Uptake Of Quantum Dots Research Articles

Delivering quantum dot-peptide bioconjugates to the cellular cytosol: escaping from the endolysosomal system

For luminescent quantum dots (QDs) to realize their full potential as intracellular labeling, imaging and sensing reagents, robust noninvasive methods for their delivery to the cellular cytosol must be developed. Our aim in this study was to explore a range of methods aimed at delivering QDs to the cytosol. We have previously shown that QDs functionalized with a polyarginine 'Tat' cell-penetrating peptide (CPP) could be specifically delivered to cells via endocytic uptake with no adverse effects on cellular proliferation. We began by assessing the long-term intracellular fate and stability of these QD-peptide conjugates. We found that the QDs remained sequestered within acidic endolysosomal vesicles for at least three days after initial uptake while the CPP appeared to remain stably associated with the QD throughout this time. We next explored techniques designed to either actively deliver QDs directly to the cytosol or to combine endocytosis with subsequent endosomal escape to the cytosol in several eukaryotic cell lines. Active delivery methods such as electroporation and nucleofection delivered only modest amounts of QDs to the cytosol as aggregates. Delivery of QDs using a variety of transfection polymers also resulted in primarily endosomal sequestration of QDs. However, in one case the commercial PULSin reagent did facilitate a modest cytosolic dispersal of QDs, but only after several days in culture and with significant polymer-induced cytotoxicity. Finally, we demonstrated that an amphiphilic peptide designed to mediate cell penetration and vesicle membrane interactions could mediate rapid QD uptake by endocytosis followed by a slower efficient endosomal release which peaked at 48 h after initial delivery. Importantly, this QD-peptide bioconjugate elicited minimal cytotoxicity in the cell lines tested.

Untargeted quantum dots in confocal microscopy of living cells

The problem of non-specific binding of quantum dots (QDs) with cells is very important but not fully understood taking into account the possible application of QDs in medical and fundamental studies. The interactions of untargeted CdSe/ZnS QDs with isolated frog muscle fibres, HeLa cells and J774 cells were investigated. The observations were performed on living cells using laser confocal microscopy (Leica TCS SL). The QDs covered with polyethylenglycol without any functional reactive groups with emission maximum at 565 nm were used in the study. This type of QDs is suggested to prevent an interaction of QDs with biological molecules. It has been shown that QDs do not enter HeLa cells, T-system and sarcoplasm of skeletal muscle fibres. However, during long-term incubation J774 cells can uptake QDs. The data obtained has demonstrated the diversity of interactions of untargeted QDs with different cell types and are important for understanding of the problems of non-selective uptake and cytotoxicity of QDs.

Fluorescence Imaging and Whole-Body Biodistribution of Near-Infrared-Emitting Quantum Dots after Subcutaneous Injection for Regional Lymph Node Mapping in Mice

This study compares fluorescence imaging to mass spectroscopy (inductively coupled plasma-mass spectroscopy, ICP-MS) for detection of quantum dots (QDs) in sentinel lymph node (LN) mapping of breast cancer. We study the accumulation of near-infrared-emitting QDs into regional LNs and their whole-body biodistribution in mice after subcutaneous injection, using in vivo fluorescence imaging and ex vivo elemental analysis by ICP-MS. We show that the QD accumulation in regional LNs is detectable by fluorescence imaging as early as 5 min post-delivery. Their concentration reaches a maximum at 4 h then decreases over a 10-day observation period. These data are confirmed by ICP-MS. The QD uptake in other organs, assessed by ICP-MS, increases steadily over time; however, its overall level remains rather low. Fluorescence imaging can be used as a non-invasive alternative to ICP-MS to follow the QD accumulation kinetics into regional LNs.

