Millimeter field-of-view miniature two-photon microscopy for brain imaging in freely moving mice.

Development of miniature two-photon microscopy (m2PM) has made it possible to observe fine structure and activity of neurons in the brain of freely moving animals. However, the imaging field-of-view of existing m2PM is still significantly smaller than that of miniature single-photon microscopy. Here we report that, through the design of low-magnification objective, large field-of-view scan lens and small tilt angle microscanner, a 2.5-g m2PM achieved a field-of-view of 1000 × 788 µm2, comparable to that of a typical single-photon miniscope. We demonstrated its capability by imaging neurons, dendrites and spines in the millimeter field-of-view, and simultaneous recording calcium activities, through a gradient-index lens, of approximately 400 neurons in the dorsal hippocampal CA1 in a freely moving mouse. Integrated with a detachable 1.2-g fast z-scanning module, it enables a 1000 × 788 × 500 µm3 volumetric neuronal imaging in the cerebral cortex. Thus, millimeter FOV m2PM provides a powerful tool for deciphering neuronal population dynamics in experimental paradigms allowing for animal's free movement.

Miniature Two-Photon Microscopic Imaging Using Dielectric Metalens.

Miniature two-photon microscopy has emerged as a powerful technique for investigating brain activity in freely moving animals. Ongoing research objectives include reducing probe weight and minimizing animal behavior constraints caused by probe attachment. Employing dielectric metalenses, which enable the use of sizable optical components in flat device structures while maintaining imaging resolution, is a promising solution for addressing these challenges. In this study, we designed and fabricated a titanium dioxide metalens with a wavelength of 920 nm and a high aspect ratio. Furthermore, a meta-optic two-photon microscope weighing 1.36 g was developed. This meta-optic probe has a lateral resolution of 0.92 µm and an axial resolution of 18.08 µm. Experimentally, two-photon imaging of mouse brain structures in vivo was also demonstrated. The flat dielectric metalens technique holds promising opportunities for high-performance integrated miniature nonlinear microscopy and endomicroscopy platforms in the biomedical field.

Miniature three-photon microscopy maximized for scattered fluorescence collection.

In deep-tissue multiphoton microscopy, diffusion and scattering of fluorescent photons, rather than ballistic emanation from the focal point, have been a confounding factor. Here we report on a 2.17-g miniature three-photon microscope (m3PM) with a configuration that maximizes fluorescence collection when imaging in highly scattering regimes. We demonstrate its capability by imaging calcium activity throughout the entire cortex and dorsal hippocampal CA1, up to 1.2 mm depth, at a safe laser power. It also enables the detection of sensorimotor behavior-correlated activities of layer 6 neurons in the posterior parietal cortex in freely moving mice during single-pellet reaching tasks. Thus, m3PM-empowered imaging allows the study of neural mechanisms in deep cortex and subcortical structures, like the dorsal hippocampus and dorsal striatum, in freely behaving animals.

Two-photon endomicroscopy with microsphere-spliced double-cladding antiresonant fiber for resolution enhancement.

We demonstrate a miniature fiber-optic two two-photon endomicroscopy with microsphere-spliced double-cladding antiresonant fiber for resolution enhancement. An easy-to-operate process for fixing microsphere permanently in an antiresonant fiber core, by arc discharge, is proposed. The flexible fiber-optic probe is integrated with a parameter of 5.8 mm × 49.1 mm (outer diameter × rigid length); the field of view is 210 µm, the resolution is 1.3 µm, and the frame rate is 0.7 fps. The imaging ability is verified using ex-vivo mouse kidney, heart, stomach, tail tendon, and in-vivo brain neural imaging.

Encoding of social novelty by sparse GABAergic neural ensembles in the prelimbic cortex.

Although the prelimbic (PrL) area is associated with social behaviors, the neural ensembles that regulate social preference toward novelty or familiarity remain unknown. Using miniature two-photon microscopy (mTPM) to visualize social behavior-associated neuronal activity within the PrL in freely behaving mice, we found that the Ca2+ transients of GABAergic neurons were more highly correlated with social behaviors than those of glutamatergic neurons. Chemogenetic suppression of social behavior-activated GABAergic neurons in the PrL disrupts social novelty behaviors. Restoring the MeCP2 level in PrL GABAergic neurons in MECP2 transgenic (MECP2-TG) mice rescues the social novelty deficits. Moreover, we identified and characterized sparsely distributed NewPNs and OldPNs of GABAergic interneurons in the PrL preferentially responsible for new and old mouse exploration, respectively. Together, we propose that social novelty information may be encoded by the responses of NewPNs and OldPNs in the PrL area, possibly via synergistic actions on both sides of the seesaw.

