Two-photon Microscopy Research Articles

Toward a brighter constellation: multiorgan neuroimaging of neural and vascular dynamics in the spinal cord and brain.

Pain comprises a complex interaction between motor action and somatosensation that is dependent on dynamic interactions between the brain and spinal cord. This makes understanding pain particularly challenging as it involves rich interactions between many circuits (e.g., neural and vascular) and signaling cascades throughout the body. As such, experimentation on a single region may lead to an incomplete and potentially incorrect understanding of crucial underlying mechanisms. We aimed to develop and validate tools to enable detailed and extended observation of neural and vascular activity in the brain and spinal cord. The first key set of innovations was targeted to developing novel imaging hardware that addresses the many challenges of multisite imaging. The second key set of innovations was targeted to enabling bioluminescent (BL) imaging, as this approach can address limitations of fluorescent microscopy including photobleaching, phototoxicity, and decreased resolution due to scattering of excitation signals. We designed 3D-printed brain and spinal cord implants to enable effective surgical implantations and optical access with wearable miniscopes or an open window (e.g., for one- or two-photon microscopy or optogenetic stimulation). We also tested the viability for BL imaging and developed a novel modified miniscope optimized for these signals (BLmini). We describe "universal" implants for acute and chronic simultaneous brain-spinal cord imaging and optical stimulation. We further describe successful imaging of BL signals in both foci and a new miniscope, the "BLmini," which has reduced weight, cost, and form-factor relative to standard wearable miniscopes. The combination of 3D-printed implants, advanced imaging tools, and bioluminescence imaging techniques offers a coalition of methods for understanding spinal cord-brain interactions. Our work has the potential for use in future research into neuropathic pain and other sensory disorders and motor behavior.

Pioglitazone Phases and Metabolic Effects in Nanoparticle-Treated Cells Analyzed via Rapid Visualization of FLIM Images.

Fluorescence lifetime imaging microscopy (FLIM) has proven to be a useful method for analyzing various aspects of material science and biology, like the supramolecular organization of (slightly) fluorescent compounds or the metabolic activity in non-labeled cells; in particular, FLIM phasor analysis (phasor-FLIM) has the potential for an intuitive representation of complex fluorescence decays and therefore of the analyzed properties. Here we present and make available tools to fully exploit this potential, in particular by coding via hue, saturation, and intensity the phasor positions and their weights both in the phasor plot and in the microscope image. We apply these tools to analyze FLIM data acquired via two-photon microscopy to visualize: (i) different phases of the drug pioglitazone (PGZ) in solutions and/or crystals, (ii) the position in the phasor plot of non-labelled poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), and (iii) the effect of PGZ or PGZ-containing NPs on the metabolism of insulinoma (INS-1 E) model cells. PGZ is recognized for its efficacy in addressing insulin resistance and hyperglycemia in type 2 diabetes mellitus, and polymeric nanoparticles offer versatile platforms for drug delivery due to their biocompatibility and controlled release kinetics. This study lays the foundation for a better understanding via phasor-FLIM of the organization and effects of drugs, in particular, PGZ, within NPs, aiming at better control of encapsulation and pharmacokinetics, and potentially at novel anti-diabetics theragnostic nanotools.

Speckle-enabled in vivo demixing of neural activity in the mouse brain.

