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The extra cellular matrix plays a major role in the biomechanical properties of tissues that impact cell behavior and fate. It is therefore crucial to mimic these complex cell-matrix interactions in 3D cell cultures. Here, two-photon polymerization is applied to produce gelatin methacryloyl (GelMA) - collagen matrixes that further enable local pO2matrix measurement, when ruthenium complexes are used as photo-activators. The fluorescence intensity of these complexes has a direct and inverse relationship with the local pO2matrix. The 3D structures reached their maximum size in cell culture conditions after 3H with a swelling factor of ~1.5. Their shape and the ruthenium fluorescence intensity of the alveoli walls stayed constant for at least 2 weeks in the absence of cells. They were used in time series to monitor the local pO2matrix adjacent to cancer cells during their division, migration and the formation of a tumor tissue mass. At the presence of these cell activities that consume O2, a significant ~3-fold increase of the ruthenium fluorescence intensity in the alveoli walls was observed. This study demonstrates that online monitoring of the local pO2matrix is possible. The ruthenium complexes provide the bio-optical sensors that are useful for further analysis of cancer and healthy cell energy metabolism in a 3D matrix that better mimics in vivo conditions and migration paths. Unraveling the cancer cell metabolic adaptations in a changing micro-environment will help the development of new therapeutic opportunities. STATEMENT OF SIGNIFICANCE: In 3D cell cultures, monitoring pericellular pO2 is as critical as controlling pH. This facility is currently missing. Here, we take advantage of the direct and inverse relationship between pO2 and the fluorescence intensity of ruthenium complexes to generate stable gelatin-collagen matrixes able to continuously monitoring the pO2 at the pericellular level. The ruthenium complexes, which are photo-activators in the two-photon polymerization of these matrixes, became covalently bind to the collagen fibers. Indeed, local O2 consumption by cancer cells during migration, mitosis and tumor mass formation caused a 3-fold increase of the ruthenium fluorescence. In the future, incorporating ruthenium complexes with other bio-optical sensors will create new drug screening platforms that monitor cell culture parameters at the pericellular level.

Multi-stimuli responsive nanogels based on biocompatible hydrophilic polymers have emerged as promising drug delivery systems to improve anticancer therapy with hydrophobic drugs, through increase of circulating-time in the bloodstream, tumor-targeting and reduction of systemic toxicity. This paper reports on the synthesis, characterization and biological perspectives of light- and thermoresponsive hyaluronic acid (HA)-based nanogels containing coumarin as the photocleavable group. Newly synthesized nanogels exhibited interesting features: formation by a temperature-triggered self-assembly process, successful incorporation of poorly water-soluble molecules, light-responsiveness as demonstrated by a significant shift in the critical aggregation temperature after light irradiation, efficient internalization by cancer cells overexpressing the CD44 receptor of HA, ability to circulate for a prolonged period of time in the bloodstream after intravenous injection in mice and considerable detection in tumor tissues. Our findings indicate that coumarin-containing HA-based nanogels may be promising delivery systems for anticancer chemotherapy.

International audience; An optical-resolution photoacoustic microscope with modulated CW laser diodes allowing multi-channel imaging is presented that can be used for both imaging biological tissues and for targeted photo-dynamic therapy (PDT) varying the optical power and exposure time. The effects of this therapy are immediately monitored in order to optimize the time of irradiation. After the description of the experimental setup, in vitro and in vivo applications are presented on a synthetic sample and on the mouse ear using hemoglobin as endogenous and methylene blue as exogenous dye for imaging and PDT, respectively.