Probing Cell‐Type‐Specific Intracellular Nanoscale Barriers Using Size‐Tuned Quantum Dots

The compartmentalization of size-tuned luminescent semiconductor nanocrystal quantum dots (QDs) in four distinctive cell lines, which would be representative of the most likely environmental exposure routes to nanoparticles in humans, is studied. The cells are fixed and permeabilized prior to the addition of the QDs, thus eliminating any cell-membrane-associated effects due to active QD uptake mechanisms or to specificity of signaling routes in different cell types, but leaving intact the putative physical subcellular barriers. All quantitative assays are performed using a high content analysis (HCA) platform, thereby obtaining robust data on large cell populations. While smaller QDs 2.1 nm in diameter enter the nuclei and localize to the nucleoli in all cell types, the rate and dynamics of their passage vary depending on the cell origin. As the QD size is increased to 4.4 nm, penetration into the cell is reduced but each cell line displays its own cutoff size thresholds reflecting cell-type-determined cytoplasmic and nuclear pore penetration specificity. These results give rise to important considerations regarding the differential compartmentalization and susceptibility of organs, tissues, and cells to nanoparticles, and may be of prime importance for biomedical imaging and drug-delivery research employing nanoparticle-based probes and systems.

Calibration of Flow Cytometry for Quantitative Quantum Dot Measurements

Observations of quantum dot (QD) labeled cells in biomedical research are mainly qualitative in nature, which limits the ability of researchers to compare results experiment-to-experiment and lab-to-lab to improve the state-of-the-art. Labeled cells are useful in a range of in vitro and in vivo assays where tracking behavior of administered cells is integral for answering research questions in areas such as tissue engineering and stem cell therapy. Before the full potential of QD based toolsets can be realized in the clinic, uptake of QDs by cells must be quantified and standardized. This unit describes a novel, simple method to assess the number of QDs per cell using flow cytometry and commercially available standards. This quick and easy method can be used by all researchers to calibrate their flow cytometry instruments and settings, and quantify QD uptake by cells for in vitro and in vivo experimentation for comparable results across QD conjugate types, cell types, research groups, lots of commercial QDs, and homemade QDs.

B-Amyloid treatment does not enhance the age-related increase in phagocytic activity of microglial cells

Microglia are the resident immune cells of the brain and are activated following insult; activated microglia are acknowledged as having both beneficial and detrimental effects. In the healthy brain, microglia are responsible for surveillance and play a role in homeostasis; it is believed that they are also responsible for the clearance of Aβ. In Alzheimer's Disease, activated microglia are associated with insoluble amyloid plaques but it is unclear whether they are involved in Aβ clearance. We investigated uptake of quantum dots by microglia, as a measure of their phagocytic activity, using flow cytometry. There was no effect of Aβ on the phagocytic activity of mixed glial cultures or bone marrow derived macrophages but it significantly increased IL-1β production in both cells. Phagocytic activity was also assessed in cells isolated from the brains of young and aged rats and the data indicated that there was a significant increase in the phagocytic activity of CD11b+ cells isolated from the brains of aged, compared with young, rats. Treatment of cells prepared from young rats with Aβ significantly increased phagocytic activity but Aβ induced no further increase in the phagocytic activity of cells isolated from the brains of aged rats. The data suggest that microglial cells in the aged brain cannot be further stimulated to increase their phagocytic activity following an additional insult with Aβ.

Aqueous‐Phase Synthesis of Highly Luminescent CdTe/ZnTe Core/Shell Quantum Dots Optimized for Targeted Bioimaging

Semiconductor quantum dots (QDs) have traditionally been synthesized in organic phase and transferred to aqueous solution by functionalizing their surface with silica, polymers, short-chain thiol ligand, or phospholipid micelles. However, these complex steps result in i) a reduction of the quantum yield (QY) of QDs, ii) partial degrdation of the QDs, and iii) a drastic increase in the hydrodynamic size of QDs, which may hinder their biomedical applications. In this work, the fabrication and applications of cysteine-capped CdTe/ZnTe QDs, which are directly synthesized in aqueous media, as optical probes for specific targeting of pancreatic and esophageal cancer cells in vitro are reported, as well as their capability for in vivo imaging. The CdTe/ZnTe QDs are synthesized in a one-pot method and capped with amino acid cysteine, which contains both carboxyl and amine functional groups on their surfaces for bioconjugation. The fabricated QDs have an ultrasmall hydrodynamic diameter (3-5 nm), possess high QY (52%), and are non-toxic to cells at experimental dosages. Confocal imaging is used to demonstrate a receptor-mediated uptake of antibody-conjugated QDs into pancreatic cancer cells in vitro. In vitro cytotoxicity studies (MTS-assay) show that the IC(50) value of these QDs is approximately 160 microg mL(-1), demonstrating low toxicity. In addition, the QDs are used for small-animal imaging where the in vivo biocompatiblity of these QDs and their clearance following systemic injection is studied.