Functional imaging of neuronal activity in awake animals, using a combination of fluorescent reporters of neuronal activity and various types of microscopy modalities, has become an indispensable tool in neuroscience. While various imaging modalities based on one-photon (1P) excitation and parallel (camera-based) acquisition have been successfully used for imaging more transparent samples, when imaging mammalian brain tissue, due to their scattering properties, two-photon (2P) microscopy systems are necessary. In 2P microscopy, the longer excitation wavelengths reduce the amount of scattering while the diffraction-limited 3D localization of excitation largely eliminates out-of-focus fluorescence. However, this comes at the cost of time-consuming serial scanning of the excitation spot and more complex and expensive instrumentation. Thus, functional 1P imaging modalities that can be used beyond the most transparent specimen are highly desirable. Here, we transform light scattering from an obstacle into a tool. We use speckles with their unique patterns and contrast, formed when fluorescence from individual neurons propagates through rodent cortical tissue, to encode neuronal activity. Spatiotemporal demixing of these patterns then enables functional recording of neuronal activity from a group of discriminable sources. For the first time, we provide an experimental, in vivo characterization of speckle generation, speckle imaging and speckle-assisted demixing of neuronal activity signals in the scattering mammalian brain tissue. We found that despite an initial fast speckle decorrelation, substantial correlation was maintained over minute-long timescales that contributed to our ability to demix temporal activity traces in the mouse brain in vivo. Informed by in vivo quantifications of speckle patterns from single and multiple neurons excited using 2P scanning excitation, we recorded and demixed activity from several sources excited using 1P oblique illumination. In our proof-of-principle experiments, we demonstrate in vivo speckle-assisted demixing of functional signals from groups of sources in a depth range of 220-320 µm in mouse cortex, limited by available speckle contrast. Our results serve as a basis for designing an in vivo functional speckle imaging modality and for maximizing the key resource in any such modality, the speckle contrast. We anticipate that our results will provide critical quantitative guidance to the community for designing techniques that overcome light scattering as a fundamental limitation in bioimaging.

Moxifloxacin promotes two-photon microscopic imaging for discriminating different stages of DSS-induced colitis on mice

BackgroundAccurate diagnosis of patients with ulcerative colitis (UC) can reduce their risk of developing colorectal cancer. This study intended to explore whether moxifloxacin, an agent with fluorescence potential, could promote two-photon microscopy (TPM) diagnosis for mice with dextran sodium sulfate (DSS)-induced colitis, which could imitate human UC. Methods32 Balb/c mice were randomly divided into 4 groups: control, acute colitis, remission colitis and chronic colitis. Fluorescence parameters, imaging performance, and tissue features of different mouse models were compared under moxifloxacin-assisted TPM and label-free TPM. ResultsExcitation wavelength of 720 nm and moxifloxacin labeling time of 2 min was optimal for moxifloxacin-assisted TPM. With moxifloxacin labeling for colonic tissues, excitation power was decreased to 1/10 of that without labeling while fluorescence intensity was increased to 10-fold of that without labeling. Photobleaching was negligible after moxifloxacin labeling and moxifloxacin fluorescence kept stable within 2 h. Compared with the control group, moxifloxacin fluorescence was reduced in the three colitis groups (P < 0.05). Meanwhile, the proportion of enhanced moxifloxacin fluorescence regions was (22.4 ± 1.6)%, (7.7 ± 1.0)%, (13.5 ± 1.7)% and (5.0 ± 1.3)% in the control, acute, remission and chronic groups respectively, with significant reduction in the three colitis groups (P < 0.05). Besides, variant tissue features of experimental colitis models were presented under moxifloxacin-assisted TPM, such as crypt opening, glandular structure, adjacent glandular space and moxifloxacin distribution. ConclusionsWith unique biological interaction between moxifloxacin and colonic mucosa, moxifloxacin-assisted TPM imaging is feasible and effective for accurate diagnosis of different stages of experimental colitis.

Calcium-activated chloride channels in pericytes shape capillary electrical signaling

