Two-photon Microscopy Research Articles

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.

Dynamic Multiplex Tissue Imaging in Inflammation Research.

Inflammation is a highly dynamic process with immune cells that continuously interact with each other and parenchymal components as they migrate through tissue. The dynamic cellular responses and interaction patterns are a function of the complex tissue environment that cannot be fully reconstructed ex vivo, making it necessary to assess cell dynamics and changing spatial patterning in vivo. These dynamics often play out deep within tissues, requiring the optical focus to be placed far below the surface of an opaque organ. With the emergence of commercially available two-photon excitation lasers that can be combined with existing imaging systems, new avenues for imaging deep tissues over long periods of time have become available. We discuss a selected subset of studies illustrating how two-photon microscopy (2PM) has helped to relate the dynamics of immune cells to their in situ function and to understand the molecular patterns that govern their behavior in vivo. We also review some key practical aspects of 2PM methods and point out issues that can confound the results, so that readers can better evaluate the reliability of conclusions drawn using this technology.

DEEP-squared: deep learning powered De-scattering with Excitation Patterning

Limited throughput is a key challenge in in vivo deep tissue imaging using nonlinear optical microscopy. Point scanning multiphoton microscopy, the current gold standard, is slow especially compared to the widefield imaging modalities used for optically cleared or thin specimens. We recently introduced “De-scattering with Excitation Patterning” or “DEEP” as a widefield alternative to point-scanning geometries. Using patterned multiphoton excitation, DEEP encodes spatial information inside tissue before scattering. However, to de-scatter at typical depths, hundreds of such patterned excitations were needed. In this work, we present DEEP2, a deep learning-based model that can de-scatter images from just tens of patterned excitations instead of hundreds. Consequently, we improve DEEP’s throughput by almost an order of magnitude. We demonstrate our method in multiple numerical and experimental imaging studies, including in vivo cortical vasculature imaging up to 4 scattering lengths deep in live mice.

High throughput detection of capillary stalling events with Bessel beam two-photon microscopy.

Brief disruptions in capillary flow, commonly referred to as capillary "stalling," have gained interest recently for their potential role in disrupting cerebral blood flow and oxygen delivery. Approaches to studying this phenomenon have been hindered by limited volumetric imaging rates and cumbersome manual analysis. The ability to precisely and efficiently quantify the dynamics of these events will be key in understanding their potential role in stroke and neurodegenerative diseases, such as Alzheimer's disease. Our study aimed to demonstrate that the fast volumetric imaging rates offered by Bessel beam two-photon microscopy combined with improved data analysis throughput allows for faster and more precise measurement of capillary stall dynamics. We found that while our analysis approach was unable to achieve full automation, we were able to cut analysis time in half while also finding stalling events that were missed in traditional blind manual analysis. The resulting data showed that our Bessel beam system was captured more stalling events compared to optical coherence tomography, particularly shorter stalling events. We then compare differences in stall dynamics between a young and old group of mice as well as a demonstrate changes in stalling before and after photothrombotic model of stroke. Finally, we also demonstrate the ability to monitor arteriole dynamics alongside stall dynamics. Bessel beam two-photon microscopy combined with high throughput analysis is a powerful tool for studying capillary stalling due to its ability to monitor hundreds of capillaries simultaneously at high frame rates.

Norepinephrine modulates calcium dynamics in cortical oligodendrocyte precursor cells promoting proliferation during arousal in mice.

