Practical strategies for robust and inexpensive imaging of aqueous-cleared tissues.

Lightsheet microscopy offers an ideal method for imaging of large (mm-cm scale) biological tissues rendered transparent via optical clearing protocols. However the diversity of clearing technologies and tissue types, and how these are adapted to the microscope can make tissue mounting complicated and somewhat irreproducible. Tissue preparation for imaging can involve glues and or equilibration in a variety of expensive and/or proprietary formulations. Here we present practical advice for mounting and capping cleared tissues in optical cuvettes for macroscopic imaging, providing a standardised 3D cell that can be imaged routinely and relatively inexpensively. We show that acrylic cuvettes cause minimal spherical aberration with objective numerical apertures less than 0.65. Furthermore, we describe methods for aligning and assessing the light sheets, discriminating fluorescence from autofluorescence, identifying chromatic artefacts due to differential scattering and removing streak artefacts such that they do not confound downstream 3D object segmentation analyses, with mouse embryo, liver and heart imaging as demonstrated examples.

Fluorescent Silica Nanoparticles to Label Metastatic Tumor Cells in Mineralized Bone Microenvironments.

During breast cancer bone metastasis, tumor cells interact with bone microenvironment components including inorganic minerals. Bone mineralization is a dynamic process and varies spatiotemporally as a function of cancer-promoting conditions such as age and diet. The functional relationship between skeletal dissemination of tumor cells and bone mineralization, however, is unclear. Standard histological analysis of bone metastasis frequently relies on prior demineralization of bone, while methods that maintain mineral are often harsh and damage fluorophores commonly used to label tumor cells. Here, fluorescent silica nanoparticles (SNPs) are introduced as a robust and versatile labeling strategy to analyze tumor cells within mineralized bone. SNP uptake and labeling efficiency of MDA-MB-231 breast cancer cells is characterized with cryo-scanning electron microscopy and different tissue processing methods. Using a 3D in vitro model of marrow-containing, mineralized bone as well as an in vivo model of bone metastasis, SNPs are demonstrated to allow visualization of labeled tumor cells in mineralized bone using various imaging modalities including widefield, confocal, and light sheet microscopy. This work suggests that SNPs are valuable tools to analyze tumor cells within mineralized bone using a broad range of bone processing and imaging techniques with the potential to increase the understanding of bone metastasis.

Inverted channel variations identified on a distal portion of a bajada in the central Atacama Desert, Chile.

In deserts, the interplay between occasional fluvial events and persistent aeolian erosion can form composite modern and relict surfaces, especially on the distal portion of alluvial fans. There, relief inversion of alluvial deposits by differential erosion can form longitudinal ridges. We identified two distinct ridge types formed by relief inversion on converging alluvial fans in the hyperarid Chilean Atacama Desert. Although they are co-located and similar in scale, the ridge types have different ages and formation histories that apparently correspond to minor paleoclimate variations. Gravel-armored ridges are remnants of deflated alluvial deposits with a bimodal sediment distribution (gravel and sand) dated to a minor pluvial phase at the end of the Late Pleistocene (~12 kyr). In contrast, younger (~9 kyr) sulfate-capped ridges formed during a minor arid phase with evaporite deposition in a pre-existing channel that armored the underlying deposits. Collectively, inverted channels at Salar de Llamara resulted from multiple episodes of surface overland flow and standing water spanning several thousand years. Based on ridge relief and age, the minimum long-term deflation rate is 0.1-0.2 m/kyr, driven primarily by wind erosion. This case study is an example of the equifinality concept whereby different processes lead to similar landforms. The complex history of the two ridge types can only be generally constrained in remotely sensed data. In situ observations are required to discern the specifics of the aqueous history, including the flow type, magnitude, sequence, and paleoenvironment. These findings have relevance for interpreting similar landforms on Mars.

Obesity-associated Adipose Stromal Cells Promote Breast Cancer Invasion Through Direct Cell Contact and ECM Remodeling.