A hyaluronic acid (HA)-based extracellular matrix (ECM) platform with independently tunable stiffness and density of cell-adhesive peptide (RGD, arginine-glycine-aspartic acid) that mimics key biochemical and mechanical features of brain matrix has been designed. We demonstrated here its utility in elucidating ECM regulation of neural progenitor cell behavior and neurite outgrowth. The analysis of neurite outgrowth in 3-D by two-photon microscopy showed several important results in the development of these hydrogels. First, the ability of neurites to extend deeply into these soft HA-based matrices even in the absence of cell-adhesive ligand further confirms the potential of HA hydrogels for central nervous system (CNS) regeneration. Second, the behavior of hippocampal neural progenitor cells differed markedly between the hydrogels with a storage modulus of 400 Pa and those with a modulus of 800 Pa. We observed an increased outgrowth and density of neurites in the softest hydrogels (G' = 400 Pa). Interestingly, cells seeded on the surface of the hydrogels functionalized with the RGD ligand experienced an optimum in neurite outgrowth as a function of ligand density. Surprinsingly, neurites preferentially progressed inside the gels in a vertical direction, suggesting that outgrowth is directed by the hydrogel structure. This work may provide design principles for the development of hydrogels to facilitate neuronal regeneration in the adult brain.

Monitoring glioma cell infiltration in the brain is critical for diagnosis and therapy. Using a new glioma Glio6 mouse model derived from human stem cells we show how diffusion tensor imaging (DTI) may predict glioma cell migration/invasion. In vivo multiparametric MRI was performed at one, two and three months of Glio6 glioma growth (Glio6 (n = 6), sham (n = 3)). This longitudinal study reveals the existence of a time window to study glioma cell/migration/invasion selectively. Indeed, at two months only Glio6 cell invasion was detected, while tumor mass formation, edema, blood-brain barrier leakage and tumor angiogenesis were detected later, at three months. To robustly confirm the potential of DTI for detecting glioma cell migration/invasion, a microscopic 3D-DTI (80 μm isotropic spatial resolution) technique was developed and applied to fixed mouse brains (Glio6 (n = 6), sham (n = 3)). DTI changes were predominant in the corpus callosum (CC), a known path of cell migration. Fractional anisotropy (FA) and perpendicular diffusivity (D⊥ ) changes derived from ex vivo microscopic 3D-DTI were significant at two months of tumor growth. In the caudate putamen an FA increase of +38% (p < 0.001) was observed, while in the CC a - 28% decrease in FA (p < 0.005) and a + 95% increase in D⊥ (p < 0.005) were observed. In the CC, DTI changes and fluorescent Glio6 cell density obtained by two-photon microscopy in the same brains were correlated (p < 0.001, r = 0.69), validating FA and D⊥ as early quantitative biomarkers to detect glioma cell migration/invasion. The origin of DTI changes was assessed by electron microscopy of the same tract, showing axon bundle disorganization. During the first two months, Glio6 cells display a migratory phenotype without being associated with the constitution of a brain tumor mass. This offers a unique opportunity to apply microscopic 3D-DTI and to validate DTI parameters FA and D⊥ as biomarkers for glioma cell invasion.

International audience; For correct analysis of anti-angiogenic therapies, we need new fluorescent probes that do not diffuse across the leaky tumour vascular endothelium before any treatment. The latter makes estimations of the functional blood volume and changes in vascular permeability uncertain after treatment. Therefore, we present here a new non diffusible fluorescent probe for two photon microscopy, which is composed of a hydrophobic push–pull dye (1) encapsulated in the apolar core of Pluronic F127–silica nanoparticles (1@F127–SiO2, d = 22 nm). In apolar media, (1) has intense emission in the red (Φf = 39% at 650 nm) and two-photon absorption properties in the NIR. The NP probe was successfully tested in vivo on mice bearing tumours without diffusion across the tumour vascular endothelium. The two photon excitation wavelength at 1000 nm is optimum for deep tissue excitation.

