Label-free, fast, 2-photon volume imaging of the organization of neurons and glia in the enteric nervous system.

The enteric nervous system (ENS), sometimes referred to as a "second brain" is a quasi-autonomous nervous system, made up of interconnected plexuses organized in a mesh-like network lining the gastrointestinal tract. Originally described as an actor in the regulation of digestion, bowel contraction, and intestinal secretion, the implications of the ENS in various central neuropathologies has recently been demonstrated. However, with a few exceptions, the morphology and pathologic alterations of the ENS have mostly been studied on thin sections of the intestinal wall or, alternatively, in dissected explants. Precious information on the three-dimensional (3-D) architecture and connectivity is hence lost. Here, we propose the fast, label-free 3-D imaging of the ENS, based on intrinsic signals. We used a custom, fast tissue-clearing protocol based on a high refractive-index aqueous solution to increase the imaging depth and allow us the detection of faint signals and we characterized the autofluorescence (AF) from the various cellular and sub-cellular components of the ENS. Validation by immunofluorescence and spectral recordings complete this groundwork. Then, we demonstrate the rapid acquisition of detailed 3-D image stacks from unlabeled mouse ileum and colon, across the whole intestinal wall and including both the myenteric and submucosal enteric nervous plexuses using a new spinning-disk two-photon (2P) microscope. The combination of fast clearing (less than 15 min for 73% transparency), AF detection and rapid volume imaging [less than 1 min for the acquisition of a z-stack of 100 planes (150*150 µm) at sub-300-nm spatial resolution] opens up the possibility for new applications in fundamental and clinical research.

Fast 3-D Imaging of Brain Organoids With a New Single-Objective Planar-Illumination Two-Photon Microscope.

Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies (EBs) enlarge the scope of investigations to multi-cellular phenomena. The highest level of complexity, brain organoids that-in many aspects-recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that uses planar two-photon (2P) excitation. Its particularity is that-unlike two- or three-lens light-sheet microscopes-it uses a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petrán spinning-disk scheme for achieving wide-field excitation. However, unlike the Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This new design, advantageous for 2P excitation, circumvents problems arising with the tandem disk from the large wavelength difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited by the light sheet is collected with the same objective and imaged onto a fast sCMOS camera. We demonstrate 3-D imaging of TO-PRO3-stained EBs and of brain organoids, uncleared and after rapid partial transparisation with triethanolamine formamide (RTF) and we compare the performance of our instrument to that of a confocal laser-scanning microscope (CLSM) having a similar numerical aperture. Our large-field 2P-spinning disk microscope permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.

Two-photon probes for in vivo multicolor microscopy of the structure and signals of brain cells.

Imaging the brain of living laboratory animals at a microscopic scale can be achieved by two-photon microscopy thanks to the high penetrability and low phototoxicity of the excitation wavelengths used. However, knowledge of the two-photon spectral properties of the myriad fluorescent probes is generally scarce and, for many, non-existent. In addition, the use of different measurement units in published reports further hinders the design of a comprehensive imaging experiment. In this review, we compile and homogenize the two-photon spectral properties of 280 fluorescent probes. We provide practical data, including the wavelengths for optimal two-photon excitation, the peak values of two-photon action cross section or molecular brightness, and the emission ranges. Beyond the spectroscopic description of these fluorophores, we discuss their binding to biological targets. This specificity allows in vivo imaging of cells, their processes, and even organelles and other subcellular structures in the brain. In addition to probes that monitor endogenous cell metabolism, studies of healthy and diseased brain benefit from the specific binding of certain probes to pathology-specific features, ranging from amyloid-ß plaques to the autofluorescence of certain antibiotics. A special focus is placed on functional in vivo imaging using two-photon probes that sense specific ions or membrane potential, and that may be combined with optogenetic actuators. Being closely linked to their use, we examine the different routes of intravital delivery of these fluorescent probes according to the target. Finally, we discuss different approaches, strategies, and prerequisites for two-photon multicolor experiments in the brains of living laboratory animals.

Combination of an optical parametric oscillator and quantum-dots 655 to improve imaging depth of vasculature by intravital multicolor two-photon microscopy.