Obesity increases the risk and worsens the prognosis for breast cancer due, in part, to altered adipose stromal cell (ASC) behavior. Whether ASCs from obese individuals increase migration of breast cancer cells relative to their lean counterparts, however, remains unclear. To test this connection, multicellular spheroids composed of MCF10A-derived tumor cell lines of varying malignant potential and lean or obese ASCs were embedded into collagen scaffolds mimicking the elastic moduli of interstitial breast adipose tissue. Confocal image analysis suggests that tumor cells alone migrate insignificantly under these conditions. However, direct cell-cell contact with either lean or obese ASCs enables them to migrate collectively, whereby obese ASCs activate tumor cell migration more effectively than their lean counterparts. Time-resolved optical coherence tomography (OCT) imaging suggests that obese ASCs facilitate tumor cell migration by mediating contraction of local collagen fibers. Matrix metalloproteinase (MMP)-dependent proteolytic activity significantly contributes to ASC-mediated tumor cell invasion and collagen deformation. However, ASC contractility is also important, as co-inhibition of both MMPs and contractility is necessary to completely abrogate ASC-mediated tumor cell migration. These findings imply that obesity-mediated changes of ASC phenotype may impact tumor cell migration and invasion with potential implications for breast cancer malignancy in obese patients.

In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation.

Rupture of the ovarian follicle releases the oocyte at ovulation, a timed event that is critical for fertilization. It is not understood how the protease activity required for rupture is directed with precise timing and localization to the outer surface, or apex, of the follicle. We hypothesized that vasoconstriction at the apex is essential for rupture. The diameter and blood flow of individual vessels and the thickness of the apical follicle wall were examined over time to expected ovulation using intravital multiphoton microscopy. Vasoconstriction of apical vessels occurred within hours preceding follicle rupture in wild-type mice, but vasoconstriction and rupture were absent in Amhr2(cre/+)SmoM2 mice in which follicle vessels lack the normal association with vascular smooth muscle. Vasoconstriction is not simply a response to reduced thickness of the follicle wall; vasoconstriction persisted in wild-type mice when thinning of the follicle wall was prevented by infusion of protease inhibitors into the ovarian bursa. Ovulation was inhibited by preventing the periovulatory rise in the expression of the vasoconstrictor endothelin 2 by follicle cells of wild-type mice. In these mice, infusion of vasoconstrictors (either endothelin 2 or angiotensin 2) into the bursa restored the vasoconstriction of apical vessels and ovulation. Additionally, infusion of endothelin receptor antagonists into the bursa of wild-type mice prevented vasoconstriction and follicle rupture. Processing tissue to allow imaging at increased depth through the follicle and transabdominal ultrasonography in vivo showed that decreased blood flow is restricted to the apex. These results demonstrate that vasoconstriction at the apex of the follicle is essential for ovulation.

Enhanced Oxygen Solubility in Metastable Water under Tension.

Despite its relevance in numerous natural and industrial processes, the solubility of molecular oxygen has never been directly measured in capillary-condensed liquid water. In this article, we measure oxygen solubility in liquid water trapped within nanoporous samples, in metastable equilibrium with a subsaturated vapor. We show that solubility increases two fold at moderate subsaturations (relative humidity ∼0.55). This evolution with relative humidity is in good agreement with a simple thermodynamic prediction using properties of bulk water, previously verified experimentally at positive pressure. Our measurement thus verifies the validity of this macroscopic thermodynamic theory to strong confinement and large negative pressures, where significant nonidealities are expected. This effect has strong implications for important oxygen-dependent chemistries in natural and technological contexts.

Comparative mechanisms of cancer cell migration through 3D matrix and physiological microtracks.

Tumor cell invasion through the stromal extracellular matrix (ECM) is a key feature of cancer metastasis, and understanding the cellular mechanisms of invasive migration is critical to the development of effective diagnostic and therapeutic strategies. Since cancer cell migration is highly adaptable to physiochemical properties of the ECM, it is critical to define these migration mechanisms in a context-specific manner. Although extensive work has characterized cancer cell migration in two- and three-dimensional (3D) matrix environments, the migration program employed by cells to move through native and cell-derived microtracks within the stromal ECM remains unclear. We previously reported the development of an in vitro model of patterned type I collagen microtracks that enable matrix metalloproteinase-independent microtrack migration. Here we show that collagen microtracks closely resemble channel-like gaps in native mammary stroma ECM and examine the extracellular and intracellular mechanisms underlying microtrack migration. Cell-matrix mechanocoupling, while critical for migration through 3D matrix, is not necessary for microtrack migration. Instead, cytoskeletal dynamics, including actin polymerization, cortical tension, and microtubule turnover, enable persistent, polarized migration through physiological microtracks. These results indicate that tumor cells employ context-specific mechanisms to migrate and suggest that selective targeting of cytoskeletal dynamics, but not adhesion, proteolysis, or cell traction forces, may effectively inhibit cancer cell migration through preformed matrix microtracks within the tumor stroma.