Delivery systems for macrophages are particularly attractive since these phagocytic cells play a important role in immunological and inflammatory responses, also acting as host cells for microorganisms that are involved in deadly infectious diseases, such as leishmaniasis. Hyaluronic acid (HA) is specifically recognized by macrophages that are known to express HA receptors. Therefore, in this study, we focused on HA-based nanogels as drug carriers for these cells. The drug delivery was validated in an in vivo study on mice using intravital two-photon laser scanning microscopy. HA derivatives were modified with a biocompatible oligo(ethylene glycol)-based thermoresponsive polymer to form nanogels. These HA conjugates were readily prepared by varying the molar mass of initial HA and the degree of substitution via radical-mediated thiol-ene chemistry in aqueous solution. The derivatives were shown to self-assemble into spherical gel particles with diameters ranging from 150 to 214 nm above 37 °C. A poorly water-soluble two-photon dye was successfully loaded into the nanogels during this self-assembly process. In vitro cellular uptake tests using a RAW 264.7 murine macrophage cell line showed successful intracellular delivery of the hydrophobic dye. After intravenous injection in mice, the nanogels circulated freely in the blood but were rapidly phagocytized within 13 min by circulating macrophages and stored in the liver and spleen, as observed by two-photon microscopy. Benefit can be thus expected in using such a delivery system for the liver and spleen macrophage-associated diseases.

International audience; The synthesis of keto-heptamethine derivatives has been expanded to various new symmetrical and asymmetrical structures, including an unprecedented di-anionic keto-polymethine. The spectroscopic behavior of these new dyes has been systematically and thoroughly investigated, revealing that the formation of hydrogen bond interactions with protic solvents is responsible for a dramatic enhancement of the fluorescence quantum yield in the far-red spectral region. The existence of these strong hydrogen-bond interactions was further confirmed by molecular dynamics simulations. These bis-dipolar polymethines exhibit large two-photon absorption (TPA) cross-sections (σ2 in GM) in the near-infrared, making them ideal candidates for NIR-to-NIR two-photon microscopy imaging applications. We demonstrate that the molecular engineering of the hydrophilic/hydrophobic balance enables targeting of different cellular components, such as cytoplasm or cell membranes. Addition of appropriate substituents provides the molecule with high-water-solubility, affording efficient two-photon probes for angiography.

Synchrotron photoactivation therapy of cisplatin relies on a synergistic effect of synchrotron X-rays and platinum and leads to tumor-cell-killing effects and reduction of the tumor blood perfusion., 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.

The keto-heptamethine family has been expanded to various symmetrical and asymmetrical structures., The synthesis of keto-heptamethine derivatives has been expanded to various new symmetrical and asymmetrical structures, including an unprecedented di-anionic keto-polymethine. The spectroscopic behavior of these new dyes has been systematically and thoroughly investigated, revealing that the formation of hydrogen bond interactions with protic solvents is responsible for a dramatic enhancement of the fluorescence quantum yield in the far-red spectral region. The existence of these strong hydrogen-bond interactions was further confirmed by molecular dynamics simulations. These bis-dipolar polymethines exhibit large two-photon absorption (TPA) cross-sections (σ 2 in GM) in the near-infrared, making them ideal candidates for NIR-to-NIR two-photon microscopy imaging applications. We demonstrate that the molecular engineering of the hydrophilic/hydrophobic balance enables targeting of different cellular components, such as cytoplasm or cell membranes. Addition of appropriate substituents provides the molecule with high-water-solubility, affording efficient two-photon probes for angiography.

The storage and catabolism of Ultrasmall SuperParamagnetic Iron Oxide (USPIO) nanoparticles were analyzed through a multiscale approach combining Two Photon Laser Scanning Microscopy (TPLSM) and High-Resolution Transmission Electron Microscopy (HRTEM) at different times after intravenous injection in an atherosclerotic ApoE(-/-) mouse model. The atherosclerotic plaque features and the USPIO heterogeneous biodistribution were revealed down from organ's scale to subcellular level. The biotransformation of the nanoparticle iron oxide (maghemite) core into ferritin, the non-toxic form of iron storage, was demonstrated for the first time ex vivo in atherosclerotic plaques as well as in spleen, the iron storage organ. These results rely on an innovative spatial and structural investigation of USPIO's catabolism in cellular phagolysosomes. This study showed that these nanoparticles were stored as non-toxic iron compounds: maghemite oxide or ferritin, which is promising for MRI detection of atherosclerotic plaques in clinics using these USPIOs. From the Clinical Editor: Advance in nanotechnology has brought new contrast agents for clinical imaging. In this article, the authors investigated the use and biotransformation of Ultrasmall Super-paramagnetic Iron Oxide (USPIO) nanoparticles for analysis of atherosclerotic plagues in Two Photon Laser Scanning Microscopy (TPLSM) and High-Resolution Transmission Electron Microscopy (HRTEM). The biophysical data generated from this study could enable the possible use of these nanoparticles for the benefits of clinical patients.