Simultaneous imaging of different cell types and structures in the mouse central nervous system (CNS) by intravital two-photon microscopy requires the characterization of fluorophores and advances in approaches to visualize them. We describe the use of a two-photon infrared illumination generated by an optical parametric oscillator (OPO) on quantum-dots 655 (QD655) nanocrystals to improve resolution of the vasculature deeper in the mouse brain both in healthy and pathological conditions. Moreover, QD655 signal can be unmixed from the DsRed2, CFP, EGFP and EYFP fluorescent proteins, which enhances the panel of multi-parametric correlative investigations both in the cortex and the spinal cord.

Phenotypic dynamics of microglial and monocyte-derived cells in glioblastoma-bearing mice.

Inflammatory cells, an integral component of tumor evolution, are present in Glioblastomas multiforme (GBM). To address the cellular basis and dynamics of the inflammatory microenvironment in GBM, we established an orthotopic syngenic model by grafting GL261-DsRed cells in immunocompetent transgenic LysM-EGFP//CD11c-EYFP reporter mice. We combined dynamic spectral two-photon imaging with multiparametric cytometry and multicolor immunostaining to characterize spatio-temporal distribution, morphology and activity of microglia and blood-derived infiltrating myeloid cells in live mice. Early stages of tumor development were dominated by microglial EYFP(+) cells invading the tumor, followed by massive recruitment of circulating LysM-EGFP(+) cells. Fluorescent invading cells were conventional XCR1(+) and monocyte-derived dendritic cells distributed in subpopulations of different maturation stages, located in different areas relative to the tumor core. The lethal stage of the disease was characterized by the progressive accumulation of EGFP(+)/EYFP(+) monocyte-derived dendritic cells. This local phenotypic regulation of monocyte subtypes marked a transition in the immune response.

An orthotopic glioblastoma mouse model maintaining brain parenchymal physical constraints and suitable for intravital two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date. Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma. Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development. Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease. The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

Six-color intravital two-photon imaging of brain tumors and their dynamic microenvironment.

The majority of intravital studies on brain tumor in living animal so far rely on dual color imaging. We describe here a multiphoton imaging protocol to dynamically characterize the interactions between six cellular components in a living mouse. We applied this methodology to a clinically relevant glioblastoma multiforme (GBM) model designed in reporter mice with targeted cell populations labeled by fluorescent proteins of different colors. This model permitted us to make non-invasive longitudinal and multi-scale observations of cell-to-cell interactions. We provide examples of such 5D (x,y,z,t,color) images acquired on a daily basis from volumes of interest, covering most of the mouse parietal cortex at subcellular resolution. Spectral deconvolution allowed us to accurately separate each cell population as well as some components of the extracellular matrix. The technique represents a powerful tool for investigating how tumor progression is influenced by the interactions of tumor cells with host cells and the extracellular matrix micro-environment. It will be especially valuable for evaluating neuro-oncological drug efficacy and target specificity. The imaging protocol provided here can be easily translated to other mouse models of neuropathologies, and should also be of fundamental interest for investigations in other areas of systems biology.

Dynamic quantitative intravital imaging of glioblastoma progression reveals a lack of correlation between tumor growth and blood vessel density.

The spatiotemporal and longitudinal monitoring of cellular processes occurring in tumors is critical for oncological research. We focused on glioblastoma multiforme (GBM), an untreatable highly vascularized brain tumor whose progression is thought to critically depend on the oxygen and metabolites supplied by blood vessels. We optimized protocols for orthotopic GBM grafting in mice that were able to recapitulate the biophysical constraints normally governing tumor progression and were suitable for intravital multiphoton microscopy. We repeatedly imaged tumor cells and blood vessels during GBM development. We established methods for quantitative correlative analyses of dynamic imaging data over wide fields in order to cover the entire tumor. We searched whether correlations existed between blood vessel density, tumor cell density and proliferation in control tumors. Extensive vascular remodeling and the formation of new vessels accompanied U87 tumor cell growth, but no strong correlation was found between local cell density and the extent of local blood vessel density irrespective of the tumor area or time points. The technique moreover proves useful for comparative analysis of mice subjected either to Bevacizumab anti-angiogenic treatment that targets VEGF or to AMD3100, an antagonist of CXCR4 receptor. Bevacizumab treatment massively reduced tumoral vessel densities but only transiently reduced U87 tumor growth rate. Again, there was no correlation between local blood vessel density and local cell density. Moreover, Bev applied only prior to tumor implantation inhibited tumor growth to the same extent as post-grafting treatment. AMD3100 achieved a potent inhibition of tumor growth without significant reduction in blood vessel density. These results indicate that in the brain, in this model, tumor growth can be sustained without an increase in blood vessel density and suggest that GBM growth is rather governed by stromal properties.

