Stimulation Emission Depleted Photoacoustic.

We demonstrate that the Stimulated Emission Depletion (STED) concept, which is usually invoked for fluorescence, can be extended to photoacoustic effects. When two-nanosecond pulses of exciting and stimulating light are synchronized, 80% of the acoustic signal generated through excited state absorption (ESA) can be quenched. Regarding the cross-sections for stimulated emission and ESA, a model gives a good order of magnitude in the depletion efficiency. The transient molecular orientation, usually measured via the fluorescence anisotropy, can be accessed in photoacoustic when STED is implemented.

Optical sectioning in optical resolution photo acoustic microscopy.

We report a novel optical resolution photoacoustic microscopy concept to obtain an axial resolution only by optical methods. The photoacoustic signal is generated through a non-radiative relaxation from a level that is populated by excited state absorption. This two-step excitation process of a single laser enables to achieve an optical sectioning without any acoustic selectivity, whereby a full optical resolution photoacoustic microscopy is obtained. We bring a proof of this concept using Rhodamine and Zinc Tetraphenylporphyrin dyes known for their efficient excited state absorption process.

Two-photon intravital imaging of lungs during anthrax infection reveals long-lasting macrophage-dendritic cell contacts.

The dynamics of the lung immune system at the microscopic level are largely unknown because of inefficient methods of restraining chest motion during image acquisition. In this study, we developed an improved intravital method for two-photon lung imaging uniquely based on a posteriori parenchymal tissue motion correction. We took advantage of the alveolar collagen pattern given by the second harmonic generation signal as a reference for frame registration. We describe here for the first time a detailed dynamic account of two major lung immune cell populations, alveolar macrophages and CD11b-positive dendritic cells, during homeostasis and infection by spores of Bacillus anthracis, the agent of anthrax. We show that after alveolar macrophages capture spores, CD11b-positive dendritic cells come in prolonged contact with infected macrophages. Dendritic cells are known to carry spores to the draining lymph nodes and elicit the immune response in pulmonary anthrax. The intimate and long-lasting contacts between these two lines of defense may therefore coordinate immune responses in the lung through an immunological synapse-like process.

STED-SPIM made simple.

We report the development of a stimulated emission depletion (STED) selective plane illumination (SPIM) microscope based on a single diode-pumped solid state (DPSS) laser that simultaneously delivers nanosecond-pulses at two wavelengths. The two wavelengths, 355 nm and 532 nm, are generated by harmonic conversion and they are used to induce respectively excitation and stimulated emission depletion. This source should allow a low-cost, compact, very efficient and simplified STED scheme since the two beams are intrinsically aligned and synchronized. Using a chromatic beam shaping device which leaves the excitation beam unaffected and produces a donut-shaped STED beam, we demonstrate a 300% reduction of the light sheet thickness, together with an enhancement of the sheet uniformity over larger field of view, at low STED power, in Coumarin dye solution.

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.

Quantitative MR estimates of blood oxygenation based on T2*: a numerical study of the impact of model assumptions.

Several MR methods have been proposed over the last decade to obtain quantitative estimates of the tissue blood oxygen saturation (StO2) using a quantification of the blood oxygen level dependent effect. These approaches are all based on mathematical models describing the time evolution of the MR signal in biological tissues in the presence of magnetic field inhomogeneities. Although the experimental results are very encouraging, possible biases induced by the model assumptions have not been extensively studied. In this study, a numerical approach was used to examine the influence on T(2)*, blood volume fraction, and StO2 estimates of possible confounding factors such as water diffusion, intravascular signal, and presence of arterial blood in the voxel. To evaluate the impact of the vessel geometry, straight cylinders and realistic data from two-photon microscopy for microvascular geometry were compared. Our results indicate that the models are sufficiently realistic, based on a good correlation between ground truth and MR estimates of StO2.

The role of fluctuations and stress on the effective viscosity of cell aggregates.

Cell aggregates are a tool for in vitro studies of morphogenesis, cancer invasion, and tissue engineering. They respond to mechanical forces as a complex rather than simple liquid. To change an aggregate's shape, cells have to overcome energy barriers. If cell shape fluctuations are active enough, the aggregate spontaneously relaxes stresses ("fluctuation-induced flow"). If not, changing the aggregate's shape requires a sufficiently large applied stress ("stress-induced flow"). To capture this distinction, we develop a mechanical model of aggregates based on their cellular structure. At stress lower than a characteristic stress tau*, the aggregate as a whole flows with an apparent viscosity eta*, and at higher stress it is a shear-thinning fluid. An increasing cell-cell tension results in a higher eta* (and thus a slower stress relaxation time t(c)). Our constitutive equation fits experiments of aggregate shape relaxation after compression or decompression in which irreversibility can be measured; we find t(c) of the order of 5 h for F9 cell lines. Predictions also match numerical simulations of cell geometry and fluctuations. We discuss the deviations from liquid behavior, the possible overestimation of surface tension in parallel-plate compression measurements, and the role of measurement duration.

Two-photon excitation and stimulated emission depletion by a single wavelength.