Fluorescent tumour imaging of type I IGF receptor in vivo: comparison of antibody-conjugated quantum dots and small-molecule fluorophore

Background:The type I insulin-like growth factor receptor (IGF1R) is a transmembrane tyrosine kinase involved in cancer proliferation, survival, and metastasis.Methods:In this study, we used two different fluorescent technologies (small-molecule fluorophores and quantum dot (QD) nanoparticles) to detect receptor expression and its downregulation by antibodies in vivo.Results:After conjugation with AVE-1642, a humanised anti-IGF1R monoclonal antibody, both QDs (705 nm) or Alexa 680 (small-molecule fluorophore) detected expression and downregulation of IGF1R in vitro. To examine their utility in vivo, either AVE-1642 conjugates were intravenously delivered to mice bearing xenograft tumours of mouse embryo fibroblasts expressing human IGF1R or MCF-7 human breast cancer cells. Quantum dot fluorescence was mainly localised to the reticuloendothelial system in several organs and engulfed by macrophages, with only very small amount of QDs detected in the xenograft tumours. Depletion of macrophages by clodronate liposomes did not alter the nonspecific uptake of QDs. In contrast, AVE-1642-conjugated Alexa 680 solely targeted to xenograft tumour and was able to detect IGF1R downregulation, with little nonspecific targeting to other tissues or organs in mice.Conclusion:Taken together, our data suggest that small-molecule fluorophores, not QDs, are suitable to detect the expression and downregulation of IGF1R in vivo.

Mechanisms of Quantum Dot Nanoparticle Cellular Uptake

Due to the superior photoemission and photostability characteristics, quantum dots (QD) are novel tools in biological and medical applications. However, the toxicity and mechanism of QD uptake are poorly understood. QD nanoparticles with an emission wavelength of 655 nm are ellipsoid in shape and consist of a cadmium/selenide core with a zinc sulfide shell. We have shown that QD with a carboxylic acid surface coating were recognized by lipid rafts but not by clathrin or caveolae in human epidermal keratinocytes (HEKs). QD were internalized into early endosomes and then transferred to late endosomes or lysosomes. In addition, 24 endocytic interfering agents were used to investigate the mechanism by which QD enter cells. Our results showed that QD endocytic pathways are primarily regulated by the G-protein-coupled receptor associated pathway and low density lipoprotein receptor/scavenger receptor, whereas other endocytic interfering agents may play a role but with less of an inhibitory effect. Lastly, low toxicity of QD was shown with the 20 nM dose in HEK at 48 h but not at 24 h by the live/dead cell assay. QD induced more actin filaments formation in the cytoplasm, which is different from the actin depolymerization by cadmium. These findings provide insight into the specific mechanism of QD nanoparticle uptake in cells. The surface coating, size, and charge of QD nanoparticles are important parameters in determining how nanoparticle uptake occurs in mammalian cells for cancer diagnosis and treatment, and drug delivery.

Elimination of Quantum Dots Cell Uptake

AbstractThe nanotechnology is undergoing enormous attention in the areas of biological research for clinical, environmental, and life sciences applications. One of the products from this new technology that attracts researcher’s attentions is the semiconductor quantum dot (QDs) nanoparticles, QDs possess incomparable advantages such as unique size-dependent physical properties, broad absorption spectrum, precise small bandwidth emission wavelength, as well as enhanced chemical and photochemical stability. The QDs can be modified for a controlled and enhanced endocytosis, enhanced cooperative binding activity, and easy introduction of multi-functionalities for medical applications such as targeted delivery and imaging. It can be used for complex studies that play very important roles in the modern biomedical researches. However, when performing the cell related assays, the non-specific cellular uptake of QDs is a major concern because they can lead to false positives or false results. In our study, we used different surface modified QDs treated with different blocking buffers to eliminate cellular uptake. The preliminary results showed that the cellular uptake of QDs can be eliminated by surface modification of the QD materials and by performing the assays in the presence of blocking buffers. As a result of the elimination of non-specific uptake of QDs the sensitivity and specificity of detection increased significantly.