Implanted adipose progenitor cells as physicochemical regulators of breast cancer.

Multipotent adipose-derived stem cells (ASCs) are increasingly used for regenerative purposes such as soft tissue reconstruction following mastectomy; however, the ability of tumors to commandeer ASC functions to advance tumor progression is not well understood. Through the integration of physical sciences and oncology approaches we investigated the capability of tumor-derived chemical and mechanical cues to enhance ASC-mediated contributions to tumor stroma formation. Our results indicate that soluble factors from breast cancer cells inhibit adipogenic differentiation while increasing proliferation, proangiogenic factor secretion, and myofibroblastic differentiation of ASCs. This altered ASC phenotype led to varied extracellular matrix (ECM) deposition and contraction thereby enhancing tissue stiffness, a characteristic feature of breast tumors. Increased stiffness, in turn, facilitated changes in ASC behavior similar to those observed with tumor-derived chemical cues. Orthotopic mouse studies further confirmed the pathological relevance of ASCs in tumor progression and stiffness in vivo. In summary, altered ASC behavior can promote tumorigenesis and, thus, their implementation for regenerative therapy should be carefully considered in patients previously treated for cancer.

Recombinant manufacturing of multispecies biolubricants.

Lubricin, a lubricating glycoprotein abundant in synovial fluid, forms a low-friction brush polymer interface in tissues exposed to sliding motion including joints, tendon sheaths, and the surface of the eye. Despite its therapeutic potential in diseases such as osteoarthritis and dry eye disease, there are few sources available. Through rational design, we developed a series of recombinant lubricin analogs that utilize the species-specific tissue-binding domains at the N- and C-termini to increase biocompatibility while replacing the central mucin domain with an engineered variant that retains the lubricating properties of native lubricin. In this study, we demonstrate the tissue binding capacity of our engineered lubricin product and its retention in the joint space of rats. Next, we present a new bioprocess chain that utilizes a human-derived cell line to produce O-glycosylation consistent with that of native lubricin and a purification strategy that capitalizes on the positively charged, hydrophobic N- and C-terminal domains. The bioprocess chain is demonstrated at 10 L scale in industry-standard equipment utilizing commonly available ion exchange, hydrophobic interaction and size exclusion chromatography resins. Finally, we confirmed the purity and lubricating properties of the recombinant biolubricant. The biomolecular engineering and bioprocessing strategies presented here are an effective means of lubricin production and could have broad applications to the study of mucins in general.

Increased Advanced Glycation Endproducts, Stiffness, and Hardness in Iliac Crest Bone From Postmenopausal Women With Type 2 Diabetes Mellitus on Insulin.