International audience; We previously reported the synthesis of gadolinium-based nanoparticles (NPs) denoted AGuIX (activation and guiding of irradiation by X-ray) NPs and demonstrated their potential as an MRI contrast agent and their efficacy as radiosensitizing particles during X-ray cancer treatment. Here we focus on the elimination kinetics of AGuIX NPs from the subcellular to whole-organ scale using original and complementary methods such as laser-induced breakdown spectroscopy (LIBS), intravital two-photon microscopy, inductively coupled plasma optical emission spectrometry (ICP-OES), transmission electron microscopy (TEM), and electrospray ionization mass spectrometry (ESI-MS). This combination of techniques allows the exact mechanism of AGuIX NPs elimination to be elucidated, including their retention in proximal tubules and their excretion as degraded or native NPs. Finally, we demonstrated that systemic AGuIX NP administration induced moderate and transient effects on renal function. These results provide useful and promising preclinical information concerning the safety of theranostic AGuIX NPs.

The uptake of Gadomer-17, as probed by fast dynamic T(1) measurements, was used to assess the vascular permeability surface-area product per leakage volume of tissue (k(Tofts)) of human glioma xenografts implanted in mice. With this approach we could discriminate between two types of glioma xenograft lines with a known difference in the perfused vascular architecture and degree of hypoxia. The T(1) data were analyzed according to the Tofts-Kermode compartment model. The fast-growing E102 tumor demonstrated a homogeneous distribution of the vascular permeability surface area across the tumor (mean k(Tofts) value = 0.18 +/- 0.05 min(-1)). The slowly growing E106 tumor showed a more heterogeneous pattern. Three perfused tumor areas with differences in vascular permeability surface area could be distinguished: a well-perfused periphery with high k(Tofts) values (0.24 +/- 0.04 min(-1)), perfused capillaries inside the tumor with low k(Tofts) values (0.108 +/- 0.026 min(-1)), and perfused capillaries adjacent to necrotic regions with high k(Tofts) values (0.29 +/- 0.10 min(-1)). On a different series of tumors, the hypoxic fractions were measured, and these were significantly higher in E106 tumors (0.14 +/- 0.05) compared to tumors of the E102 line (0.03 +/- 0.02).

The combination of localized 2D 1H MR correlation spectroscopy and Hadamard encoding allows the simultaneous acquisition of multiple volumes of interest without an increase in the experimental duration, compared to single-voxel acquisition. In the present study, 2D correlation spectra were acquired simultaneously within 20 to 40 min in two voxels located in each hemisphere of the rat brain. An intervoxel distance of 20% of the voxel size was sufficient to limit spatial contamination. The following cerebral metabolites gave detectable crosspeaks: N-acetylaspartate, the glutamate/glutamine pool, aspartate, phosphoethanolamine, glucose, glutathione, taurine, myo-inositols, lactate, threonine, ¿-aminobutyric acid, and alanine. Most of the metabolites were measured without contamination of other resonances. two-dimensional correlation 1H MRS