The brain vasculature, composed of penetrating arterioles (PAs) giving rise to the dense capillary network, facilitates continuous energy substrate delivery to neurons and glia by tightly controlling blood flow. The capillaries are covered by perivascular cells known as pericytes, which exhibit varying morphological and physiological properties based on their location in relation to the terminal arteriole, and these contribute to blood flow control within the capillary network in distinct ways. Thin-strand pericytes —the focus of this study—are located in the deep capillary bed. Serendipitously, we have discovered a robust chloride current during perforated patch clamp experiments on these thin-strand pericytes. We hypothesized that this current is mediated by calcium-activated chloride channels (CaCCs), which may influence electrical signaling and thereby regulate blood flow. Our data reveal that this robust current is dependent on external calcium concentration and can be blocked by voltage-gated calcium channel blockers, depletion of endoplasmic reticulum calcium stores, and calcium-activated chloride channel blockers. This suggests a mechanism wherein calcium entry into the cytosol via voltage-gated channels and intracellular calcium release govern the activity of CaCCs in the thin-strand pericyte membrane. Using two-photon microscopy, we further observed that blocking CaCCs led to PA dilation and hyperemia. Together, our data indicate a novel role for CaCCs in thin-strand pericytes, where they modulate electrical signaling across the capillary bed in response to intracellular calcium activity. By providing a depolarizing signal countering hyperpolarizing electrical signaling, these channels contribute to the intricate regulation of blood flow and vessel dilation. This work was supported by the NIH (grants 1R01AG066645, 5R01NS115401, 1DP2NS121347-01), and the American Heart Association (19IPLOI34660108). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Compounding Effects of Aging and Sex on Cerebrovascular Endothelial Mitochondria

Despite the increasing burden of age-related neurological diseases such as vascular cognitive impairment and dementia or Alzheimer’s disease, the underlying mechanisms of these pathologies are not clear. Nonetheless, structural and functional changes in the microvasculature represent probable pathological aspects of these age-associated diseases. The silent and cumulative changes in endothelial mitochondria with subsequently compromised cerebral vascular function are thought to play a central role in neurological disease development. We tested the hypothesis that mitochondrial morphological changes in endothelial cells facilitate the distinct aging- and sex-related differences in the cerebral microvascular proteome and energetics. Mito-dendra2 (mt-D2) labeled endothelial mitochondria were imaged in vivo via a cranial window using two-photon microscopy at 875 nm in young and old, male and female, mito-Dendra2 mice (YM, YF, OM, and OF, respectively). Mt-D2 associated signal was analyzed in 3D stacks using the Huygens Pro CE 23.04.0p3 object analyzer (160 images, originated from 4-5 mt-D2 mice/groups). We also measured mRNA copy numbers of mitochondrial fission and fusion genes in isolated, cortical microvessels (&lt;70 μm) from YM, YF, OM, and OF C57Bl/6J mice (n=7-11/age/sex) using ddPCR. Kruskal-Wallis test with Dwass-Steel-Critchlow-Fligner post-hoc comparisons were used to compare mt-D2 signal among YM, YF, OM, and OF, whereas Mann-Whitney U test determined changes across age and sex strata. p&lt;0.05 was considered statistically significant. mRNA copy numbers were normalized to housekeeping genes (GAPDH, β-actin, and HPRT-1) and compared with one-way ANOVA based on data distribution. Analysis of four object parameters revealed that object surface was smaller in old vs. young that was significant in YM vs. OM. We found significantly greater corrected lateral width in OM vs. YM, OF vs. YF, and in YF vs. YM. The lateral aspect ratio was significantly greater in YM vs. OM, in YF vs. OF, as well as in YM vs. YF. Accordingly, the axial sphericity of the principal box was significantly greater in OM vs. YM, in OF vs. YF, and in YF vs. YM. Normalized mRNA copy numbers of mitochondrial fission and fusion genes were not statistically different in our samples for DRP-1, Fis-1 or Mfn-1/2, Opa-1, respectively. These results suggest that aging driven morphological differences, such as the shortening of endothelial mt-D2 signal are, at least in part, facilitated by altered fission/fusion protein abundances and/or activity rather than transcriptional changes in our samples. Although we are still analyzing other morphological parameters, by assessing the effect sizes and their physiological relevance, we postulate that these morphological differences contribute to the vulnerability of the aged cerebral microvasculature to injury. AG083567, 3P30GM145498-01, U54GM104940, HL148836. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Contractile pericyte-mediated cerebrovascular deficits in a mouse model of CADASIL