Individuals with type 2 diabetes mellitus (T2DM) have a greater risk of bone fracture compared with those with normal glucose tolerance (NGT). In contrast, individuals with impaired glucose tolerance (IGT) have a lower or similar risk of fracture. Our objective was to understand how progressive glycemic derangement affects advanced glycation endproduct (AGE) content, composition, and mechanical properties of iliac bone from postmenopausal women with NGT (n = 35, age = 65 ± 7 years, HbA1c = 5.8% ± 0.3%), IGT (n = 26, age = 64 ± 5 years, HbA1c = 6.0% ± 0.4%), and T2DM on insulin (n = 25, age = 64 ± 6 years, HbA1c = 9.1% ± 2.2%). AGEs were assessed in all samples using high-performance liquid chromatography to measure pentosidine and in NGT/T2DM samples using multiphoton microscopy to spatially resolve the density of fluorescent AGEs (fAGEs). A subset of samples (n = 14 NGT, n = 14 T2DM) was analyzed with nanoindentation and Raman microscopy. Bone tissue from the T2DM group had greater concentrations of (i) pentosidine versus IGT (cortical +24%, p = 0.087; trabecular +35%, p = 0.007) and versus NGT (cortical +40%, p = 0.003; trabecular +35%, p = 0.004) and (ii) fAGE cross-link density versus NGT (cortical +71%, p < 0.001; trabecular +44%, p < 0.001). Bone pentosidine content in the IGT group was lower than in the T2DM group and did not differ from the NGT group, indicating that the greater AGE content observed in T2DM occurs with progressive diabetes. Individuals with T2DM on metformin had lower cortical bone pentosidine compared with individuals not on metformin (-35%, p = 0.017). Cortical bone from the T2DM group was stiffer (+9%, p = 0.021) and harder (+8%, p = 0.039) versus the NGT group. Bone tissue AGEs, which embrittle bone, increased with worsening glycemic control assessed by HbA1c (Pen: R2  = 0.28, p < 0.001; fAGE density: R2  = 0.30, p < 0.001). These relationships suggest a potential mechanism by which bone fragility may increase despite greater tissue stiffness and hardness in individuals with T2DM; our results suggest that it occurs in the transition from IGT to overt T2DM. © 2022 American Society for Bone and Mineral Research (ASBMR).

Endothelial cells metabolically regulate breast cancer invasion toward a microvessel.

Breast cancer metastasis is initiated by invasion of tumor cells into the collagen type I-rich stroma to reach adjacent blood vessels. Prior work has identified that metabolic plasticity is a key requirement of tumor cell invasion into collagen. However, it remains largely unclear how blood vessels affect this relationship. Here, we developed a microfluidic platform to analyze how tumor cells invade collagen in the presence and absence of a microvascular channel. We demonstrate that endothelial cells secrete pro-migratory factors that direct tumor cell invasion toward the microvessel. Analysis of tumor cell metabolism using metabolic imaging, metabolomics, and computational flux balance analysis revealed that these changes are accompanied by increased rates of glycolysis and oxygen consumption caused by broad alterations of glucose metabolism. Indeed, restricting glucose availability decreased endothelial cell-induced tumor cell invasion. Our results suggest that endothelial cells promote tumor invasion into the stroma due, in part, to reprogramming tumor cell metabolism.

Testing Rover Science Protocols to Identify Possible Biosignatures on Mars: Achieving Sampling Goals Under a Highly Constrained Time Line.

At a Mars analog site in Utah, we tested two science operation methods for data acquisition and decision-making protocols: a scenario where the tactical day is preplanned, but major adjustments may still be made before plan delivery; and a scenario in which the sol path must largely be planned before a given tactical planning day and very few adjustments to the plan may be made. The goal was to provide field-tested insight into operations planning for rover missions where science operations must facilitate the efficient choice of sampling locations at a site relevant to searching for habitability and biosignatures. Results of the test indicate that preplanning sol paths did not result in a sol cost savings nor did it improve science return or optimal biologically relevant sample collection. In addition because facies variations in an environment can be subtle and evident only at scales below orbital resolution, acquiring systematic observations is crucial. We also noted that while spectral data provided insight into the chemical components as a whole at this site, they did not provide a guide to targets for which the traverse should be altered. Finally, strategic science planning must include a special effort to account for terrain.

Orbital and In-Situ Investigation of Periodic Bedrock Ridges in Glen Torridon, Gale Crater, Mars.

Gale crater, the field site for NASA's Mars Science Laboratory Curiosity rover, contains a diverse and extensive record of aeolian deposition and erosion. This study focuses on a series of regularly spaced, curvilinear, and sometimes branching bedrock ridges that occur within the Glen Torridon region on the lower northwest flank of Aeolis Mons, the central mound within Gale crater. During Curiosity's exploration of Glen Torridon between sols ∼2300-3080, the rover drove through this field of ridges, providing the opportunity for in situ observation of these features. This study uses orbiter and rover data to characterize ridge morphology, spatial distribution, compositional and material properties, and association with other aeolian features in the area. Based on these observations, we find that the Glen Torridon ridges are consistent with an origin as wind-eroded bedrock ridges, carved during the exhumation of Mount Sharp. Erosional features like the Glen Torridon ridges observed elsewhere on Mars, termed periodic bedrock ridges (PBRs), have been interpreted to form transverse to the dominant wind direction. The size and morphology of the Glen Torridon PBRs are consistent with transverse formative winds, but the orientation of nearby aeolian bedforms and bedrock erosional features raise the possibility of PBR formation by a net northeasterly wind regime. Although several formation models for the Glen Torridon PBRs are still under consideration, and questions persist about the nature of PBR-forming paleowinds, the presence of PBRs at this site provides important constraints on the depositional and erosional history of Gale crater.