Pathophysiologic parameters of the functional neovasculature and the blood-brain barrier of 9L-glioma in rat brain were measured noninvasively by dynamic 1H magnetic resonance imaging studies of gadolinium (Gd)-DTPA uptake. Changes of apparent [Gd-DTPA] uptake in time (CT[t]) were analyzed in a slice through the center of 10 9L-gliomas using fast T1 measurements. The distribution of the contrast agent was spatially correlated with the distribution of perfused microvessels as determined by immunohistochemical analysis. This method permits a distinction between perfused and nonperfused microvessels with a disrupted blood-brain barrier. In transverse slices of the whole tumor, a spatial correlation was observed between CT maps and the two-dimensional distribution of perfused microvessels. In the next step, Gd-DTPA uptake rates were spatially related to the perfused microvessel density (Np) or vascular surface area (Sp). In tumor voxels with perfused microvessels, a linear correlation was found between Gd-DTPA uptake rate constants (k values) and Np or Sp. No correlation was observed between k values and the total microvessel density. These are the first data that show a relation between Gd-DTPA uptake rates and parameters of the functional neovasculature in 9L-glioma growing in rat brain. Now that Gd-DTPA uptake studies can be related to parameters of the functional neovasculature, they may be used more efficiently as a prognostic tool before or during therapy.

The aim of this study was to evaluate the effect of carbogen breathing on the physiological profile of human glioma xenografts. Near infrared spectroscopy was used to investigate changes in oxy- and deoxyhemoglobin concentrations in tumor blood. Oxygen tension changes in tumor tissue were evaluated by (19)F-MR relaxometry, using perfluoro-15-crown-5-ether, and modifications of tumor blood perfusion (TBP) were analyzed by fast dynamic (1)H-MR imaging of Gd-DTPA uptake. Finally, changes of the bioenergetic status and pH of tumor cells were analyzed by (31)P-MRS. After 5 to 8 min of carbogen breathing, the average oxygen tension increase in tumor tissue was 4.6 +/- 1.3 mm Hg, which is in agreement with an increase of the oxyhemoglobin concentration in tumor blood (Delta[O(2)Hb] = 9. 2 +/- 3 microM). However, simultaneously the TBP was reduced, the bioenergetic status was diminished, and pH was decreased. As 100% O(2) breathing alone did not result in a detectable increase of oxyhemoglobin in tumor blood, the increase of the tumor oxygenation by carbogen appears to be mediated by its CO(2) content. This component may cause a nutrient-limited decrease of oxidative energy metabolism, indirectly via a steal-effect and/or by inhibition of the glycolytic rate resulting from tissue acidification. Magn Reson Med 42:490-499, 1999.

To assess angiogenesis noninvasively in a C6 rat brain tumor model, the rapid-steady-state- T1 (RSST1) magnetic resonance imaging (MRI) method was used for microvascular blood volume fraction (BVf) quantification with a novel contrast agent gadolinium per (3,6 anhydro) α-cyclodextrin (Gd-ACX). In brain tissue contralateral to the tumor, equal BVfs were obtained with Gd-ACX and the clinically approved gadoterate meglumine (Gd-DOTA). Contrary to Gd-DOTA, which leaks out of the tumor vasculature, Gd-ACX was shown to remain vascular in the tumor tissue allowing quantification of the tumor BVf. We sought to confirm the obtained tumor BVf using an independent method: instead of using a ‘standard’ two-dimensional histologic method, we study here how vascular morphometry combined with a stereological technique can be used for three-dimensional assessment of the vascular volume fraction ( VV). The VV is calculated from the vascular diameter and length density. First, the technique is evaluated on simulated data and the healthy rat brain vasculature and is then applied to the same C6 tumor vasculature previously quantified by RSST1-MRI with Gd-ACX. The mean perfused VV and the BVf obtained by MRI in tumor regions are practically equal and the technique confirms the spatial heterogeneity revealed by MRI.

International audience; Fluorescent staining of astrocytes without damaging or interfering with normal brain functions is essential for intravital microscopy studies. Current methods involved either transgenic mice or local intracerebral injection of sulforhodamine 101. Transgenic rat models rarely exist, and in mice, a backcross with GFAP transgenic mice may be difficult. Local injections of fluorescent dyes are invasive. Here, we propose a non-invasive, specific and ubiquitous method to stain astrocytes in vivo. This method is based on iv injection of sulforhodamine dyes and is applicable on rats and mice from postnatal age to adulthood. The astrocytes staining obtained after iv injection was maintained for nearly half a day and showed no adverse reaction on astrocytic calcium signals or electroencephalographic recordings in vivo. The high contrast of the staining facilitates the image processing and allows to quantify 3D morphological parameters of the astrocytes and to characterize their network. Our method may become a reference for in vivo staining of the whole astrocytes population in animal models of neurological disorders.