Small vessel disease (SVD) of the brain underlies 25% of stroke cases and is the most common cause of vascular cognitive impairment. The most common monogenic form of SVD is CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), caused by mutations of the NOTCH3 protein which is widely expressed in mural cells (vascular smooth muscle cells [SMCs] and pericytes). Cerebral blood flow (CBF) deficits present early in CADASIL, attributable in part to NOTCH3-mediated extracellular matrix dysregulation, which alters heparin-binding epidermal growth factor-like (HB-EGF) signaling in SMCs and endothelial cells (ECs). Pericytes also robustly express NOTCH3 and are a relatively underexplored cell type in the context of CADASIL. Pericytes proximal to the penetrating arteriole (PA), known as contractile pericytes, express α-smooth muscle actin (αSMA) and can contract to rapidly regulate capillary diameter and thus blood flow in the downstream capillary tree. We conducted two-photon laser scanning microscopy (2PLSM) of blood flow and pericyte contractile responses with CADASIL Notch3R169C transgenic and control mice. Our data indicates that contractile pericyte-mediated control of blood flow is impaired prior to other known CBF deficits, thus raising the tantalizing possibility that these pericytes could be a target for early clinical intervention in CADASIL. While PA responses to stimulation of capillary electrical signaling remain intact, both blood flow as well as capillary diameter of contractile pericyte-covered vessels are diminished. Taken together, this work will establish pericytes as a novel site of dysfunction and provide a new candidate mechanism by which CBF is diminished early in CADASIL pathogenesis for potential therapeutic intervention. 5T32GM008181-33 (AV), 1DP2OD02944801 (TL), 1R01AG066645 (TL), 5R01NS115401 (TL). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

An antagonist-based two-photon fluorogenic probe for imaging metabotropic glutamate receptor 5 in neuronal cells

The expression of metabotropic glutamate receptor 5 (mGluR5) is subject to developmental regulation and undergoes significant changes in neuropsychiatric disorders and diseases. Visualizing mGluR5 by fluorescence imaging is a highly desired innovative technology for biomedical applications. Nevertheless, there are substantial problems with the chemical probes that are presently accessible. In this study, we have successfully developed a two-photon fluorogenic probe, mGlu-5-TP, based on the structure of mGluR5 antagonist 6-methyl-2-(phenylethynyl)pyridine (MPEP). Due to this antagonist-based probe selectively recognizes mGluR5, high expression of mGluR5 on living SH-SY5Y human neuroblastoma cells has been detected during intracellular inflammation triggered by lipopolysaccharides (LPS). Of particular significance, the probe can be employed along with two-photon fluorescence microscopy to enable real-time visualization of the mGluR5 in Aβ fiber-treated neuronal cells, thereby establishing a connection to the progression of Alzheimer's disease (AD). These results revealed that the probe can be a valuable imaging tool for studying mGluR5-related diseases in the nervous system.

Resident macrophages in kidney medulla are critical in cleaning intratubular sedimentary particles

During the passage of the glomerulus filtrate through the renal tubular system, various microscopic sedimentary particles, including mineral crystals resulting from urine concentration, are generated. Dislodging these particles in the intratubular compartment is critical to ensure free flow of filtrate and the final formation of urine. However, it is generally assumed that particle removal is solely attributed to urine flush and whether there is other devoted mechanism is unknown. In this study, we uncovered that most macrophages in the kidney medulla of mice constitutively formed transepithelial protrusions and were “sampling” urine contents, as revealed by high-resolution confocal microscopy. This phenomenon was less pronounced in the cortex. The transepithelial protrusions of medulla macrophages were formed in a transcellular route instead of between epithelial cells, so the overall junctional architecture of tubular epithelium was intact. Juxtatubular macrophages were effcient in sequestering and phagocytosing intraluminal particles through their transepithelial protrusions, and would make transmigration to the tubule lumen to "escort" the excretion of sediment particles, as documented by live two-photon microscopy recording. In accordance, abundant particle-carrying macrophages could be detected in the urine. As such, specifically depleting renal macrophages precipitated kidney stone formation in a hyperoxaluria model. Moreover, naïve mice with underrepresentation of renal macrophages were prone to developing various intratubular sediments. RNA-seq analyses unveiled that medulla macrophages were enriched with genes associated with cell adhesion relative to cortex macrophages. Mechanistically, we found that integrin β1-mediated ligation to the tubular epithelium was crucial to the transepithelial behaviors of medulla macrophages, as evidenced by both pharmacological and genetic approaches. This study unveils a previously unappreciated role of renal medulla macrophages in keeping the tubular system unobstructed by removing intratubular particles. These findings may pave way for developing novel therapeutics for nephrolithiasis. National Natural Science Foundation of China (32170894, 31970898 and 82170441) and the Natural Science Foundation of Zhejiang Province (LZ22C080001). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Label-free single cell metabolic imaging of neutrophil activation

