imaging of live cells and bulk specimens with low photodamage and phototoxicity. Combining light-sheet illumination with super-resolution imaging is expected to resolve subcellular structures. Actually, such kind of super-resolution light-sheet microscopy was recently demonstrated using a single-molecule localization algorithm. However, the imaging depth and temporal resolution of this method are limited owing to the requirements of precise single molecule localization and reconstruction. In this work, we present two-photon super-resolution light-sheet imaging via stochastic optical fluctuation imaging (2PLS-SOFI), which acquires high spatiotemporal resolution and excellent optical sectioning ability. 2PLS-SOFI is based on non-linear excitation of fluctuation/blinking probes using our recently developed fast two-photon three-axis digital scanned light-sheet microscope (2P3A-DSLM), which enables both deep penetration and thin sheet of light. Overall, 2PLS-SOFI demonstrates up to 3-fold spatial resolution enhancement compared with conventional two-photon light-sheet (2PLS) microscopy and about 40-fold temporal resolution enhancement compared with individual molecule localization-selective plane illumination microscopy (IML-SPIM). Therefore, 2PLS-SOFI is promising for 3D long-term, deep-tissue imaging with high spatiotemporal resolution.

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 LL5ß/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.