Particle Size, Surface Coating, and PEGylation Influence the Biodistribution of Quantum Dots in Living Mice

This study evaluates the influence of particle size, PEGylation, and surface coating on the quantitative biodistribution of near-infrared-emitting quantum dots (QDs) in mice. Polymer- or peptide-coated 64Cu-labeled QDs 2 or 12 nm in diameter, with or without polyethylene glycol (PEG) of molecular weight 2000, are studied by serial micropositron emission tomography imaging and region-of-interest analysis, as well as transmission electron microscopy and inductively coupled plasma mass spectrometry. PEGylation and peptide coating slow QD uptake into the organs of the reticuloendothelial system (RES), liver and spleen, by a factor of 6-9 and 2-3, respectively. Small particles are in part renally excreted. Peptide-coated particles are cleared from liver faster than physical decay alone would suggest. Renal excretion of small QDs and slowing of RES clearance by PEGylation or peptide surface coating are encouraging steps toward the use of modified QDs for imaging living subjects.

Genetically Modifiable Flagella as Templates for Silica Fibers: From Hybrid Nanotubes to 1D Periodic Nanohole Arrays

AbstractBacterial flagellum is a protein nanotube that is helically self‐assembled from thousands of a protein subunit called flagellin. The solvent‐exposed domain of each flagellin on the flagella is genetically modifiable, in that a foreign peptide can be genetically inserted into this domain, leading to the high‐density display of this foreign peptide on the surface of flagella. In this work, wild‐type and genetically engineered flagella (inner diameter of ∼2 nm and outer diameter of ∼14 nm) detached from the surface of Salmonella bacterial cells are used as templates to site‐specifically form silica sheaths on the flagellar surface, resulting in the formation of double‐layered silica/flagella nanotubes. The flagella templates inside the silica/flagella nanotubes can be removed to obtain silica nanotubes by calcining the nanotubes at high temperature (550°C). Further calcination of the silica nanotubes at a higher temperature (800 °C) leads to the formation of a periodic nanohole array along the silica fibers with a center‐to‐center nanohole spacing of ∼79 nm. It is demonstrated that the double‐layered silica‐flagella nanotubes can be used for selective CdTe quantum dot uptake into the inner channels or selective Au nanoparticle coating on the outer wall of the nanotubes due to the different chemistry between inner flagellum core (protein) and outer silica wall of the nanotubes. It is also found that flagella displaying different peptides result in different morphologies of the silica nanotubes. This work suggests that the monodisperse diameter and genetically tunable surface chemistry of the flagella can be exploited for the fabrication of silica nanotubes with uniform diameter and controllable morphologies as well as silica nanofibers decorated with periodic nanohole arrays.

Enhanced intratumoral uptake of quantum dots concealed within hydrogel nanoparticles

Effective nanomedical devices for tumor imaging and drug delivery are not yet available. Inan attempt to construct a more functional device for tumor imaging, we have embeddedquantum dots (which have poor circulatory behavior) within hydrogel nanoparticles made ofpoly-N-isopropylacrylamide. We found that the hydrogel encapsulated quantumdots are more readily taken up by cultured tumor cells. Furthermore, in amelanoma model, hydrogel encapsulated quantum dots also preferentiallyaccumulate in the tumor tissue compared with normal tissue and have∼16-fold greater intratumoral uptake compared to non-derivatized quantum dots. Our resultssuggest that these derivatized quantum dots, which have greatly improved tumorlocalization, may enhance cancer monitoring and chemotherapy.