Abstract Upon activation, neutrophils combat infiltrating pathogens through a variety of effector functions, including oxidative burst, phagocytosis, and neutrophil extracellular traps (NETs). Metabolic pathways within neutrophils can be pharmacologically affected to modify immune outcomes. However, there is a lack of techniques to assess neutrophil metabolism in a label-free, non-destructive manner at single cell level. To address this, human neutrophil metabolism was investigated using two-photon microscopy of intrinsic fluorophores reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, and flavin adenine dinucleotide, FAD. We image the optical redox ratio (ORR), [NAD(P)H / (NAD(P)H + FAD)] and the fluorescence lifetimes of NAD(P)H. Single-cell metabolic profiles of neutrophils were generated using an automatic cell segmentation pipeline. We found that activation of neutrophils either pharmacologically or by pathogen infection induced a significant metabolic change characterized by an increase in ORR and decrease in NAD(P)H lifetime, indicating an increase of the NAD(P)H pool, which suggests increases in glycolysis and the pentose phosphate pathway. Neutrophil activation was confirmed by measuring effector functions including oxidative burst. Importantly, identical metabolic changes were detected in the neutrophils of transgenic zebrafish larvae following activation, confirming the validity of these findings in vivo.

Aberrant cortical activity, functional connectivity, and neural assembly architecture after photothrombotic stroke in mice.

Despite substantial progress in mapping the trajectory of network plasticity resulting from focal ischemic stroke, the extent and nature of changes in neuronal excitability and activity within the peri-infarct cortex of mice remains poorly defined. Most of the available data have been acquired from anesthetized animals, acute tissue slices, or infer changes in excitability from immunoassays on extracted tissue, and thus may not reflect cortical activity dynamics in the intact cortex of an awake animal. Here, in vivo two-photon calcium imaging in awake, behaving mice was used to longitudinally track cortical activity, network functional connectivity, and neural assembly architecture for 2 months following photothrombotic stroke targeting the forelimb somatosensory cortex. Sensorimotor recovery was tracked over the weeks following stroke, allowing us to relate network changes to behavior. Our data revealed spatially restricted but long-lasting alterations in somatosensory neural network function and connectivity. Specifically, we demonstrate significant and long-lasting disruptions in neural assembly architecture concurrent with a deficit in functional connectivity between individual neurons. Reductions in neuronal spiking in peri-infarct cortex were transient but predictive of impairment in skilled locomotion measured in the tapered beam task. Notably, altered neural networks were highly localized, with assembly architecture and neural connectivity relatively unaltered a short distance from the peri-infarct cortex, even in regions within 'remapped' forelimb functional representations identified using mesoscale imaging with anaesthetized preparations 8 weeks after stroke. Thus, using longitudinal two-photon microscopy in awake animals, these data show a complex spatiotemporal relationship between peri-infarct neuronal network function and behavioral recovery. Moreover, the data highlight an apparent disconnect between dramatic functional remapping identified using strong sensory stimulation in anaesthetized mice compared to more subtle and spatially restricted changes in individual neuron and local network function in awake mice during stroke recovery.