International audience; Our purpose is to test if Pluronic® fluorescent nanomicelles can be used for in vivo two-photon imaging of both the normal and the tumor vasculature. The nanomicelles were obtained after encapsulating a hydrophobic two-photon dye: di-stryl benzene derivative, in Pluronic block copolymers. Their performance with respect to imaging depth, blood plasma staining, and diffusion across the tumor vascular endothelium is compared to a classic blood pool dye Rhodamin B dextran (70 kDa) using two-photon microscopy. Pluronic nanomicelles show, like Rhodamin B dextran, a homogeneous blood plasma staining for at least 1 h after intravenous injection. Their two-photon imaging depth is similar in normal mouse brain, using 10 times less injected mass. In contrast with Rhodamin B dextran, no extravasation is observed in leaky tumor vessels due to their large size: 20-100 nm. In conclusion, Pluronic nanomicelles can be used as a blood pool dye, even in leaky tumor vessels. The use of Pluronic block copolymers is a valuable approach for encapsulating two-photon fluorescent dyes that are hydrophobic and not suitable for intravenous injection.

International audience; 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.

International audience; A novel water-soluble Ru(ii) complex has been prepared, which represents a promising new class of selective two-photon sensitizers for use in photodynamic therapy within a confined space.

International audience; Cerebral edema is one of the main acute complications arising after irradiation of brain tumors. Microbeam radiation therapy (MRT), an innovative experimental radiotherapy technique using spatially fractionated synchrotron x-rays, has been shown to spare radiosensitive tissues such as mammal brains. The aim of this study was to determine if cerebral edema occurs after MRT using diffusion-weighted MRI and microgravimetry. Prone Swiss nude mice's heads were positioned horizontally in the synchrotron x-ray beam and the upper part of the left hemisphere was irradiated in the antero-posterior direction by an array of 18 planar microbeams (25 mm wide, on-center spacing 211 mm, height 4 mm, entrance dose 312 Gy or 1000 Gy). An apparent diffusion coefficient (ADC) was measured at 7 T 1, 7, 14, 21 and 28 days after irradiation. Eventually, the cerebral water content (CWC) was determined by microgravimetry. The ADC and CWC in the irradiated (312 Gy or 1000 Gy) and in the contralateral non-irradiated hemispheres were not significantly different at all measurement times, with two exceptions: (1) a 9% ADC decrease (p < 0.05) was observed in the irradiated cortex 1 day after exposure to 312 Gy, (2) a 0.7% increase (p < 0.05) in the CWC was measured in the irradiated hemispheres 1 day after exposure to 1000 Gy. The results demonstrate the presence of a minor and transient cellular edema (ADC decrease) at 1 day after a 312 Gy exposure, without a significant CWC increase. One day after a 1000 Gy exposure, the CWC increased, while the ADC remained unchanged and may reflect the simultaneous presence of cellular and vasogenic edema. Both types of edema disappear within a week after microbeam exposure which may confirm the normal tissue sparing effect of MRT.

Since the early nineties, multiphoton microscopy has become a powerful tool to investigate morphological and physiological parameters in vivo or on thick ex vivo sections. To stain structures of interest many dyes have been developed and two-photon properties (cross section, excitation and emission spectra) of existing ones have been characterized. Recently, our team has shown a new property of sulforhodamine B (SRB). This dye has the ability to bind specifically elastic fibers. The observation of elastin using its endofluorescence properties was already widely described but required long exposition delays up to 10s and the imaging depth was limited to approximately 50 μm. With a multiphoton microscope and SRB, it is possible to observe elastic fibers directly in the living animal or on thick tissue sections with a micrometric spatial resolution in less than one second per image with an imaging depth of ~ 200 μm. Moreover, with an appropriate set of filters, we can acquire simultaneously the SRB and the second harmonic generation (SHG) signals of collagen fibers. Here, we report various applications of this new staining method on different arterial rings. The layers of the arterial wall, as well as, the elastic lamellae are observed and are numbered. With the addition of a nuclear stain such as the Hoechst 33342, a more accurate morphological study of the arterial walls can be accomplished. Finally, an intravital observation of the saphenous artery morphology is presented.

