High-precision angle error compensation method for a Dove prism scanning system based on galvanometers.

Dove prisms suffer from angle and shift errors due to inevitable errors in manufacturing and installation, limiting their applicability in tasks requiring high-precision scanning. These errors, particularly angle errors, can significantly deform and ruin the intended scanning trajectory. Here, we propose a method for compensating the angle errors in Dove prisms using galvanometers. The method first determines the angle error by analyzing the distorted scanning trajectory. Subsequently, by synchronizing the galvanometers with the Dove prism rotation, the galvanometers dynamically correct the angle error at each rotation angle. This approach eliminates the need for complex mechanical adjustment mechanisms and offers a convenient calibration process. Our experiments demonstrate that the angle error can be adjusted to be below 17 µrad under the described conditions. By enabling high-precision scanning, this method has the potential to broaden the application scenarios of Dove prisms in various fields.

RecV recombinase system for in vivo targeted optogenomic modifications of single cells or cell populations.

Brain circuits comprise vast numbers of interconnected neurons with diverse molecular, anatomical and physiological properties. To allow targeting of individual neurons for structural and functional studies, we created light-inducible site-specific DNA recombinases based on Cre, Dre and Flp (RecVs). RecVs can induce genomic modifications by one-photon or two-photon light induction in vivo. They can produce targeted, sparse and strong labeling of individual neurons by modifying multiple loci within mouse and zebrafish genomes. In combination with other genetic strategies, they allow intersectional targeting of different neuronal classes. In the mouse cortex they enable sparse labeling and whole-brain morphological reconstructions of individual neurons. Furthermore, these enzymes allow single-cell two-photon targeted genetic modifications and can be used in combination with functional optical indicators with minimal interference. In summary, RecVs enable spatiotemporally precise optogenomic modifications that can facilitate detailed single-cell analysis of neural circuits by linking genetic identity, morphology, connectivity and function.

Multiparameter mobile blood analysis for complete blood count using contrast-enhanced defocusing imaging and machine vision.

Blood analysis through complete blood count is the most basic medical test for disease diagnosis. Conventional blood analysis requires bulky and expensive laboratory facilities and skilled technicians, limiting the universal medical use of blood analysis outside well-equipped laboratory environments. Here, we propose a multiparameter mobile blood analyzer combined with label-free contrast-enhanced defocusing imaging (CEDI) and machine vision for instant and on-site diagnostic applications. We designed a low-cost and high-resolution miniature microscope (size: 105 mm × 77 mm × 64 mm, weight: 314 g) that comprises a pair of miniature aspheric lenses and a 415 nm LED for blood image acquisition. The analyzer, adopting CEDI, can obtain both the refractive index distributions of the white blood cell (WBC) and hemoglobin spectrophotometric information, enabling the analyzer to supply rich blood parameters, including the five-part WBC differential count, red blood cell (RBC) count, and mean corpuscular hemoglobin (MCH) quantification with machine vision algorithms and the Lambert-Beer law. We have shown that our assay can analyze a blood sample within 10 minutes without complex staining, and measurements (30 samples) from the analyzer have a strong linear correlation with clinical reference values (significance level of 0.0001). This study provides a miniature, light weight, low-cost, and easy-to-use blood analysis technique that overcomes the challenge of simultaneously realizing FWD count, RBC count, and MCH analysis using a mobile device and has great potential for integrated surveillance of various epidemic diseases, including coronavirus infection, invermination, and anemia, especially in low- and middle-income countries.

Anatomical Evidence for Parasympathetic Innervation of the Renal Vasculature and Pelvis.

BACKGROUND: The kidneys critically contribute to body homeostasis under the control of the autonomic nerves, which enter the kidney along the renal vasculature. Although the renal sympathetic and sensory nerves have long been confirmed, no significant anatomic evidence exists for renal parasympathetic innervation. METHODS: We identified cholinergic nerve varicosities associated with the renal vasculature and pelvis using various anatomic research methods, including a genetically modified mouse model and immunostaining. Single-cell RNA sequencing (scRNA-Seq) was used to analyze the expression of AChRs in the renal artery and its segmental branches. To assess the origins of parasympathetic projecting nerves of the kidney, we performed retrograde tracing using recombinant adeno-associated virus (AAV) and pseudorabies virus (PRV), followed by imaging of whole brains, spinal cords, and ganglia. RESULTS: We found that cholinergic axons supply the main renal artery, segmental renal artery, and renal pelvis. On the renal artery, the newly discovered cholinergic nerve fibers are separated not only from the sympathetic nerves but also from the sensory nerves. We also found cholinergic ganglion cells within the renal nerve plexus. Moreover, the scRNA-Seq analysis suggested that acetylcholine receptors (AChRs) are expressed in the renal artery and its segmental branches. In addition, retrograde tracing suggested vagus afferents conduct the renal sensory pathway to the nucleus of the solitary tract (NTS), and vagus efferents project to the kidney. CONCLUSIONS: Cholinergic nerves supply renal vasculature and renal pelvis, and a vagal brain-kidney axis is involved in renal innervation.