Aberrant cortical activity, functional connectivity, and neural assembly architecture after photothrombotic stroke in mice

Despite substantial progress in mapping the trajectory of network plasticity resulting from focal ischemic stroke, the extent and nature of changes in neuronal excitability and activity within the peri-infarct cortex of mice remains poorly defined. Most of the available data have been acquired from anesthetized animals, acute tissue slices, or infer changes in excitability from immunoassays on extracted tissue, and thus may not reflect cortical activity dynamics in the intact cortex of an awake animal. Here, in vivo two-photon calcium imaging in awake, behaving mice was used to longitudinally track cortical activity, network functional connectivity, and neural assembly architecture for 2 months following photothrombotic stroke targeting the forelimb somatosensory cortex. Sensorimotor recovery was tracked over the weeks following stroke, allowing us to relate network changes to behavior. Our data revealed spatially restricted but long-lasting alterations in somatosensory neural network function and connectivity. Specifically, we demonstrate significant and long-lasting disruptions in neural assembly architecture concurrent with a deficit in functional connectivity between individual neurons. Reductions in neuronal spiking in peri-infarct cortex were transient but predictive of impairment in skilled locomotion measured in the tapered beam task. Notably, altered neural networks were highly localized, with assembly architecture and neural connectivity relatively unaltered a short distance from the peri-infarct cortex, even in regions within ‘remapped’ forelimb functional representations identified using mesoscale imaging with anaesthetized preparations 8 weeks after stroke. Thus, using longitudinal two-photon microscopy in awake animals, these data show a complex spatiotemporal relationship between peri-infarct neuronal network function and behavioral recovery. Moreover, the data highlight an apparent disconnect between dramatic functional remapping identified using strong sensory stimulation in anaesthetized mice compared to more subtle and spatially restricted changes in individual neuron and local network function in awake mice during stroke recovery.

PD-1 knockout on cytotoxic primary murine CD8+ T cells improves their motility in retrovirus infected mice.

Cytotoxic T lymphocyte (CTL) motility is an important feature of effective CTL responses and is impaired when CTLs become exhausted, e.g. during chronic retroviral infections. A prominent T cell exhaustion marker is programmed cell death protein 1 (PD-1) and antibodies against the interaction of PD-1 and PD-ligand 1 (PD-L1) are known to improve CTL functions. However, antibody blockade affects all PD-1/PD-L1-expressing cell types, thus, the observed effects cannot be attributed selectively to CTLs. To overcome this problem, we performed CRISPR/Cas9 based knockout of the PD-1 coding gene PDCD1 in naïve Friend Retrovirus (FV)-specific CTLs. We transferred 1,000 of these cells into mice where they proliferated upon FV-infection. Using intravital two-photon microscopy we visualized CTL motility in the bone marrow and evaluated cytotoxic molecule expression by flow cytometry. Knockout of PDCD1 improved the CTL motility at 14 days post infection and enhanced the expression of cytotoxicity markers. Our data show the potential of genetic tuning of naive antiviral CTLs and might be relevant for future designs of improved T cell-mediated therapies.

A sterilizable platform based on crosslinked xanthan gum for controlled-release of polymeric micelles: Ocular application for the delivery of neuroprotective compounds to the posterior eye segment

TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate) polymeric micelles show interesting properties for ocular administration thanks to their solubilization capability, nanometric size and tissue penetration ability. However, micelles formulations are generally characterized by low viscosity, poor adhesion and very short retention time at the administration site. Therefore, the idea behind this work is the preparation and characterization of a crosslinked film based on xanthan gum that contains TPGS micelles and is capable of controlling their release. The system was loaded with melatonin and cyclosporin A, neuroprotective compounds to be delivered to the posterior eye segment. Citric acid and heating at different times and temperatures were exploited as crosslinking approach, giving the possibility to tune swelling, micelles release and drug release. The biocompatibility of the platform was confirmed by HET-CAM assay. Ex vivo studies on isolated porcine ocular tissues, conducted using Franz cells and two-photon microscopy, demonstrated the potential of the xanthan gum-based platform and enlightened micelles penetration mechanism. Finally, the sterilization step was approached, and a process to simultaneously crosslink and sterilize the platform was developed.