International audience; 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.

International audience; 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.

International audience; 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. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc.

International audience; PURPOSE: The purpose of this study was to assess the early effects of microbeam irradiation on the vascular permeability and volume in the parietal cortex of normal nude mice using two-photon microscopy and immunohistochemistry. METHODS AND MATERIALS: The upper part of the left hemisphere of 55 mice was irradiated anteroposteriorly using 18 vertically oriented beams (width 25 microm, interdistance 211 microm; peak entrance doses: 312 or 1000 Gy). At different times after microbeam exposure, the microvasculature in the cortex was analyzed using intravital two-photon microscopy after intravascular injection of fluorescein isothiocyanate (FITC)-dextrans and sulforhodamine B (SRB). Changes of the vascular volume were observed at the FITC wavelength over a maximum depth of 650 mum from the dura. The vascular permeability was detected as extravasations of SRB. RESULTS: For all times (12 h to 1 month) after microbeam irradiation and for both doses, the FITC-dextran remained in the vessels. No significant change in vascular volume was observed between 12 h and 3 months after irradiation. Diffusion of SRB was observed in microbeam irradiated regions from 12 h until 12 days only after a 1000 Gy exposure. CONCLUSION: No radiation damage to the microvasculature was detected in normal brain tissue after a 312 Gy microbeam irradiation. This dose would be more appropriate than 1000 Gy for the treatment of brain tumors using crossfired microbeams.

We report on the design, synthesis and optical properties of new two-photon absorbing multi-branched chromophores, based on a pyridine core. Measurements of TPA using the two-photon fluorescence method in the fs regime indicate that these chromophores exhibit two-photon absorptivity. The results indicate that a change of the substitution of the electron-acceptor core influences significantly the TPA cross sections. Moreover, upon protonation, the TPA properties are greatly enhanced in the 700-900 nm range. Thus, these molecules can serve as pH-sensitive TPA dyes. Preliminary results related to biological applications will be presented.

Knowledge of the volume of blood per unit volume of brain tissue is important for understanding brain function in health and disease. We describe a novel method using two-photon laser scanning microscopy to obtain the local blood volume in the cortex of the anesthetized mouse. We infused fluorescent dyes in the circulating blood and imaged the blood vessels, including the capillaries, to a depth of 400 microns below the dura at the brain surface. Blood volume was calculated by normalizing the total fluorescence measured at each depth. This method, which dispenses with form recognition, is rapid and only weakly sensitive to background noise; it could be extended to measure the leakiness of the blood vessels.

Purpose To investigate the responses of two experimental rat tumors to single and fractionated X-ray doses whether or not combined with Motexafin gadolinium (MGd), and the distribution of MGd in R3327-MATLyLu (MLL) tumors using MRI. Methods L44 lung tumor in BN rats and MLL prostate tumor in Copenhagen rats were grown subcutaneously. MGd at concentrations of 8.7 to 25.1 μmol/kg was administered 2 h before or just before treatments with single and fractionated X-ray doses. Tumor volume growth delay was the endpoint used. The two-dimensional distribution of the MGd concentration in time was analyzed simultaneously in slices through the center of MLL tumors using MRI. Directly after the MRI experiments, tumor sections were stained for cytoplasm, nuclei, and microvessel endothelium. Results MGd at different concentrations administered a few minutes or 2 h before X-ray doses produced no radiation enhancement in the two tumor models. The MGd concentration as determined by MRI was maximal 5 min after injection and decreased slowly thereafter. In a representative section at the center of the MLL tumor, the microvessel density is nearly homogeneous and correlates with a nearly homogeneous MGd distribution. Hardly any MGd is taken up in underlying muscle tissue. Conclusions No radiosensitization was observed for the different irradiation regimens. The distribution of MGd is nearly homogeneous in the MLL tumor and hardly any MGd is taken up in underlying muscles. Our negative results on radiosensitivity in our two tumor models raise questions about the efficacy of MGd as a general radiosensitizing agent.

