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High-speed translaminar imaging reveals region-specific sensory and motor-evoked activity in behaving GFAP-GCaMP6f mice a Schematic showing the translaminar imaging approach. An 0.7 mm × 0.7 mm × 0.7 mm glass reflective microprism was inserted at the lateral interface between the spinal gray and white matter at the L4-L5 spinal level without tissue removal (Methods). The microprism’s reflective hypotenuse tilts the microscope’s imaging plane by 90 degrees allowing high-speed measurements across laminae. Imaging was performed at 75 μm or 125 μm focal depth from the vertical microprism face. Scale bar, 0.5 mm. b Predicted extent and borders of spinal laminae across the 700 μm field of view (FOV) for 75 μm (top) and 125 μm focal depths (bottom). c Example fluorescence images showing the translaminar blood vessel pattern in a GFAP-GCaMP6f mouse at 75 μm and 125 μm focal depths four weeks after microprism implantation. Scale bars, 100 μm. d Average intensity projection image from a different GFAP-GCaMP6f mouse at 75 μm focal depth overlaid with 10 μm × 10 μm ROIs. Only ROIs with at least 50% (yellow) or 75% ∆F/F (orange) in response to a noxious tail pinch (p > 500 g; duration: 1.5 s ± 0.5 s) are shown. In this example, the tail pinch did not evoke a locomotor response. Scale bar, 100 μm. e Noxious tail pinch evoked activity across tissue depth and time for the example recording shown in d. Each row depicts the percent of active ROIs (≥50% ∆F/F) for a given tissue depth. The corresponding pressure stimulus, locomotor activity, and average calcium transient across the FOV are shown above the activity heat map. Running speed was recorded by placing the animal on a spherical treadmill (Supplementary Movie 3). The vertical dashed line indicates pinch onset. f Population data showing the average percent of active ROIs across tissue depths. Significant activity occurred in central FOV regions, corresponding to the upper spinal laminae. g Population data showing average calcium response onset latency for pinch-only and run-only trials. h Population data showing individual ROI and average calcium transient duration for pinch-only trials. i Activity map from the same animal as in d for a spontaneous run trial without pressure stimulus application. Same focal depth as in d. Scale bar, 100 μm. j Running-evoked astrocyte activity across tissue depth and time (Supplementary Movie 4). The pressure sensor readout, locomotor activity, and average calcium signal across the FOV are shown above the activity heat map. The vertical dashed lines indicate run onset (Methods). k Population data showing the average percent of active ROIs across tissue depths for run trials. Significant activity occurred in FOV regions corresponding to deep dorsal horn laminae involved in motor processing. l Population data showing individual ROI and average calcium transient duration for run-only trials (2–4 s-long runs with ≥25 s rest period before the run). The data in f, g (left), h are from 1105 ROIs, 18 recordings, and 5 mice. The data in g (right), k, l are from 855 ROIs, 8 recordings, and 2 mice. All shown data were acquired four weeks after microprism implantation. Two-sided paired t-tests determined P values, and all bar plots are presented as mean ± s.e.m. The box and whisker plots mark the median and the 25th and 75th percentiles, and the whiskers cover the minimum and maximum of the data. All images are representatives from one sample. Images with similar properties were obtained across multiple independent samples. Source data are provided as a Source Data file. Panel 4a image reproduced with permission from ref. ³⁵.
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While the spinal cord is known to play critical roles in sensorimotor processing, including pain-related signaling, corresponding activity patterns in genetically defined cell types across spinal laminae have remained challenging to investigate. Calcium imaging has enabled cellular activity measurements in behaving rodents but is currently limited...
Citations
... Imaging cellular and molecular activity across space and time has emerged as a crucial approach in many fields such as neuroscience 1 , cell biology 2 , pathology 3 , and developmental biology 4 . Recent developments in modern genetically encoded fluorescent probes [5][6][7] and advanced imaging techniques [8][9][10] have enabled observation of a wide range of signals including calcium ions, ATP, neurotransmitters, neuromodulators, and other molecules, expanding greatly the breadth and depth of scientific studies. However, with the rapid growth of data generation and the revealing of complex spatiotemporal activity patterns such as heterogeneous spatial footprints and various propagations (see top row of Figure 3), quantifying and understanding the data has become a limiting factor. ...