Large depth-of-field fluorescence microscopy based on deep learning supported by Fresnel incoherent correlation holography.

Fluorescence microscopy plays an irreplaceable role in biomedicine. However, limited depth of field (DoF) of fluorescence microscopy is always an obstacle of image quality, especially when the sample is with an uneven surface or distributed in different depths. In this manuscript, we combine deep learning with Fresnel incoherent correlation holography to describe a method to obtain significant large DoF fluorescence microscopy. Firstly, the hologram is restored by the Auto-ASP method from out-of-focus to in-focus in double-spherical wave Fresnel incoherent correlation holography. Then, we use a generative adversarial network to eliminate the artifacts introduced by Auto-ASP and output the high-quality image as a result. We use fluorescent beads, USAF target and mouse brain as samples to demonstrate the large DoF of more than 400µm, which is 13 times better than that of traditional wide-field microscopy. Moreover, our method is with a simple structure, which can be easily combined with many existing fluorescence microscopic imaging technology.

Chemical Sectioning for Immunofluorescence Imaging.

Immunofluorescence (IF) is a powerful investigative tool in biological research and medical diagnosis, whereas conventional imaging methods are always conflict between speed, contrast/resolution, and specimen volume. Chemical sectioning (CS) is an effective method to overcome the conflict, which works by chemically manipulating the off/on state of fluorescent materials and turning on only the extremely superficial surface fluorescence of tissues to realize the sectioning capacity of wide-field imaging. However, the current mechanism of CS is only applicable to samples labeled with pH-sensitive fluorescent proteins and still cannot fulfill samples immunolabeled with frequently used commercial fluorescent dyes. Here, immunofluorescence chemical sectioning (IF-CS) is described to present an off/on mechanism for Alexa dyes by complexation reactions, allowing CS imaging of IF labeled tissues. IF-CS enables IF freeing from out-of-focus interference in wide-field imaging and satisfying with multicolor imaging. IF-CS demonstrates the utility of the 3D submicron-resolution imaging of large immunolabeled tissues on the wide-field block-face system. IF-CS may remarkably facilitate systematic studies of refined subcellular architectures of endogenous proteins in intact biological systems.

Cell-type-specific and projection-specific brain-wide reconstruction of single neurons.

We developed a dual-adeno-associated-virus expression system that enables strong and sparse labeling of individual neurons with cell-type and projection specificity. We demonstrated its utility for whole-brain reconstruction of midbrain dopamine neurons and striatum-projecting cortical neurons. We further extended the labeling method for rapid reconstruction in cleared thick brain sections and simultaneous dual-color labeling. This labeling system may facilitate the process of generating mesoscale single-neuron projectomes of mammalian brains.

Aberration-corrected three-dimensional non-inertial scanning for femtosecond lasers.

Large aberrations are induced by non-collimated light when the convergence or divergence of the incident beam on the back-pupil plane of the objective lens is adjusted for 3D non-inertial scanning. These aberrations significantly degrade the focus quality and decrease the peak intensity of the femtosecond laser focal spot. Here, we describe an aberration-corrected 3D non-inertial scanning method for femtosecond lasers based on a digital micromirror device (DMD) that is used for both beam scanning and aberration correction. An imaging setup is used to detect the focal spot in the 3D space, and an iterative optimization algorithm is used to optimize the focal spot. We demonstrate the application of our proposed approach in two-photon imaging. With correction for the 200-µm out-of-focal plane, the optical axial resolution improves from 7.67 to 3.25 µm, and the intensity of the fluorescence signal exhibits an almost fivefold improvement when a 40× objective lens is used. This aberration-corrected 3D non-inertial scanning method for femtosecond lasers offers a new approach for a variety of potential applications, including nonlinear optical imaging, microfabrication, and optical storage.

Extending the 3D scanning range of DMD-based scanners for femtosecond lasers.