Dynamic Magnetic Resonance Imaging (T1-mapping) using the Gadolinium complex Gadomer-17, was applied to characterize the vascular permeability of human glioma xenografts implanted in nude mice. We aimed at measuring permeability differences in two types of glioma xenograft lines with a known difference in perfusion status. The T1-data could be analyzed according to the Tofts-Kermode compartmental model for modeling tracer kinetics to vascular permeability. This vascular permeability was mapped as the permeability surface area product per unit of leakage volume (k). The two tumor types displayed different k-maps. For the fast growing E102 tumor, we observed a homogeneous distribution of the vascular permeability across the tumor with a mean k-value of (0.207 plus or minus 0.027) min-1. However, for the slowly growing E106 tumor, we could distinguish four different regions with different permeability characteristics: a well-perfused rim [k equals (0.30 plus or minus 0.09) min-1], regions at the inner side of the tumor with lower permeability [k equals (0.130 plus or minus 0.019) min-1], regions at the inner side of the tumor around necrotic regions demonstrating locally increased permeability [k equals (0.33 plus or minus 0.11) min-1], and necrotic regions.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

In recent years, two-photon laser scanning microscopy (TPLSM), especially intravital, has emerged as an essential toolin biomedical research and has become a routine technique to track biological processes down to the cellular level in living animals. In this context, fuorophores presenting electronic transitions of the “intramolecular charge transfer” (ICT) type are considered as a particularly appealing class of molecules. The peculiar linear (environmental sensitivity, olvatochromism) and nonlinear (two-photon absorption, second harmonic generation) spectroscopic properties ssociated with ICT transitions have been taken advantage of for the development of functional materials, including H orpolarity probesand biosensors with one or two-photon induced fuorescence. These fuorophores are inherently lipophilic, because of their extended conjugated carbon backbone. Thus, major efforts have been put into the improvement of their solubility in biological media and their biocompatibility. In this context, it has been shown by Müllen et al. that controlled growth of a star-shaped polymer shell around a central fuorophore is a particularly effective strategy. Moreover, we have recently provided proof of the principle that such structures are valuable candidates for the ectorization of twophoton fuorophores into cells. We showed that the resulting structures display good-to-very-good conservation of their twophoton brightness in aqueous media, with emission wavelengths up to 570 nm. This result was however not fully optimal for in vivo two-photon imaging applications, since it is desirable that the probe features maximal Fuorescence in the biological window, i.e. at a wavelength between 700–1000 nm.; International audience; A water soluble Lemke chromophore derivative shows an unusual polarity dependence of its emission efficiency, leading to strong red-NIR fluorescence in water when fitted with appropriate water-solubilizing polymer chains. In this edge article, the synthesis of the chromophore is described. The dependence of its fluorescence on solvent polarity is investigated experimentally and rationalized on the basis of ab initio calculations. Finally, we demonstrate that this chromophore is a valuable candidate for in vivo two-photon imaging of cerebral vasculature, with two-photon absorption and emission in the biological transparency window.

International audience; 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.

An improved two-photon microscopy technique for imaging and quantifying blood brain barrier (BBB) leakage in the cortex of mice is presented. Local BBB leakage was induced using synchrotron X-ray micro-beams. Two fluorescent dyes were simultaneously infused intravenously, one of high molecular weight (FITC-dextran) and one of low molecular weight (sulforhodamine B, SRB). By separation of the two fluorescence spectra, and matched subtraction, images were obtained in which the vasculature was invisible and the low molecular weight dye in the parenchyma could be imaged with high resolution. An algorithm is presented for quantifying the extravasation without the need of vessel recognition.