Synergistic effect of cisplatin and synchrotron irradiation on F98 gliomas growing in nude mice.

Among brain tumors, glioblastoma multiforme appears as one of the most aggressive forms of cancer with poor prognosis and no curative treatment available. Recently, a new kind of radio-chemotherapy has been developed using synchrotron irradiation for the photoactivation of molecules with high-Z elements such as cisplatin (PAT-Plat). This protocol showed a cure of 33% of rats bearing the F98 glioma but the efficiency of the treatment was only measured in terms of overall survival. Here, characterization of the effects of the PAT-Plat on tumor volume and tumor blood perfusion are proposed. Changes in these parameters may predict the overall survival. Firstly, changes in tumor growth of the F98 glioma implanted in the hindlimb of nude mice after the PAT-Plat treatment and its different modalities have been characterized. Secondly, the effects of the treatment on tumor blood perfusion have been observed by intravital two-photon microscopy. Cisplatin alone had no detectable effect on the tumor volume. A reduction of tumor growth was measured after a 15 Gy synchrotron irradiation, but the whole therapy (15 Gy irradiation + cisplatin) showed the largest decrease in tumor growth, indicating a synergistic effect of both synchrotron irradiation and cisplatin treatment. A high number of unperfused vessels (52%) were observed in the peritumoral area in comparison with untreated controls. In the PAT-Plat protocol the transient tumor growth reduction may be due to synergistic interactions of tumor-cell-killing effects and reduction of the tumor blood perfusion.

Three-dimensional imaging of small intestine morphology using non-linear optical microscopy and endogenous signals.

Two-photon microscopy (2PM) has become a gold standard for deep-tissue observations in the living animal as well as on thick samples. Using 2PM, the endofluorescence properties of biomolecules have shown an interesting potential for the imaging of tissues without any staining. In this short communication, we report a method to observe the different layers of mouse small intestine explants with subcellular resolution and without any staining or clearing. This method allows rapid observations of samples with little to no preparation thanks to the endofluorescence properties of biomolecules such as NAD(P)H or flavins and second-harmonic generation. Finally, we show different three-dimensional reconstructions of the mouse small intestine anatomy obtained with this approach to show the potential of this method in morphological studies.

Tolerance of arteries to microplanar X-ray beams.

PURPOSE: The purpose is to evaluate effects of a new radiotherapy protocol, microbeam radiation therapy, on the artery wall. In previous studies on animal models, it was shown that capillaries recover well from hectogray doses of X-rays delivered in arrays of narrow (< or = 50 microm) beams with a minimum spacing of 200 microm. Here, short- and long-term effects of comparable microplanar beam configurations on the saphenous artery of the mouse hind leg were analyzed in situ by use of nonlinear optics and compared with histopathologic findings. METHODS AND MATERIALS: The left hind leg of normal mice including the saphenous artery was irradiated by an array of 26 microbeams of synchrotron X-rays (50 microm wide, spaced 400 microm on center) with peak entrance doses of 312 Gy and 2,000 Gy. RESULTS: The artery remained patent, but narrow arterial smooth muscle cell layer segments that were in the microplanar beam paths became atrophic and fibrotic in a dose-dependent pattern. The wide tunica media segments between those paths hypertrophied, as observed in situ by two-photon microscopy and histopathologically. CONCLUSIONS: Clinical risks of long-delayed disruption or occlusion of nontargeted arteries from microbeam radiation therapy will prove less than corresponding risks from broad-beam radiosurgery, especially if peak doses are kept below 3 hectograys.

Short-term effects of synchrotron irradiation on vasculature and tissue in healthy mouse brain.

The purpose of this study is to measure the effects of a tomographic synchrotron irradiation on healthy mouse brain. The cerebral cortexes of healthy nude mice were irradiated with a monochromatic synchrotron beam of 79 keV at a dose of 15 Gy in accordance with a protocol of photoactivation of cisplatin previously tested in our laboratory. Forty-eight hours, one week and one month after irradiation, the blood brain barrier (BBB) permeability was measured in the irradiated area with intravital multiphoton microscopy using fluorescent dyes with molecular weights of 4 and 70 kDa. Vascular parameters and gliosis were also assessed using quantitative immunohistochemistry. No extravasation of the fluorescent dyes was observed in the irradiated area at any measurement time (48 h, 1 week, 1 month). It appears that the BBB remains impermeable to molecules with a molecular weight of 4 kDa and above. The vascular density and vascular surface were unaffected by irradiation and no gliosis was induced. These findings suggest that a 15 Gy/79 keV synchrotron irradiation does not induce important damage on brain vasculature and tissue on the short term following irradiation.