Is a Linear or a Walkabout Protocol More Efficient When Using a Rover to Choose Biologically Relevant Samples in a Small Region of Interest?

We conducted a field test at a potential Mars analog site to provide insight into planning for future robotic missions such as Mars 2020, where science operations must facilitate efficient choice of biologically relevant sampling locations. We compared two data acquisition and decision-making protocols currently used by Mars Science Laboratory: (1) a linear approach, where sites are examined as they are encountered and (2) a walkabout approach, in which the field site is first examined with remote rover instruments to gain an understanding of regional context followed by deployment of time- and power-intensive contact and sampling instruments on a smaller subset of locations. The walkabout method was advantageous in terms of both the time required to execute and a greater confidence in results and interpretations, leading to enhanced ability to tailor follow-on observations to better address key science and sampling goals. This advantage is directly linked to the walkabout method's ability to provide broad geological context earlier in the science analysis process. For Mars 2020, and specifically for small regions to be explored (e.g., <1 km2), we recommend that the walkabout approach be considered where possible, to provide early context and time for the science team to develop a coherent suite of hypotheses and robust ways to test them.

Extraformational sediment recycling on Mars.

Extraformational sediment recycling (old sedimentary rock to new sedimentary rock) is a fundamental aspect of Earth's geological record; tectonism exposes sedimentary rock, whereupon it is weathered and eroded to form new sediment that later becomes lithified. On Mars, tectonism has been minor, but two decades of orbiter instrument-based studies show that some sedimentary rocks previously buried to depths of kilometers have been exposed, by erosion, at the surface. Four locations in Gale crater, explored using the National Aeronautics and Space Administration's Curiosity rover, exhibit sedimentary lithoclasts in sedimentary rock: At Marias Pass, they are mudstone fragments in sandstone derived from strata below an erosional unconformity; at Bimbe, they are pebble-sized sandstone and, possibly, laminated, intraclast-bearing, chemical (calcium sulfate) sediment fragments in conglomerates; at Cooperstown, they are pebble-sized fragments of sandstone within coarse sandstone; at Dingo Gap, they are cobble-sized, stratified sandstone fragments in conglomerate derived from an immediately underlying sandstone. Mars orbiter images show lithified sediment fans at the termini of canyons that incise sedimentary rock in Gale crater; these, too, consist of recycled, extraformational sediment. The recycled sediments in Gale crater are compositionally immature, indicating the dominance of physical weathering processes during the second known cycle. The observations at Marias Pass indicate that sediment eroded and removed from craters such as Gale crater during the Martian Hesperian Period could have been recycled to form new rock elsewhere. Our results permit prediction that lithified deltaic sediments at the Perseverance (landing in 2021) and Rosalind Franklin (landing in 2023) rover field sites could contain extraformational recycled sediment.

Temperature alters solute transport in growth plate cartilage measured by in vivo multiphoton microscopy.

Solute delivery to avascular cartilaginous plates is critical to bone elongation, and impaired transport of nutrients and growth factors in cartilage matrix could underlie many skeletal abnormalities. Advances in imaging technology have revolutionized our ability to visualize growth plates in vivo, but quantitative methods are still needed. We developed analytical standards for measuring solute delivery, defined by amount and rate of intravenous tracer entry, in murine growth plates using multiphoton microscopy. We employed an acute temperature model because of its well-established impact on bone circulation and tested the hypothesis that solute delivery changes positively with limb temperature when body core and respiration are held constant (36 degrees C, 120 breaths/min). Tibial growth plates were surgically exposed in anesthetized 5-wk-old mice, and their hindlimbs were immersed in warm (36 degrees C) or cool (23 degrees C) saline (n = 6/group). After 30 min of thermal equilibration, we administered an intracardiac injection of fluorescein (50 microl, 0.5%) and captured sequentially timed growth plate images spanning 10 min at standardized depth. Absolute growth plate fluorescence was normalized to vascular concentrations for interanimal comparisons. As predicted, more fluorescein infiltrated growth plates at 36 degrees C, with standardized values nearly double those at 23 degrees C. Changing initial limb temperature did not alter baseline values, suggesting a sustained response period. These data validate the sensitivity of our system and have relevance to strategies for enhancing localized delivery of therapeutic agents to growth plates of children. Applications of this technique include assessment of solute transport in models of growth plate dysfunction, particularly chondrodysplasias with matrix irregularities.