Quantum Dots for Tracking Dendritic Cells and Priming an Immune Response In Vitro and In Vivo

Dendritic cells (DCs) play a key role in initiating adaptive immune response by presenting antigen to T cells in lymphoid organs. Here, we investigate the potential of quantum dots (QDs) as fluorescent nanoparticles for in vitro and in vivo imaging of DCs, and as a particle-based antigen-delivery system to enhance DC-mediated immune responses. We used confocal, two-photon, and electron microscopies to visualize QD uptake into DCs and compared CD69 expression, T cell proliferation, and IFN-γ production by DO11.10 and OT-II T cells in vivo in response to free antigen or antigen-conjugated to QDs. CD11c+ DCs avidly and preferentially endocytosed QDs, initially into small vesicles near the plasma membrane by an actin-dependent mechanism. Within 10 min DCs contained vesicles of varying size, motion, and brightness distributed throughout the cytoplasm. At later times, endocytosed QDs were compartmentalized inside lysosomes. LPS-induced maturation of DCs reduced the rate of endocytosis and the proportion of cells taking up QDs. Following subcutaneous injection of QDs in an adjuvant depot, DCs that had endocytosed QDs were visualized up to 400 µm deep within draining lymph nodes. When antigen-conjugated QDs were used, T cells formed stable clusters in contact with DCs. Antigen-conjugated QDs induced CD69 expression, T cell proliferation, and IFN-γ production in vivo with greater efficiency than equivalent amounts of free antigen. These results establish QDs as a versatile platform for immunoimaging of dendritic cells and as an efficient nanoparticle-based antigen delivery system for priming an immune response.

The impact of different nanoparticle surface chemistry and size on uptake and toxicity in a murine macrophage cell line

This study investigated the uptake, kinetics and cellular distribution of different surface coated quantum dots (QDs) before relating this to their toxicity. J774.A1 cells were treated with organic, COOH and NH 2 (PEG) surface coated QDs (40 nM). Model 20 nm and 200 nm COOH-modified coated polystyrene beads (PBs) were also examined (50 μg ml − 1 ). The potential for uptake of QDs was examined by both fixed and live cell confocal microscopy as well as by flow cytometry over 2 h. Both the COOH 20 nm and 200 nm PBs were clearly and rapidly taken up by the J774.A1 cells, with uptake of 20 nm PBs being relatively quicker and more extensive. Similarly, COOH QDs were clearly taken up by the macrophages. Uptake of NH 2 (PEG) QDs was not detectable by live cell imaging however, was observed following 3D reconstruction of fixed cells, as well as by flow cytometry. Cells treated with organic QDs, monitored by live cell imaging, showed only a small amount of uptake in a relatively small number of cells. This uptake was insufficient to be detected by flow cytometry. Imaging of fixed cells was not possible due to a loss in cell integrity related to cytotoxicity. A significant reduction ( p < 0.05) in the fluorescent intensity in a cell-free environment was found with organic QDs, NH 2 (PEG) QDs, 20 nm and 200 nm PBs at pH 4.0 (indicative of an endosome) after 2 h, suggesting reduced stability. No evidence of exocytosis was found over 2 h. These findings confirm that surface coating has a significant influence on the mode of NP interaction with cells, as well as the subsequent consequences of that interaction.

Cyclic tensile strain increases interactions between human epidermal keratinocytes and quantum dot nanoparticles

The effects of quantum dots (QD) on cell viability have gained increasing interest due to many recent developments utilizing QD for pharmaceutical and biomedical applications. The potential use of QD nanoparticles as diagnostic, imaging, and drug delivery agents has raised questions about their potential for cytotoxicity. The objective of this study was to investigate the effects of applied strain on QD uptake by human epidermal keratinocytes (HEK). It was hypothesized that introduction of a 10% average strain to cell cultures would increase QD uptake. HEK were seeded at a density of 150,000 cells/mL on collagen-coated Flexcell culture plates (Flexcell Intl.). QD were introduced at a concentration of 3 nM and a 10% average strain was applied to the cells. After 4h of cyclic strain, the cells were examined for cell viability, QD uptake, and cytokine production. The results indicate that addition of strain results in an increase in cytokine production and QD uptake, resulting in irritation and a negative impact on cell viability. Application of physiological load conditions can increase cell membrane permeability, thereby increasing the concentration of QD nanoparticles in cells.