Oligodendrocytes, the myelinating cells of the central nervous system (CNS), are generated from oligodendrocyte precursor cells (OPCs) that express neurotransmitter receptors. However, the mechanisms that affect OPC activity in vivo and the physiological roles of neurotransmitter signaling in OPCs are unclear. In this study, we generated a transgenic mouse line that expresses membrane-anchored GCaMP6s in OPCs and used longitudinal two-photon microscopy to monitor OPC calcium (Ca2+) dynamics in the cerebral cortex. OPCs exhibit focal and transient Ca2+ increases within their processes that are enhanced during locomotion-induced increases in arousal. The Ca2+ transients occur independently of excitatory neuron activity, rapidly decline when OPCs differentiate and are inhibited by anesthesia, sedative agents or noradrenergic receptor antagonists. Conditional knockout of α1A adrenergic receptors in OPCs suppresses spontaneous and locomotion-induced Ca2+ increases and reduces OPC proliferation. Our results demonstrate that OPCs are directly modulated by norepinephrine in vivo to enhance Ca2+ dynamics and promote population homeostasis.

Cell-specific alterations in autophagy-lysosomal activity near the chronically implanted microelectrodes

Intracortical microelectrodes that can record and stimulate brain activity have become a valuable technique for basic science research and clinical applications. However, long-term implantation of these microelectrodes can lead to progressive neurodegeneration in the surrounding microenvironment, characterized by elevation in disease-associated markers. Dysregulation of autophagy-lysosomal degradation, a major intracellular waste removal process, is considered a key factor in the onset and progression of neurodegenerative diseases. It is plausible that similar dysfunctions in autophagy-lysosomal degradation contribute to tissue degeneration following implantation-induced focal brain injury, ultimately impacting recording performance. To understand how the focal, persistent brain injury caused by long-term microelectrode implantation impairs autophagy-lysosomal pathway, we employed two-photon microscopy and immunohistology. This investigation focused on the spatiotemporal characterization of autophagy-lysosomal activity near the chronically implanted microelectrode. We observed an aberrant accumulation of immature autophagy vesicles near the microelectrode over the chronic implantation period. Additionally, we found deficits in autophagy-lysosomal clearance proximal to the chronic implant, which was associated with an accumulation of autophagy cargo and a reduction in lysosomal protease level during the chronic period. Furthermore, our evidence demonstrates reactive astrocytes have myelin-containing lysosomes near the microelectrode, suggesting its role of myelin engulfment during acute implantation period. Together, this study sheds light on the process of brain tissue degeneration caused by long-term microelectrode implantation, with a specific focus on impaired intracellular waste degradation.

Navigating chimeric antigen receptor-engineered natural killer cells as drug carriers via three-dimensional mapping of the tumor microenvironment

Chimeric antigen receptor (CAR)-modified natural killer (NK) cells are recognized as promising immunotherapeutic agents for cancer treatment. However, the efficacy and trafficking of CAR-NK cells in solid tumors are hindered by the complex barriers present in the tumor microenvironment (TME). We have developed a novel strategy that utilizes living CAR-NK cells as carriers to deliver anticancer drugs specifically to the tumor site. We also introduce a time-lapse method for evaluating the efficacy and tumor specificity of CAR-NK cells using a two-photon microscope in live mouse models and three-dimensional (3D) tissue slide cultures. Our results demonstrate that CAR-NK cells exhibit enhanced antitumor immunity when combined with photosensitive chemicals in both in vitro and in vivo tumor models. Additionally, we have successfully visualized the trafficking, infiltration, and accumulation of drug-loaded CAR-NK cells in deeply situated TME using non-invasive intravital two-photon microscopy. Our findings highlight that tumor infiltration of CAR-NK cells can be intravitally monitored through the two-photon microscope approach. In conclusion, our study demonstrates the successful integration of CAR-NK cells as drug carriers and paves the way for combined cellular and small-molecule therapies in cancer treatment. Furthermore, our 3D platform offers a valuable tool for assessing the behavior of CAR cells within solid tumors, facilitating the development and optimization of immunotherapeutic strategies with clinical imaging approaches.

Polarization-sensitive Two-photon Microscopy for a Label-free Amyloid Structural Characterization.

