Imaging the microcirculation is becoming increasingly important in assessing life-threatening disease states. To address this issue in a highly light absorbing and light scattering tissue, we use laser scanning multiphoton microscopy and fluorescent 655-nm 5000-MW methoxy-PEGylated quantum dots to image the functional microcirculation deep in mouse hind limb skeletal muscle. Using this approach, we are able to minimize in vivo background tissue autofluorescence and visualize complete 3-D microvascular units, including feeding arterioles, capillary networks, and collecting venules to depths of 150 to 200 microm. In CD1 mice treated with lipopolysaccharide to model an endotoxemic response to bacterial infection, we find that these quantum dots accumulate at microvascular bifurcations and extravasate from the microcirculation in addition to accumulating in organs (liver, spleen, lung, and kidney). The quantum dots are cleared from the circulation with a first-order elimination rate constant seven times greater than under normal conditions, 1.6+/-0.06 compared to 0.23+/-0.05 h(-1), P<0.05, thereby reducing the imaging time window. In vitro experiments using TNFalpha treated isolated leukocytes suggest that circulating monocytes (phagocytes) increased their nonspecific uptake of quantum dots when activated. In combination with multiphoton microscopy, quantum dots provide excellent in vivo imaging contrast of deep microvascular structures.
"In contrast to fluorescent confocal microscopy or spinning-disc microscopy where a single photon is used to excite a given fluorophore, in multiphoton microscopy (MPM), two photons of longer wavelength are used to simultaneously excite a sample fluorophore . The advantage of MPM is that longer wavelengths allow for deeper sample penetration of both tissues  and fluids with less light scattering, less photobleaching and loss of fluorescent signal, as may occur with confocal microscopy. This occurs because multiphoton excitation is non-linear and limited to a small femtoliter focal volume at the focal point of the laser beam; whereas, one photon imaging creates a large cone of excitation within the sample. "
[Show abstract][Hide abstract] ABSTRACT: Altered fibrin clot architecture is increasingly associated with cardiovascular diseases; yet, little is known about how fibrin networks are affected by small molecules that alter fibrinogen structure. Based on previous evidence that S-nitrosoglutathione (GSNO) alters fibrinogen secondary structure and fibrin polymerization kinetics, we hypothesized that GSNO would alter fibrin microstructure.
Accordingly, we treated human platelet-poor plasma with GSNO (0.01-3.75 mM) and imaged thrombin induced fibrin networks using multiphoton microscopy. Using custom designed computer software, we analyzed fibrin microstructure for changes in structural features including fiber density, diameter, branch point density, crossing fibers and void area. We report for the first time that GSNO dose-dependently decreased fibrin density until complete network inhibition was achieved. At low dose GSNO, fiber diameter increased 25%, maintaining clot void volume at approximately 70%. However, at high dose GSNO, abnormal irregularly shaped fibrin clusters with high fluorescence intensity cores were detected and clot void volume increased dramatically. Notwithstanding fibrin clusters, the clot remained stable, as fiber branching was insensitive to GSNO and there was no evidence of fiber motion within the network. Moreover, at the highest GSNO dose tested, we observed for the first time, that GSNO induced formation of fibrin agglomerates.
Taken together, low dose GSNO modulated fibrin microstructure generating coarse fibrin networks with thicker fibers; however, higher doses of GSNO induced abnormal fibrin structures and fibrin agglomerates. Since GSNO maintained clot void volume, while altering fiber diameter it suggests that GSNO may modulate the remodeling or inhibition of fibrin networks over an optimal concentration range.
PLoS ONE 08/2012; 7(8):e43660. DOI:10.1371/journal.pone.0043660 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We review the syntheses, optical properties, and biological applications of cadmium selenide (CdSe) and cadmium selenide-zinc sulfide (CdSe-ZnS) quantum dots (QDs) and gold (Au) and silver (Ag) nanoparticles (NPs). Specifically, we selected the syntheses of QDs and Au and Ag NPs in aqueous and organic phases, size- and shape-dependent photoluminescence (PL) of QDs and plasmon of metal NPs, and their bioimaging applications. The PL properties of QDs are discussed with reference to their band gap structure and various electronic transitions, relations of PL and photoactivated PL with surface defects, and blinking of single QDs. Optical properties of Ag and Au NPs are discussed with reference to their size- and shape-dependent surface plasmon bands, electron dynamics and relaxation, and surface-enhanced Raman scattering (SERS). The bioimaging applications are discussed with reference to in vitro and in vivo imaging of live cells, and in vivo imaging of cancers, tumor vasculature, and lymph nodes. Other aspects of the review are in vivo deep tissue imaging, multiphoton excitation, NIR fluorescence and SERS imaging, and toxic effects of NPs and their clearance from the body.
[Show abstract][Hide abstract] ABSTRACT: Quantum dots (QDs) have primarily been developed as fluorescent probes with unique optical properties. We herein demonstrate an extension of these QD utilities to photoacoustic (PA) and photothermal (PT) microscopy, using a nanosecond pulse laser excitation (420-900 nm, 8 ns, 10(-3)-10 J/cm(2)). The laser-induced PA, PT and accompanying bubble formation phenomena were studied with an advanced multifunctional microscope, which integrates fluorescence, PA, PT imaging, and PT thermolens modules. It was demonstrated that QDs, in addition to being excellent fluorescent probes, can be used as PA and PT contrast agents and sensitizers, thereby providing an opportunity for multimodal high resolution (300 nm) PA-PT-fluorescent imaging as well as PT therapy. Further improvements for this technology are suggested by increasing the conversion of laser energy in PT, PA, and bubble phenomena in hybrid multilayer QDs that have optimized absorption, thermal, and acoustic properties.
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