Folate-mediated tumor cell uptake of quantum dots entrapped in lipid nanoparticles

Quantum dots (QDs) are fluorescent semiconductor nanocrystals with superior optical properties compared to organic dyes currently undergoing rapid development for biological applications, particularly in fluorescence imaging. The folate receptor, overexpressed in a broad spectrum of malignant tumors, is an attractive target for selective delivery of imaging agents to tumor cells. This study examines nanoparticles containing QDs entrapped in a lipid shell, and post-loaded with a folate-lipid conjugate for targeting to mouse and human tumor cells expressing the folate receptor. Hydrophobic QDs were mixed with 1,2 dipalmitoyl-sn-glycero-3 phosphocholine and methoxy–polyethylene–glycol–distearoyl–phosphatidyl–ethanolamine (mPEG-DSPE) generating a nanoparticle referred to as lipodot, with a mean diameter size of ∼ 100 nm. Folate-derivatized PEG-DSPE was post-loaded into the lipodots at 0.5% lipid molar concentration. Mouse J6456 lymphoma cells (J6456-FR) and human head and neck KB cancer cells (KB-FR), up-regulated for their folate receptors, were incubated with folate-targeted and non-targeted lipodots in vitro. Using fluorescence microscopy, it was found that only folate-targeted lipodots were taken up by tumor cells. Confocal depth scanning showed substantial internalization. Confirming the specificity of folate-targeted lipodots, binding and internalization were inhibited by free folate, and no uptake was found in a folate-receptor negative cell line. Selective binding and uptake of folate-targeted lipodots by J6456-FR cells was also observed in vivo after intra-peritoneal injection in mice bearing ascitic J6456-FR tumors based on FACS analysis and confocal imaging of harvested cells from the peritoneal cavity. Folate-targeted lipodots represent an attractive approach for tumor cell labeling both in vitro and in vivo.

Surface Coatings Determine Cytotoxicity and Irritation Potential of Quantum Dot Nanoparticles in Epidermal Keratinocytes

Quantum dot (QD) nanoparticles have potential applications in nanomedicine as drug delivery vectors and diagnostic agents, but the skin toxicity and irritation potential of QDs are unknown. Human epidermal keratinocytes (HEKs) were used to assess if QDs with different surface coatings would cause differential effects on HEK cytotoxicity, proinflammatory cytokine release, and cellular uptake. Commercially available QDs of two different sizes, QD 565 and QD 655, with neutral (polyethylene glycol (PEG)), cationic (PEG-amine), or anionic (carboxylic acid) coatings were utilized. Live cell imaging and transmission electron microscopy were used to determine that all QDs localized intracellularly by 24 hours, with evidence of QD localization in the nucleus. Cytotoxicity and release of the proinflammatory cytokines IL-1beta, IL-6, IL-8, IL-10, and tumor necrosis factor-alpha were assessed at 24 and 48 hours. Cytotoxicity was observed for QD 565 and QD 655 coated with carboxylic acids or PEG-amine by 48 hours, with little cytotoxicity observed for PEG-coated QDs. Only carboxylic acid-coated QDs significantly increased release of IL-1beta, IL-6, and IL-8. These data indicate that QD surface coating is a primary determinant of cytotoxicity and immunotoxicity in HEKs, which is consistent across size. However, uptake of QDs by HEKs is independent of surface coating.

Fluorescence Intensity and Intermittency as Tools for Following Dopamine Bioconjugate Processing in Living Cells

CdSe/ZnS quantum dots (QDs) conjugated to biomolecules that quench their fluorescence, particularly dopamine, have particular spectral properties that allow determination of the number of conjugates per particle, namely, photoenhancement and photobleaching. In this work, we quantify these properties on a single-particle and ensemble basis in order to evaluate their usefulness as a tool for indicating QD uptake, breakdown, and processing in living cells. This creates a general framework for the use of fluorescence quenching and intermittency to better understand nanoparticle-cell interactions.

Endotoxemia increases the clearance of mPEGylated 5000-MW quantum dots as revealed by multiphoton microvascular imaging

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.