Super-resolved optical microscopy using stimulated emission depletion (STED) is now a mature method for imaging fluorescent samples at scales beyond the diffraction limit. Nevertheless the practical implementation of STED microscopy is complex and costly, especially since it requires laser beams with different wavelengths for excitation and depletion. In this paper, we propose using a single wavelength to induce both processes. We studied stimulated emission depletion of 4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4H-pyran (DCM) dye with a laser delivering a single wavelength in the near infrared. Fluorescence was excited by two photon absorption with a femtosecond pulse, then depleted by one photon stimulated emission with a stretched pulse. Time-resolved fluorescence decay measurements were performed to determine the depletion efficiency and to prove that fluorescence quenching is not affected by side effects. Numerical simulations show that this method can be applied to super-resolved microscopy.

Polarization Angle Dependence of Optical Gain in a Hybrid Structure of Alexa-Flour 488/M13 Bacteriophage.

We measured optical modal gain of a dye-virus hybrid structure using a variable stripe length method, where Alexa-fluor-488 dye was coated on a virus assembly of M13 bacteriophage. Inspired by the structural periodicity of the wrinkle-like virus assembly, the edge emission of amplified spontaneous emission was measured for increasing excited optical stripe length, which was aligned to be either parallel or perpendicular to the wrinkle alignment. We found that the edge emission showed a strong optical anisotropy, and a spectral etalon also appeared in the gain spectrum. These results can be attributed to the corrugated structure, which causes a similar effect to a DFB laser, and we also estimated effective cavity lengths.

Improved optical slicing by stimulated emission depletion light sheet microscopy.

Three-dimensional microscopy is mandatory for biological investigation. We describe a stimulated emission depletion selective plane illumination microscope (STED-SPIM) that provides both ease of implementation and an efficient optical slicing. This self-aligned system is based on a single diode-pumped solid-state laser and phase masks made of simple cover glass. A three-fold reduction of the light sheet thickness is achieved together with an enhancement of the sheet uniformity. This method is validated by using fluorescent microspheres and thick slices of fixed and clarified mouse brain to provide an enhanced imaging of Alzheimer's disease models.

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.

Fluorescent Pluronic nanodots for in vivo two-photon imaging.

We report the synthesis of new nanosized fluorescent probes based on bio-compatible polyethylene-polypropylene glycol (Pluronic) materials. In aqueous solution, mini-emulsification of Pluronic with a high fluorescent di-stryl benzene-modified derivative, exhibiting a two-photon absorption cross section as high as 2500 Goeppert-Mayer units at 800 nm, leads to nanoparticles exhibiting a hydrodynamic radius below 100 nm. We have demonstrated that these new probes with luminescence located in the spectral region of interest for bio-imaging (the yellow part of the visible spectrum) allow deep (500 microm) bio-imaging of the mice brain vasculature. The dose injected during our experiments is ten times lower when compared to the classical commercial rhodamine-B isothicyanate-Dextran system but gives similar results to homogeneous blood plasma staining. The mean fluorescent signal intensity stayed constant during more than 1 h.

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.

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.

A direct method for measuring mouse capillary cortical blood volume using multiphoton laser scanning microscopy.

Knowledge of the blood volume per unit volume of brain tissue is important for understanding brain function in health and disease. We describe a direct method using two-photon laser scanning microscopy to obtain in vivo the local capillary blood volume in the cortex of anesthetized mouse. We infused fluorescent dyes in the circulating blood and imaged the blood vessels, including the capillaries, to a depth of 600 microm below the dura at the brain surface. Capillary cortical blood volume (CCBV) was calculated without any form recognition and segmentation, by normalizing the total fluorescence measured at each depth and integrating the collected intensities all over the stack. Theoretical justifications are presented and numerical simulations were performed to validate this method which was weakly sensitive to background noise. Then, CCBV had been estimated on seven healthy mice between 2%+/-0.3% and 2.4%+/-0.4%. We showed that this measure of CCBV is reproductible and that this method is highly sensitive to the explored zones in the cortex (vessel density and size). This method, which dispenses with form recognition, is rapid and would allow to study in vivo temporal and highly resolute spatial variations of CCBV under different conditions or stimulations.

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.

Label-free imaging of bone multiscale porosity and interfaces using third-harmonic generation microscopy.

Interfaces provide the structural basis of essential bone functions. In the hierarchical structure of bone tissue, heterogeneities such as porosity or boundaries are found at scales ranging from nanometers to millimeters, all of which contributing to macroscopic properties. To date, however, the complexity or limitations of currently used imaging methods restrict our understanding of this functional integration. Here we address this issue using label-free third-harmonic generation (THG) microscopy. We find that the porous lacuno-canalicular network (LCN), revealing the geometry of osteocytes in the bone matrix, can be directly visualized in 3D with submicron precision over millimetric fields of view compatible with histology. THG also reveals interfaces delineating volumes formed at successive remodeling stages. Finally, we show that the structure of the LCN can be analyzed in relation with that of the extracellular matrix and larger-scale structures by simultaneously recording THG and second-harmonic generation (SHG) signals relating to the collagen organization.

In vivo two-photon microscopy study of short-term effects of microbeam irradiation on normal mouse brain microvasculature.

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.