Subtraction method for intravital two-photon microscopy: intraparenchymal imaging and quantification of extravasation in mouse brain cortex.

Brain pathologies, including stroke and tumors, are associated with a variable degree of breakdown of the blood-brain barrier (BBB), which can usefully be studied in animal models. We describe a new optical technique for quantifying extravasation in the cortex of the living mouse and for imaging intraparenchymal tissue. Leakiness of the BBB was induced by microbeam x-irradiation. Two fluorescent dyes were simultaneously infused intravenously, one of high molecular weight (fluorescein-labeled dextran, 70 kDa, green fluorescence) and one of low molecular weight (sulforhodamine B, 559 Da, red fluorescence). A two-photon microscope, directed through a cranial window, obtained separate images of the two dyes in the cortex. The gains of the two channels were adjusted so that the signals coming from within the vessels were equal. Subtraction of the image of the fluorescein-dextran from that of the sulforhodamine B gave images in which the vasculature was invisible and the sulforhodamine B in the parenchyma could be imaged with high resolution. Algorithms are presented for rapidly quantifying the extravasation without the need for shape recognition and for calculating the permeability of the BBB. Sulforhodamine B labeled certain intraparenchymal cells; these cells, and other details, were best observed using the subtraction method.

In vivo staining of neocortical astrocytes via the cerebral microcirculation using sulforhodamine B.

Staining and imaging glial cells in vivo while observing the microvasculature could help understand brain physiology, namely neuronal-glial-vascular communication. Two-photon excitation microscopy provides a means to monitor these interactions at the cellular level in living animals, but the cells of interest must be fluorescent. Injecting dyes intravenously is a rapid and quasi noninvasive method to stain cells in the brain. It necessitates that the dye is soluble in the blood plasma and crosses the blood brain barrier (BBB). We demonstrate here, using two-photon imaging, that sulforhodamine B (SRB) crosses the BBB and stains in vivo, specifically mouse astrocytes. This is confirmed by experiments on primary neurons and astrocytes cultures showing the preferential SRB staining of the latter. SRB is rapidly eliminated from the blood, which allows repeated injections in longitudinal studies.

Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation.

Because of the spreading of nonlinear microscopies in biology, there is a strong demand for specifically engineered probes in these applications. Herein, we report on the imaging properties in living cells and nude mice brains of recently developed water soluble blue fluorophores that show efficient diffusion through cell membranes and blood-brain barriers. They are characterized by two-photon absorption cross-sections of 100-150 Goeppert-Mayer range in the near IR and fluorescence efficiencies of up to 72% in water. They were found to stain homogeneously the cytoplasm of cultured living cells within minutes. Moreover, their diffusion times and fluorescence characteristics in the cytoplasm suggest a hydrophobic association with intracellular membranes. Their intracellular fluorescent decays were found to be almost mono-exponential, a very favorable feature for fluorescence lifetime imaging. Two photon images of living cells were obtained with a good signal to noise ratio using laser powers in the sub-milliwatt range. This allows continuous imaging without significant photobleaching for tens of minutes. In addition, these fluorophores allowed in vivo three-dimensional two-photon imaging of mice cortex vasculatures and extra vasculature structures, with no sign of toxicity.

In vivo imaging of elastic fibers using sulforhodamine B.

Until now, the imaging of elastic fibers was restricted to tissue sections using the endofluorescence properties of elastin or histological dyes. Methods to study their morphology in vivo and in situ have been lacking. We present and characterize a new application of a fluorescent dye for two-photon microscopy: sulforhodamine B (SRB), which is shown to specifically stain elastic fibers in vivo. SRB staining of elastic fibers is demonstrated to be better than using elastin endofluorescence for two-photon microscopy. Our imaging method of elastic fibers is shown to be suitable for simultaneous imaging with both other fluorescent intravital dyes and second-harmonic generation (SHG). We illustrate these findings with intravital imaging of elastic and collagen fibers in muscle epimysium and endomysium and in blood vessel walls. We expect SRB staining to become a key method to study elastic fibers in vivo.