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue penetration, efficient operation in densely packed systems, and reduced angular photoselection of fluorophores. Thus, the introduction of polarization analysis in two-photon fluorescence microscopy (2PFM) provides a more precise determination of molecular organization in a sample compared to standard imaging methods based on linear optical processes. In this work, we focus on polarization-sensitive 2PFM (ps-2PFM) and its application in the determination of molecular ordering within complex bio-structures-amyloid spherulites. Neurodegenerative diseases such as Alzheimer's or Parkinson's are often diagnosed through the detection of amyloids-protein aggregates formed due to an impaired protein misfolding process. Exploring their structure leads to a better understanding of their creation pathway and consequently, to developing more sensitive diagnostic methods. This paper presents the ps-2PFM adapted for the determination of local fibril ordering inside the bovine insulin spherulites and spherical amyloidogenic protein aggregates. Moreover, we prove that the proposed technique can resolve the three-dimensional organization of fibrils inside the spherulite.

OS09.1.AIN VIVOTWO-PHOTON TUMOR IMAGING IN AN ORTHOTOPIC MEDULLOBLASTOMA MOUSE MODEL

Abstract BACKGROUND Medulloblastoma represents the most common primary brain malignancy in pediatric patients and is associated with neuro-cognitive impairment and frequent relapses after aggressive multimodal therapy. Adoptive immunotherapies such as CAR T-cell therapy are currently being investigated, showing promising results in preclinical models but lacking efficacy in first-in-human trials. Refined in vivo models to investigate reasons for treatment failures are therefore urgently needed. METHODS We developed a xenogeneic orthotopic medulloblastoma (MB) model in mice by combining a chronic cerebellar window with repetitive intravital two-photon laser scanning microscopy. Red tdTomato-fluorescent DAOY (SHH MB) tumor cells expressing EphA2 were implanted intracranially into the cerebellum of immunodeficient mice (n = 7), and tumor cell formation was followed by in vivo-microscopy. Intravenous fluorescein isothiocyanate (FITC)-dextran was used for intravascular plasma staining. EphA-directed CAR T-cells were injected into the adjacent brain parenchyma. RESULTS Chronic cranial window implantation was well tolerated and allowed repetitive visualization of the mouse cerebellum. After intracranial tumor cell implantation, continuous tumor growth could be evaluated with epifluorescence as well as 2-photon laser scanning microscopy. Tumor formation was identified as early as day 5 in every mouse and could continuously be followed up to 35 days until mice succumbed due to tumor burden. Window quality and fluorescence intensity remained high until the end of the experiments. Studies on GFP-expressing CAR T-cell injection directed against EphA2 are currently ongoing. CONCLUSIONS We herein establish a robust orthotopic medulloblastoma mouse model that allows repetitive in vivo tracking of fluorescence-expressing tumor cells, CAR T-cells and blood vessels over weeks. Such models may be used to study medulloblastoma tumor growth under the influence of different immunotherapies and reveal interactions between tumor cells and CAR T-cell therapies on a single-cell level.

Invited Session IV: Studies of the visual cortex with sub-millimeter resolution: Imaging the visual brain at high spatiotemporal resolution.

Since the discovery of orientation-selective neurons in the cat primary visual cortex (V1), how the mammalian nervous system computes the orientation of visual stimuli has been a flagship question in neuroscience. With the recent advances in in vivo imaging of neural activity using genetically encoded indicators and two-photon fluorescence microscopy, we can now achieve synapse-resolving functional imaging and kilohertz imaging of membrane voltage in the mouse V1. I will describe our recent advancements in imaging visual processing at high spatiotemporal resolution, as well as two studies on the orientation selectivity of neurons in the mouse visual pathway.

Gallic Acid Ameliorates Cognitive Impairment Caused by Sleep Deprivation through Antioxidant Effect.