Digital micromirror devices (DMDs) have shown their potential in 2-photon imaging and microfabrication as diffractive scanners for femtosecond lasers. However, the scanning range of a DMD-based scanner is decreased by the spatial filter (SF) used to block undesired diffraction orders. Instead of an SF, we present a method of introducing and correcting aberration (ICA) to reduce the effects of these undesired diffraction orders. In ICA, aberrations are introduced by optical elements, and only the aberration of the desired diffraction order is corrected by adding a compensatory phase to the scanning phase. The scanning ranges in the y and z directions can be nearly doubled when the SF is removed. We demonstrate that ICA can be conveniently applied to a previously constructed DMD-based 2-photon microscope, and the field of view can be extended at different axial positions.

3D high resolution generative deep-learning network for fluorescence microscopy imaging.

Microscopic fluorescence imaging serves as a basic tool in many research areas including biology, medicine, and chemistry. With the help of optical clearing, large volume imaging of a mouse brain and even a whole body has been enabled. However, constrained by the physical principles of optical imaging, volume imaging has to balance imaging resolution and speed. Here, we develop a new, to the best of our knowledge, 3D deep learning network based on a dual generative adversarial network (dual-GAN) framework for recovering high-resolution (HR) volume images from high speed acquired low-resolution (LR) volume images. The proposed method does not require a precise image registration process and meanwhile guarantees the predicted HR volume image faithful to its corresponding LR volume image. The results demonstrated that our method can recover ${20} {\times} /1.0\text-{\rm NA}$20×/1.0-NA volume images from coarsely registered ${5} {\times} /0.16\text-{\rm NA}$5×/0.16-NA volume images collected by light-sheet microscopy. This method would provide great potential in applications which require high resolution volume imaging.

NeuroGPS-Tree: automatic reconstruction of large-scale neuronal populations with dense neurites.

The reconstruction of neuronal populations, a key step in understanding neural circuits, remains a challenge in the presence of densely packed neurites. Here we achieved automatic reconstruction of neuronal populations by partially mimicking human strategies to separate individual neurons. For populations not resolvable by other methods, we obtained recall and precision rates of approximately 80%. We also demonstrate the reconstruction of 960 neurons within 3 h.

High-resolution femtosecond laser beam shaping via digital holography.

We propose a method to achieve high-resolution femtosecond laser beam shaping (HR-FLBS) via digital holography based on a digital mirror device (DMD) and optimized optical design. By programming the hologram on the DMD, we captured several patterns such as light spots, decorated letters of "HUST" (Huazhong University of Science and Technology), and a series of concentric annuli whose fine structure approaches the optical diffraction limit. The experimental results confirm that the proposed method can shape the amplitude and phase of a femtosecond laser arbitrarily with high resolution.

Simultaneous two-plane, two-photon imaging based on spatial multiplexing.

The two-photon microscope is a powerful tool in life science. Conventional two-photon microscopy can image only a plane of a particular axial position at a time. Axial scanning can get the volumetric information, but it gets signals from different axial positions serially, which means that the exposure time at every plane is limited. Here we demonstrate a novel method, to the best of our knowledge, that can simultaneously record images from two planes at different xyz positions. The demultiplexing of the signal is realized using a confocal strategy. The experimental results show that it can be used for simultaneous two-photon imaging at two focal planes with little cross talk.

Primary Auditory Cortex is Required for Anticipatory Motor Response.

The ability of the brain to predict future events based on the pattern of recent sensory experience is critical for guiding animal's behavior. Neocortical circuits for ongoing processing of sensory stimuli are extensively studied, but their contributions to the anticipation of upcoming sensory stimuli remain less understood. We, therefore, used in vivo cellular imaging and fiber photometry to record mouse primary auditory cortex to elucidate its role in processing anticipated stimulation. We found neuronal ensembles in layers 2/3, 4, and 5 which were activated in relationship to anticipated sound events following rhythmic stimulation. These neuronal activities correlated with the occurrence of anticipatory motor responses in an auditory learning task. Optogenetic manipulation experiments revealed an essential role of such neuronal activities in producing the anticipatory behavior. These results strongly suggest that the neural circuits of primary sensory cortex are critical for coding predictive information and transforming it into anticipatory motor behavior.

Parallel pathways convey olfactory information with opposite polarities in Drosophila.