Responsive regularity of neurons in the hindlimb region of primary somatosensory cortex to different temperature thermal needle stimulation of "Zusanli"(ST36) in mice.

To study the regularity of central response to thermal needle stimulation of "Zusanli" (ST36) at different temperature, and to analyze the temperature difference of central responses. Six male C57BL/6j adult mice were used in the present study. For observing activities of neurons in the hindlimb region of left primary somatosensory cortex (S1HL, A/P=0.46 mm, M/L=1.32 mm, D/V=-0.14 mm) by using a fast high-resolution miniature two-photon microscopy (FHIRM-TPM), the mice were anesthetized with 3% isoflurane (inhalation), with its head fixed in a stereotaxic apparatus, then, adeno-associated virus (AAV-hSyn-GCaMP6f-WPRE-hGHpA, for showing intracellular calcium transients in neurons transfected) was injected into the left S1HL region using a micro-syringe after scalp surgical operation. The mice's right ST36 were stimulated using internal thermal needles with the temperature being 43 ℃, or 45 ℃, or 47 ℃, separately. Image J software and MATLAB 2020b software were used to process the image data of neuronal calcium activity (Ca2+ signaling) in the left S1HL region, including the instant maximum calcium peak value (ΔF/F) in 2 s, instant calcium spike frequency in 2 s, short-term calcium peak value (ΔF/F) in 3.5 min, short-term calcium spike frequency in 3.5 min, calcium peak duration in 3.5 min, maximum calcium peak value (ΔF/F) at the 1st , 2nd and 3rd min, and calcium spike frequency at the 1st, 2nd and 3rd min after thermal needle stimulation. In comparison with the normal temperature needle stimulation, the instant intracellular maximum calcium peak value, instant calcium spike frequency, short-term maximum calcium peak value, short-term calcium spike frequency, and calcium peak duration of S1HL neurons in response to 43 ℃, 45 ℃ and 47 ℃ internal thermal needle stimulation of ST36 were significantly increased (P<0.001, P<0.01). Comparison among the 43 ℃, 45 ℃ and 47 ℃ thermal needle stimulation showed that the 45 ℃ thermal needle stimulation was obviously superior to 43 ℃ and 47 ℃ thermal needle stimulation in increasing instant calcium spike frequency, short-term calcium spike frequency and calcium peak duration of S1HL neurons (P<0.001, P<0.01). The 47 ℃ thermal needle stimulation was stronger than 43 ℃ and 45 ℃ thermal needle stimulation in increasing the instant maximum calcium peak value (P<0.001). The maximum calcium peak value was apparently higher (P<0.001) at the 2nd min than that at the 1st and 3rd min after 43 ℃, 45 ℃ and 47 ℃ thermal needle stimulation. No significant differences were found in the short-term maximum calcium peak value among the 3 thermal needle stimulation and in the calcium spike frequency among the 3 time points after 43 ℃, 45 ℃ and 47 ℃ thermal needle stimulation. S1HL neurons respond to all 43 ℃, 45 ℃ and 47 ℃ thermal needle stimulation of ST36 in mice, while more actively to 45 ℃ thermal needle stimulation.