Sleep deprivation (SD) has a profound impact on the central nervous system, resulting in an array of mood disorders, including depression and anxiety. Despite this, the dynamic alterations in neuronal activity during sleep deprivation have not been extensively investigated. While some researchers propose that sleep deprivation diminishes neuronal activity, thereby leading to depression. Others argue that short-term sleep deprivation enhances neuronal activity and dendritic spine density, potentially yielding antidepressant effects. In this study, a two-photon microscope was utilized to examine the calcium transients of anterior cingulate cortex (ACC) neurons in awake SD mice in vivo at 24-hour intervals. It was observed that SD reduced the frequency and amplitude of Ca2+ transients while increasing the proportions of inactive neurons. Following the cessation of sleep deprivation, neuronal calcium transients demonstrated a gradual recovery. Moreover, whole-cell patch-clamp recordings revealed a significant decrease in the frequency of spontaneous excitatory post-synaptic current (sEPSC) after SD. The investigation also assessed several oxidative stress parameters, finding that sleep deprivation substantially elevated the level of malondialdehyde (MDA), while simultaneously decreasing the expression of Nuclear Factor erythroid 2-Related Factor 2 (Nrf2) and activities of Superoxide dismutase (SOD) in the ACC. Importantly, the administration of gallic acid (GA) notably mitigated the decline of calcium transients in ACC neurons. GA was also shown to alleviate oxidative stress in the brain and improve cognitive impairment caused by sleep deprivation. These findings indicate that the calcium transients of ACC neurons experience a continuous decline during sleep deprivation, a process that is reversible. GA may serve as a potential candidate agent for the prevention and treatment of cognitive impairment induced by sleep deprivation.

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.

SNR enhanced high-speed two-photon microscopy using a pulse picker and time gating detection

Two-photon microscopy (TPM) is an attractive biomedical imaging method due to its large penetration depth and optical sectioning capability. In particular, label-free autofluorescence imaging offers various advantages for imaging biological samples. However, relatively low intensity of autofluorescence leads to low signal-to-noise ratio (SNR), causing practical challenges for imaging biological samples. In this study, we present TPM using a pulse picker to utilize low pulse repetition rate of femtosecond pulsed laser to increase the pulse peak power of the excitation source leading to higher emission of two-photon fluorescence with the same average illumination power. Stronger autofluorescence emission allowed us to obtain higher SNR images of arterial and liver tissues. In addition, by applying the time gating detection method to the pulse signals obtained by TPM, we were able to significantly reduce the background noise of two-photon images. As a result, our TPM system using the pulsed light source with a 19 times lower repetition rate allowed us to obtain the same SNR image more than 19 times faster with the same average power. Although high pulse energy can increase the photobleaching, we also observed that high-speed imaging with low total illumination energy can mitigate the photobleaching effect to a level similar to that of conventional illumination with a high repetition rate. We anticipate that this simple approach will provide guidance for SNR enhancement with high-speed imaging in TPM as well as other nonlinear microscopy.

Elimination of senescent cells by treatment with Navitoclax/ABT263 reverses whole brain irradiation-induced blood-brain barrier disruption in the mouse brain.

Whole brain irradiation (WBI), a commonly employed therapy for multiple brain metastases and as a prophylactic measure after cerebral metastasis resection, is associated with a progressive decline in neurocognitive function, significantly impacting the quality of life for approximately half of the surviving patients. Recent preclinical investigations have shed light on the multifaceted cerebrovascular injury mechanisms underlying this side effect of WBI. In this study, we aimed to test the hypothesis that WBI induces endothelial senescence, contributing to chronic disruption of the blood-brain barrier (BBB) and microvascular rarefaction. To accomplish this, we utilized transgenic p16-3MR mice, which enable the identification and selective elimination of senescent cells. These mice were subjected to a clinically relevant fractionated WBI protocol (5 Gy twice weekly for 4 weeks), and cranial windows were applied to both WBI-treated and control mice. Quantitative assessment of BBB permeability and capillary density was performed using two-photon microscopy at the 6-month post-irradiation time point. The presence of senescent microvascular endothelial cells was assessed by imaging flow cytometry, immunolabeling, and single-cell RNA-sequencing (scRNA-seq). WBI induced endothelial senescence, which associated with chronic BBB disruption and a trend for decreased microvascular density in the mouse cortex. In order to investigate the cause-and-effect relationship between WBI-induced senescence and microvascular injury, senescent cells were selectively removed from animals subjected to WBI treatment using Navitoclax/ABT263, a well-known senolytic drug. This intervention was carried out at the 3-month post-WBI time point. In WBI-treated mice, Navitoclax/ABT263 effectively eliminated senescent endothelial cells, which was associated with decreased BBB permeability and a trend for increased cortical capillarization. Our findings provide additional preclinical evidence that senolytic treatment approaches may be developed for prevention of the side effects of WBI.