... Within the spinal cord, individual laminae contain unique neuronal populations, exhibit distinct connectivity patterns, and serve specialized functional roles 41,42 . A recently introduced translaminar imaging approach offers the capability for rapid measurements across spinal laminae, providing an opportunity to explore and comprehend the interactions and dynamics inherent to these distinct laminae 8 . Yet, the absence of a mature analysis tool for this novel imaging technique requires researchers to manually partition the field of view (FOV) into equally sized ROIs for analysis. ...
Optical recording of intricate molecular dynamics is becoming an indispensable technique for biological studies, accelerated by the development of new or improved biosensors and microscopy technology. This creates major computational challenges to extract and quantify biologically meaningful patterns embedded within complex and rich data sources. Here, we introduce Activity Quantification and Analysis (AQuA2), a fast, accurate, and versatile data analysis platform built upon advanced machine learning techniques. AQuA2 decomposes complex live imaging-based datasets into elementary signaling events, allowing accurate and unbiased quantification of molecular activities and identification of consensus functional units. We demonstrate applications of AQuA2 across a range of biosensors (calcium, norepinephrine, ATP, acetylcholine, dopamine), cell types (astrocytes, oligodendrocytes, microglia, neurons), organs (brains and spinal cords), animal models (zebrafish and mouse), and imaging modalities (confocal, two-photon, light sheet). As exemplar findings, we show how AQuA2 identified drug-dependent interactions between neurons and astroglia, and distinct sensorimotor signal propagation patterns in the mouse spinal cord.
... Recent innovations have allowed for population recordings using GCaMPs in the mouse dorsal spinal cord. 14,15 However, these proxy events have time constants that are typically greater than 100 ms, 16 which do not accurately capture high-frequency spiking dynamics. In addition, myelinated axonal tracts surrounding the spinal gray matter typically limit optical access to all but sensory-related regions located in the superficial dorsal horn, currently precluding studies of motor circuit activity in the ventral horn of behaving mammals. ...
... Therefore, to provide a stable base for the probe and its recording backstage, we adopted previously reported chronic imaging platforms, which are capable of accommodating the comparatively larger weight and torque from wearable microscopes and comfortably exceed the requirement of our recording approach. 15,40 In this study, we did not pursue precise targeting in mediallateral directions during implantation. Instead, our priority was to avoid vasculature damage, and therefore the dorsal entry sites (among animals) landed in the range of 50-250 mm from midline. ...
... It is therefore not known how the tuning of individual ALT neurons is altered or preserved during the induction and maintenance of persistent pain states, or whether nociceptive and neuropathic pain signals are transmitted from the spinal cord to the brain by the same or different ensembles of neurons. In vivo and ex vivo cellular imaging techniques can now be applied to the superficial dorsal horn of the spinal cord, allowing direct visualization of population sensory responses in spinal projection neurons and interneurons 26,[30][31][32][33][34][35][36] . We have adapted this approach to allow repeated imaging cellular function in vivo over prolonged time scales, and applied it to track how sensory coding in spinal output circuitry is transformed during the development and maintenance of persistent neuropathic pain. ...
Pain hypersensitivity arises from the plasticity of peripheral and spinal somatosensory neurons, which modifies nociceptive input to the brain and alters pain perception. We utilized chronic calcium imaging of spinal dorsal horn neurons to determine how the representation of somatosensory stimuli in the anterolateral tract, the principal pathway transmitting nociceptive signals to the brain, changes between distinct pain states. In healthy conditions, we identify stable, narrowly tuned outputs selective for cooling or warming, and a neuronal ensemble activated by intense/noxious thermal and mechanical stimuli. Induction of an acute peripheral sensitization with capsaicin selectively and transiently retunes nociceptive output neurons to encode low-intensity stimuli. In contrast, peripheral nerve injury-induced neuropathic pain results in a persistent suppression of innocuous spinal outputs coupled with activation of a normally silent population of high-threshold neurons. These results demonstrate the differential modulation of specific spinal outputs to the brain during nociceptive and neuropathic pain states.