Non-Invasive Skin Imaging Assessment of Human Stress During Head-Down Bed Rest Using a Portable Handheld Two-Photon Microscope.

Spaceflight presents a series of physiological and pathological challenges to astronauts resulting from ionizing radiation, microgravity, isolation, and other spaceflight hazards. These risks cause a series of aging-related diseases associated with increased oxidative stress and mitochondria dysfunction. The skin contains many autofluorescent substances, such as nicotinamide adenine dinucleotide phosphate (NAD(P)H), keratin, melanin, elastin, and collagen, which reflect physiological and pathological changes in vivo. In this study, we used a portable handheld two-photon microscope to conduct high-resolution in vivo skin imaging on volunteers during 15 days of head-down bed rest. The two-photon microscope, equipped with a flexible handheld scanning head, was used to measure two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) images of the left forearm, left front chest, and forehead of volunteers. Changes in TPEF, SHG, and the extended SHG-to-AF(TPEF) aging index of the dermis (SAAID) were measured. It was found that TPEF intensity increased during bed rest and was restored to normal levels after recovery. Meanwhile, SHG increased slightly during bed rest, and the skin aging index increased. Moreover, we found the skin TPEF signals of the left forearm were significantly negatively associated with the oxidative stress marker malondialdehyde (MDA) and DNA damage marker 8-hydroxy-2'-desoxyguanosine (8-OHdG) values of subjects during head-down bed rest. Meanwhile, the SHG signals were also significantly negatively correlated with MDA and 8-OHDG. A significant negative correlation between the extended SAAID of the left chest and serum antioxidant superoxide dismutase (SOD) levels was also found. These results demonstrate that skin autofluorescence signals can reflect changes in human oxidant status. This study provides evidence for in-orbit monitoring of changes in human stress using a portable handheld two-photon microscope for skin imaging.

Angiorganoid: vitalizing the organoid with blood vessels.

The emergence of the organoid simulates the native organs and this mini organ offers an excellent platform for probing multicellular interaction, disease modeling and drug discovery. Blood vessels constitute the instructive vascular niche which is indispensable for organ development, function and regeneration. Therefore, it is expected that the introduction of infiltrated blood vessels into the organoid might further pump vitality and credibility into the system. While the field is emerging and growing with new concepts and methodologies, this review aims at presenting various sources of vascular ingredients for constructing vascularized organoids and the paired methodology including de- and recellularization, bioprinting and microfluidics. Representative vascular organoids corresponding to specific tissues are also summarized and discussed to elaborate on the next generation of organoid development.

MouseVenue3D: A Markerless Three-Dimension Behavioral Tracking System for Matching Two-Photon Brain Imaging in Free-Moving Mice.

Understanding the connection between brain and behavior in animals requires precise monitoring of their behaviors in three-dimensional (3-D) space. However, there is no available three-dimensional behavior capture system that focuses on rodents. Here, we present MouseVenue3D, an automated and low-cost system for the efficient capture of 3-D skeleton trajectories in markerless rodents. We improved the most time-consuming step in 3-D behavior capturing by developing an automatic calibration module. Then, we validated this process in behavior recognition tasks, and showed that 3-D behavioral data achieved higher accuracy than 2-D data. Subsequently, MouseVenue3D was combined with fast high-resolution miniature two-photon microscopy for synchronous neural recording and behavioral tracking in the freely-moving mouse. Finally, we successfully decoded spontaneous neuronal activity from the 3-D behavior of mice. Our findings reveal that subtle, spontaneous behavior modules are strongly correlated with spontaneous neuronal activity patterns.

Sparse deconvolution improves the resolution of live-cell super-resolution fluorescence microscopy.

A main determinant of the spatial resolution of live-cell super-resolution (SR) microscopes is the maximum photon flux that can be collected. To further increase the effective resolution for a given photon flux, we take advantage of a priori knowledge about the sparsity and continuity of biological structures to develop a deconvolution algorithm that increases the resolution of SR microscopes nearly twofold. Our method, sparse structured illumination microscopy (Sparse-SIM), achieves ~60-nm resolution at a frame rate of up to 564 Hz, allowing it to resolve intricate structures, including small vesicular fusion pores, ring-shaped nuclear pores formed by nucleoporins and relative movements of inner and outer mitochondrial membranes in live cells. Sparse deconvolution can also be used to increase the three-dimensional resolution of spinning-disc confocal-based SIM, even at low signal-to-noise ratios, which allows four-color, three-dimensional live-cell SR imaging at ~90-nm resolution. Overall, sparse deconvolution will be useful to increase the spatiotemporal resolution of live-cell fluorescence microscopy.