A mechanistic computational framework to investigate the hemodynamic fingerprint of the blood oxygenation level-dependent signal.

Blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) is one of the most used imaging techniques to map brain activity or to obtain clinical information about human cortical vasculature, in both healthy and disease conditions. Nevertheless, BOLD fMRI is an indirect measurement of brain functioning triggered by neurovascular coupling. The origin of the BOLD signal is quite complex, and the signal formation thus depends, among other factors, on the topology of the cortical vasculature and the associated hemodynamic changes. To understand the hemodynamic evolution of the BOLD signal response in humans, it is beneficial to have a computational framework available that virtually resembles the human cortical vasculature, and simulates hemodynamic changes and corresponding MRI signal changes via interactions of intrinsic biophysical and magnetic properties of the tissues. To this end, we have developed a mechanistic computational framework that simulates the hemodynamic fingerprint of the BOLD signal based on a statistically defined, three-dimensional, vascular model that approaches the human cortical vascular architecture. The microvasculature is approximated through a Voronoi tessellation method and the macrovasculature is adapted from two-photon microscopy mice data. Using this computational framework, we simulated hemodynamic changes-cerebral blood flow, cerebral blood volume, and blood oxygen saturation-induced by virtual arterial dilation. Then we computed local magnetic field disturbances generated by the vascular topology and the corresponding blood oxygen saturation changes. This mechanistic computational framework also considers the intrinsic biophysical and magnetic properties of nearby tissue, such as water diffusion and relaxation properties, resulting in a dynamic BOLD signal response. The proposed mechanistic computational framework provides an integrated biophysical model that can offer better insights regarding the spatial and temporal properties of the BOLD signal changes.

All-synchronized picosecond pulses and time-gated detection improve the spatial resolution of two-photon STED microscopy in brain tissue imaging.

Super-resolution in two-photon excitation (2PE) microscopy offers new approaches for visualizing the deep inside the brain functions at the nanoscale. In this study, we developed a novel 2PE stimulated-emission-depletion (STED) microscope with all-synchronized picosecond pulse light sources and time-gated fluorescence detection, namely, all-pulsed 2PE-gSTED microscopy. The implementation of time-gating is critical to excluding undesirable signals derived from brain tissues. Even in a case using subnanosecond pulses for STED, the impact of time-gating was not negligible; the spatial resolution in the image of the brain tissue was improved by approximately 1.4 times compared with non time-gated image. This finding demonstrates that time-gating is more useful than previously thought for improving spatial resolution in brain tissue imaging. This microscopy will facilitate deeper super-resolution observation of the fine structure of neuronal dendritic spines and the intracellular dynamics in brain tissue.

Fully refractive telecentric f-theta microscope based on adaptive elements for 3D raster scanning of biological tissues.