... Imaging system miniaturization has advanced rapidly in past years, bringing about technological developments like miniaturized fluorescence microscopes 20,21 , lensless microscopy [22][23][24] , chip-scale metalens microscopes 25,26 , etc. Among these, metasurface or metalenses, with flat and ultrathin architectures composed of subwavelength units 27,28 , have shown massive potential in providing miniaturized solutions for diverse information processing [29][30][31][32] or imaging applications 33 39,45 . ...
Quantitative phase imaging (QPI) based on the transport-of-intensity equation (TIE) is a powerful technique in label-free microscopy. The image stack required for a successful TIE-QPI is traditionally obtained by translating the object or image plane, and the optical elements used in the conventional TIE-QPI systems are usually bulky and cumbersome. Stable and compact TIE-QPI methods capable of non-motion optical zooming can significantly facilitate applications that demand portability. Here, we propose a non-motion TIE-QPI method based on a dispersive metalens. The dispersive nature of the metalens is utilized to provide a spectral focal tuning. With fixed object and image planes, seven through-focus intensity images are captured by changing the illumination wavelength. The QPI performance is validated by retrieving the surface phase profiles of a microlens array and a phase resolution target, showing a high phase detection accuracy (deviation less than 0.03 wavelength). Subsequently, we established a compact meta-microscope by integrating the metalens with a commercially available CMOS image sensor, which shows good performance in microscopic imaging of unstained bio-samples. Our approach, based on the large-dispersive metalens, facilitates a compact and robust QPI system for optical metrology and label-free microscopy.
... It is therefore not known how the tuning of individual ALT neurons is altered or preserved during the induction and maintenance of persistent pain states, or whether nociceptive and neuropathic pain signals are transmitted from the spinal cord to the brain by the same or different ensembles of neurons. In vivo and ex vivo cellular imaging techniques can now be applied to the superficial dorsal horn of the spinal cord, allowing direct visualization of population sensory responses in spinal projection neurons and interneurons 26,[30][31][32][33][34][35][36] . We have adapted this approach to allow repeated imaging cellular function in vivo over prolonged time scales, and applied it to track how sensory coding in spinal output circuitry is transformed during the development and maintenance of persistent neuropathic pain. ...
Nonlinear optical microscopy enables non-invasive imaging in scattering samples with cellular resolution. The spinal cord connects the brain with the periphery and governs fundamental behaviors such as locomotion and somatosensation. Because of dense myelination on the dorsal surface, imaging to the spinal grey matter is challenging, even with two-photon microscopy. Here we show that three-photon excited fluorescence (3PEF) microscopy enables multicolor imaging at depths of up to ~550 μm into the mouse spinal cord, in vivo . We quantified blood flow across vessel types along the spinal vascular network. We then followed the response of neurites and microglia after occlusion of a surface venule, where we observed depth-dependent structural changes in neurites and interactions of perivascular microglia with vessel branches upstream from the clot. This work establishes that 3PEF imaging enables studies of functional dynamics and cell type interactions in the top 550 μm of the murine spinal cord, in vivo .
Quantitative phase imaging (QPI) based on the transport-of-intensity equation (TIE) is a powerful technique in label-free microscopy. The image stack required for a successful TIE-QPI is traditionally obtained by translating the object or image plane, and the optical elements used in the conventional TIE-QPI systems are usually bulky and cumbersome. Stable and compact TIE-QPI methods capable of non-motion optical zooming can significantly facilitate applications that demand portability. Here, we propose a non-motion TIE-QPI method based on a dispersive metalens. The dispersive nature of the metalens is utilized to provide a spectral focal tuning. With fixed object and image planes, seven through-focus intensity images are captured by changing the illumination wavelength. The QPI performance is validated by retrieving the surface phase profiles of a microlens array and a phase resolution target, showing a high phase detection accuracy (deviation less than 0.03 wavelength). Subsequently, we established a compact meta-microscope by integrating the metalens with a commercially available CMOS image sensor, which shows good performance in microscopic imaging of unstained bio-samples. Our approach, based on the large-dispersive metalens, facilitates a compact and robust QPI system for optical metrology and label-free microscopy.
Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics’ agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, this status report reviews an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion report, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed, and provide an outlook for the future directions.