Dynamics of a disinhibitory prefrontal microcircuit in controlling social competition.

Social competition plays a pivotal role in determining individuals' social status. While the dorsomedial prefrontal cortex (dmPFC) is essential in regulating social competition, it remains unclear how information is processed within its local networks. Here, by applying optogenetic and chemogenetic manipulations in a dominance tube test, we reveal that, in accordance with pyramidal (PYR) neuron activation, excitation of the vasoactive intestinal polypeptide (VIP) or inhibition of the parvalbumin (PV) interneurons induces winning. The winning behavior is associated with sequential calcium activities initiated by VIP and followed by PYR and PV neurons. Using miniature two-photon microscopic (MTPM) and optrode recordings in awake mice, we show that VIP stimulation directly leads to a two-phased activity pattern of both PYR and PV neurons-rapid suppression followed by activation. The delayed activation of PV implies an embedded feedback tuning. This disinhibitory VIP-PV-PYR motif forms the core of a dmPFC microcircuit to control social competition.

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.

An optimized acetylcholine sensor for monitoring in vivo cholinergic activity.

The ability to directly measure acetylcholine (ACh) release is an essential step toward understanding its physiological function. Here we optimized the GRABACh (GPCR-activation-based ACh) sensor to achieve substantially improved sensitivity in ACh detection, as well as reduced downstream coupling to intracellular pathways. The improved version of the ACh sensor retains the subsecond response kinetics, physiologically relevant affinity and precise molecular specificity for ACh of its predecessor. Using this sensor, we revealed compartmental ACh signals in the olfactory center of transgenic flies in response to external stimuli including odor and body shock. Using fiber photometry recording and two-photon imaging, our ACh sensor also enabled sensitive detection of single-trial ACh dynamics in multiple brain regions in mice performing a variety of behaviors.

Miniature Fluorescence Microscopy for Imaging Brain Activity in Freely-Behaving Animals.

An ultimate goal of neuroscience is to decipher the principles underlying neuronal information processing at the molecular, cellular, circuit, and system levels. The advent of miniature fluorescence microscopy has furthered the quest by visualizing brain activities and structural dynamics in animals engaged in self-determined behaviors. In this brief review, we summarize recent advances in miniature fluorescence microscopy for neuroscience, focusing mostly on two mainstream solutions - miniature single-photon microscopy, and miniature two-photon microscopy. We discuss their technical advantages and limitations as well as unmet challenges for future improvement. Examples of preliminary applications are also presented to reflect on a new trend of brain imaging in experimental paradigms involving body movements, long and complex protocols, and even disease progression and aging.

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.

Fast, long-term, super-resolution imaging with Hessian structured illumination microscopy.

To increase the temporal resolution and maximal imaging time of super-resolution (SR) microscopy, we have developed a deconvolution algorithm for structured illumination microscopy based on Hessian matrixes (Hessian-SIM). It uses the continuity of biological structures in multiple dimensions as a priori knowledge to guide image reconstruction and attains artifact-minimized SR images with less than 10% of the photon dose used by conventional SIM while substantially outperforming current algorithms at low signal intensities. Hessian-SIM enables rapid imaging of moving vesicles or loops in the endoplasmic reticulum without motion artifacts and with a spatiotemporal resolution of 88 nm and 188 Hz. Its high sensitivity allows the use of sub-millisecond excitation pulses followed by dark recovery times to reduce photobleaching of fluorescent proteins, enabling hour-long time-lapse SR imaging of actin filaments in live cells. Finally, we observed the structural dynamics of mitochondrial cristae and structures that, to our knowledge, have not been observed previously, such as enlarged fusion pores during vesicle exocytosis.

Robust hollow-fiber-pigtailed 930 nm femtosecond Nd:fiber laser for volumetric two-photon imaging.

We demonstrate a robust high power 930 nm femtosecond Nd:fiber laser system with hollow-core photonic bandgap fiber (HC-PBGF) as the output delivery, which can be easily integrated into compact two-photon microscopy system for bio-imaging. The whole laser system can deliver up to 17.4 nJ, 220-fs pulses at 930 nm with repetition rate of 46 MHz. In this paper, this laser was demonstrated as the light source for volumetric imaging of zebrafish blood vessel.

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.