Various techniques in microscopy are based on point-wise acquisition, which provides advantages in acquiring sectioned images, for example in confocal or two-photon microscopy. The advantages come along with the need to perform three-dimensional scanning, which is often realized by mechanical movement achieved by stage-scanning or piezo-based scanning in the axial direction. Lateral scanning often employs galvo-mirrors, leading to a reflective setup and hence to a folded beam path. In this paper, we introduce a fully refractive microscope capable of three-dimensional scanning, which employs the combination of an adaptive lens, an adaptive prism, and a tailored telecentric f-theta objective. Our results show that this microscope is capable to perform flexible three-dimensional scanning, with low scan-induced aberrations, at a uniform resolution over a large tuning range of X=Y=6300 μ m and Z=480 μ m with only transmissive components. We demonstrate the capabilities at the example of volumetric measurements on the transgenic fluorescence of the thyroid of a zebrafish embryo and mixed pollen grains. This is the first step towards flexible aberration-free volumetric smart microscopy of three-dimensional samples like embryos and organoids, which could be exploited for the demands in both lateral and axial dimensions in biomedical samples without compromising image quality.

Ethanol Causes Cell Death and Neuronal Differentiation Defect During Initial Neurogenesis of the Neural Retina by Disrupting Calcium Signaling in Human Retinal Organoids.

Fetal Alcohol Syndrome (FAS) affects a significant proportion, exceeding 90%, of afflicted children, leading to severe ocular aberrations such as microphthalmia and optic nerve hypoplasia. During the early stages of pregnancy, the commencement of neural retina neurogenesis represents a critical period for human eye development, concurrently exposing the developing retinal structures to the highest risk of prenatal ethanol exposure due to a lack of awareness. Despite the paramount importance of this period, the precise influence and underlying mechanisms of short-term ethanol exposure on the developmental process of the human neural retina have remained largely elusive. In this study, we utilize the human embryonic stem cells derived retinal organoids (hROs) to recapitulate the initial retinal neurogenesis and find that 1% (v/v) ethanol slows the growth of hROs by inducing robust cell death and retinal ganglion cell differentiation defect. Bulk RNA-seq analysis and two-photon microscope live calcium imaging reveal altered calcium signaling dynamics derived from ethanol-induced down-regulation of RYR1 and CACNA1S. Moreover, the calcium-binding protein RET, one of the downstream effector genes of the calcium signaling pathway, synergistically integrates ethanol and calcium signals to abort neuron differentiation and cause cell death. To sum up, our study illustrates the effect and molecular mechanism of ethanol on the initial neurogenesis of the human embryonic neural retina, providing a novel interpretation of the ocular phenotype of FAS and potentially informing preventative measures for susceptible populations.

A general design of pyridinium-based fluorescent probes for enhancing two-photon microscopy

Two-photon absorbing fluorescent probes have emerged as powerful imaging tools for subcellular-level monitoring of biological substances and processes, offering advantages such as deep light penetration, minimal photodamage, low autofluorescence, and high spatial resolution. However, existing two-photon absorbing probes still face several limitations, such as small two-photon absorption cross-section, poor water solubility, low membrane permeability, and potentially high toxicity. Herein, we report three small-molecule probes, namely MSP-1arm, Lyso-2arm, and Mito-3arm, composed of a pyridinium center (electron-acceptor) and various methoxystyrene “arms” (electron-donor). These probes exhibit excellent fluorescence quantum yield and decent aqueous solubility. Leveraging the inherent intramolecular charge transfer and excitonic coupling effect, these complexes demonstrate excellent two-photon absorption in the near-infrared region. Notably, Lyso-2arm and Mito-3arm exhibit distinct targeting abilities for lysosomes and mitochondria, respectively. In two-photon microscopy experiments, Mito-3arm outperforms a commercial two-photon absorbing dye in 2D monolayer HeLa cells, delivering enhanced resolution, broader NIR light excitation window, and higher signal-to-noise ratio. Moreover, the two-photon bioimaging of 3D human forebrain organoids confirms the successful deep tissue imaging capabilities of both Lyso-2arm and Mito-3arm. Overall, this work presents a rational design strategy in developing competent two-photon-absorbing probes by varying the number of conjugated “arms” for bioimaging applications.

Corrigendum: Sensorless wavefront correction in two-photon microscopy across different turbidity scales

Corrigendum: Sensorless wavefront correction in two-photon microscopy across different turbidity scales