In insects, olfactory information received by peripheral olfactory receptor neurons (ORNs) is conveyed from the antennal lobes (ALs) to higher brain regions by olfactory projection neurons (PNs). Despite the knowledge that multiple types of PNs exist, little is known about how these different neuronal pathways work cooperatively. Here we studied the Drosophila GABAergic mediolateral antennocerebral tract PNs (mlPNs), which link ipsilateral AL and lateral horn (LH), in comparison with the cholinergic medial tract PNs (mPNs). We examined the connectivity of mlPNs in ALs and found that most mlPNs received inputs from both ORNs and mPNs and participated in AL network function by forming gap junctions with other AL neurons. Meanwhile, mlPNs might innervate LH neurons downstream of mPNs, exerting a feedforward inhibition. Using dual-color calcium imaging, which enables a simultaneous monitoring of neural activities in two groups of PNs, we found that mlPNs exhibited robust odor responses overlapping with, but broader than, those of mPNs. Moreover, preferentially down-regulation of GABA in most mlPNs caused abnormal courtship and aggressive behaviors in male flies. These findings demonstrate that in Drosophila, olfactory information in opposite polarities are carried coordinately by two parallel and interacted pathways, which could be essential for appropriate behaviors.

Large-scale localization of touching somas from 3D images using density-peak clustering.

BACKGROUND: Soma localization is an important step in computational neuroscience to map neuronal circuits. However, locating somas from large-scale and complicated datasets is challenging. The challenges primarily originate from the dense distribution of somas, the diversity of soma sizes and the inhomogeneity of image contrast. RESULTS: We proposed a novel localization method based on density-peak clustering. In this method, we introduced two quantities (the local density ρ of each voxel and its minimum distance δ from voxels of higher density) to describe the soma imaging signal, and developed an automatic algorithm to identify the soma positions from the feature space (ρ, δ). Compared with other methods focused on high local density, our method allowed the soma center to be characterized by high local density and large minimum distance. The simulation results indicated that our method had a strong ability to locate the densely positioned somas and strong robustness of the key parameter for the localization. From the analysis of the experimental datasets, we demonstrated that our method was effective at locating somas from large-scale and complicated datasets, and was superior to current state-of-the-art methods for the localization of densely positioned somas. CONCLUSIONS: Our method effectively located somas from large-scale and complicated datasets. Furthermore, we demonstrated the strong robustness of the key parameter for the localization and its effectiveness at a low signal-to-noise ratio (SNR) level. Thus, the method provides an effective tool for the neuroscience community to quantify the spatial distribution of neurons and the morphologies of somas.

Compensation of spatial dispersion of an acousto-optic deflector with a special Keplerian telescope.

Compensation of spatial dispersion caused by the acousto-optic deflector (AOD) when using a femtosecond laser is difficult across the whole scanning range of the system, and this is a significant impediment to its use. In conventional methods, the dispersion of the AOD was compensated only when it was at a particular position, while at other positions, the quality of the light beam was reduced. We developed a novel method for compensating the spatial dispersion within the entire scanning range using a special Keplerian telescope. Our experimental results show that the residual dispersion of the AOD is compensated sufficiently, and the focal spots of the laser reach the diffraction limit within a 40-MHz ultrasound bandwidth.

Monitoring the Oxygen Dynamics of Brain Tissue In Vivo by Fast Acousto-Optic Scanning Microscopy: A Proposed Instrument.

The function of the brain neural circuit is highly dependent on oxygen supply. Imaging the precise oxygen distribution and dynamics are critical for understanding the relationship between neuronal activity and oxygen dynamics of the nearby capillaries. Here, we develop fast acousto-optic scanning two-photon microscopy. Combined with oxygen probes, such as PtP-C343, we can monitor oxygen dynamics at the submicron level by this real-time microscopy. In this fast acousto-optic scanning microscopy, an acousto-optic deflector (AOD), an inertia-less scanner, is used to scan the femtosecond laser. A cylindrical lens is used to compensate the 'cylindrical lens effect' of AOD and a prism is used to compensate the chromatic dispersion of AOD. An electro-optical modulator (EOM) and a sCMOS camera are gated to measure the phosphorescence lifetime. With a 40× water objective lens, this set-up can image a 100 µm × 100 µm field of view at a speed of 20 frames per second and a 25 µm × 8 µm field of view at a speed of 500 frames per second. This real-time two-photon microscopy is expected to be a good tool for observing and recording the precise rapid oxygen dynamics in the cerebral cortex, which will facilitate studies of oxygen metabolism in neurosciences.

Experimentally exploiting the violation of the Lagrange invariant for resolution improvement.

It is experimentally demonstrated that even though the numerical aperture in the object space is fixed, the resolution of an imaging system still can be improved by adjusting the parameters in the image space. This strategy cannot be realized until the discovery of the violation of the Lagrange invariant in a kind of self-interference holography. With the violation, parameters in the image space can escape the constraint of the object space for resolution improvement. Experiments that directly confirm this new ability are implemented and results agree well with the theoretical prediction. Additionally, better performance on frequency recording and finer details beyond the diffraction limit have been recorded with this method.