Compressive sensing-based multi-focus line-scanning two-photon microscopy for fast 3D imaging

Fast 3D volume imaging methods have been playing increasingly important roles in biological studies. In this article, we present the design and characterization of a multi-focus line-scanning two-photon microscope. Specifically, a digital micromirror device (DMD) is employed to generate a randomly distributed focus array on a plane (i.e., x-z) via binary holography. Next, a galvanometric mirror scans the focus array in a direction normal to the plane (i.e., y-axis) over the imaging volume. For sparse samples, e.g., neural networks in a brain, 1-3 foci are used together with compressive sensing algorithm to achieve a volume imaging rate of 15.5 volumes/sec over 77 × 120 × 40 µm3. High-resolution optical cross-sectional images on selected planes and regions can be generated by sequentially scanning the laser focus generated on the x-z plane with good imaging speeds (e.g., 107 frames/sec over 80 × 120 × 40 µm3). In the experiments, microbeads, pollens, and mouse brain slices have been imaged to characterize the point spread function and volume image rate and quality at different sampling ratios. The results show that the multi-focus line-scanning microscope presents a fast and versatile 3D imaging platform for deep tissue imaging and dynamic live animal studies.

Neural Tracing Protein-Functionalized Nanoparticles Capable of Fast Retrograde Axonal Transport in Live Neurons.

Neural tracing proteins like horseradish peroxidase-conjugated wheat germ agglutinin (WGA-HRP) can target the central nervous system (CNS) through anatomic retrograde transport without crossing the blood-brain barrier (BBB). Conjugating WGA-HRP to nanoparticles may enable the creation of BBB-bypassing nanomedicine. Microfluidics and two-photon confocal microscopy is applied to screen nanocarriers for transport efficacy and gain mechanistic insights into their interactions with neurons. Protein modification of gold nanoparticles alters their cellular uptake at the axonal terminal and activates fast retrograde transport. Trajectory analysis of individual endosomes carrying the nanoparticles reveals a run-and-pause pattern along the axon with endosomes carrying WGA-HRP-conjugated gold nanoparticles exhibiting longer run duration and faster instantaneous velocity than those carrying nonconjugated nanoparticles. The results offer a mechanistic explanation of the different axonal transport dynamics as well as a cell-based functional assay of neuron-targeted nanoparticles with the goal of developing BBB-bypassing nanomedicine for the treatment of nervous system disorders.

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung.

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in naïve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

Intravital observation of high-scattering and dense-labeling hepatic tissues using multi-photon fluorescence microscopy.

Achieving high-resolution and large-depth microscopic imaging in vivo under conditions characterized by high-scattering and dense-labeling, as commonly encountered in the liver, poses a formidable challenge. Here, through the optimization of multi-photon fluorescence excitation window, tailored to the unique optical properties of the liver, intravital microscopic imaging of hepatocytes and hepatic blood vessels with high spatial resolution was attained. It's worth noting that resolution degradation caused by tissue scattering of excitation light was mitigated by accounting for moderate tissue self-absorption. Leveraging high-quality multi-photon fluorescence microscopy, we discerned structural and functional alterations in hepatocytes during drug-induced acute liver failure. Furthermore, a reduction in indocyanine green metabolism rates associated with acute liver failure was observed using NIR-II fluorescence macroscopic imaging.

Integrated Microprism and Microelectrode Array for Simultaneous Electrophysiology and Two-Photon Imaging across All Cortical Layers.

Cerebral neural electronics play a crucial role in neuroscience research with increasing translational applications such as brain-computer interfaces for sensory input and motor output restoration. While widely utilized for decades, the understanding of the cellular mechanisms underlying this technology remains limited. Although two-photon microscopy (TPM) has shown great promise in imaging superficial neural electrodes, its application to deep-penetrating electrodes is technically difficult. Here, a novel device integrating transparent microelectrode arrays with glass microprisms, enabling electrophysiology recording and stimulation alongside TPM imaging across all cortical layers in a vertical plane, is introduced. Tested in Thy1-GCaMP6 mice for over 4 months, the integrated device demonstrates the capability for multisite electrophysiological recording/stimulationand simultaneous TPM calcium imaging. As a proof of concept, the impact of microstimulation amplitude, frequency, and depth on neural activation patterns is investigated using the setup. With future improvements in material stability and single unit yield, this multimodal tool greatly expands integrated electrophysiology and optical imaging from the superficial brain to the entire cortical column, opening new avenues for neuroscience research and neurotechnology development.