A Diverse Array of Fluvial Depositional Systems in Arabia Terra: Evidence for mid-Noachian to Early Hesperian Rivers on Mars.

Branching to sinuous ridges systems, hundreds of kilometers in length and comprising layered strata, are present across much of Arabia Terra, Mars. These ridges are interpreted as depositional fluvial channels, now preserved as inverted topography. Here we use high-resolution image and topographic data sets to investigate the morphology of these depositional systems and show key examples of their relationships to associated fluvial landforms. The inverted channel systems likely comprise indurated conglomerate, sandstone, and mudstone bodies, which form a multistory channel stratigraphy. The channel systems intersect local basins and indurated sedimentary mounds, which we interpret as paleolake deposits. Some inverted channels are located within erosional valley networks, which have regional and local catchments. Inverted channels are typically found in downslope sections of valley networks, sometimes at the margins of basins, and numerous different transition morphologies are observed. These relationships indicate a complex history of erosion and deposition, possibly controlled by changes in water or sediment flux, or base-level variation. Other inverted channel systems have no clear preserved catchment, likely lost due to regional resurfacing of upland areas. Sediment may have been transported through Arabia Terra toward the dichotomy and stored in local and regional-scale basins. Regional stratigraphic relations suggest these systems were active between the mid-Noachian and early Hesperian. The morphology of these systems is supportive of an early Mars climate, which was characterized by prolonged precipitation and runoff.

Endothelial cells promote 3D invasion of GBM by IL-8-dependent induction of cancer stem cell properties.

Rapid growth and perivascular invasion are hallmarks of glioblastoma (GBM) that have been attributed to the presence of cancer stem-like cells (CSCs) and their association with the perivascular niche. However, the mechanisms by which the perivascular niche regulates GBM invasion and CSCs remain poorly understood due in part to a lack of relevant model systems. To simulate perivascular niche conditions and analyze consequential changes of GBM growth and invasion, patient-derived GBM spheroids were co-cultured with brain endothelial cells (ECs) in microfabricated collagen gels. Integrating these systems with 3D imaging and biochemical assays revealed that ECs increase GBM invasiveness and growth through interleukin-8 (IL-8)-mediated enrichment of CSCs. Blockade of IL-8 inhibited these effects in GBM-EC co-cultures, while IL-8 supplementation increased CSC-mediated growth and invasion in GBM-monocultures. Experiments in mice confirmed that ECs and IL-8 stimulate intracranial tumor growth and invasion in vivo. Collectively, perivascular niche conditions promote GBM growth and invasion by increasing CSC frequency, and IL-8 may be explored clinically to inhibit these interactions.

Valley network morphology in the greater Meridiani Planum region, Mars.

The Greater Meridiani Planum region on Mars is a key locale for a diverse range of fluvial landforms. Valley networks in this region have a range of geomorphologic styles that include negative relief, positive relief, or some combination of both along their lengths. Using high-resolution ~5-6 m/pixel orbital images in ArcGIS Desktop software, we mapped previously under-recognized fine-scale valley networks within the Greater Meridiani Planum region and recorded their geomorphic characteristics as feature attributes. The objectives in using the mapped features are to 1) document the full range of valley network morphologic types in the region, 2) document changes in morphologic types both on a regional scale and along the valley network segments, and 3) to use the mapped features along with other geologic information from previous studies to better understand landscape evolution in the Greater Meridiani Planum region.

Core-shell silica nanoparticles as fluorescent labels for nanomedicine.

Progress in biomedical imaging depends on the development of probes that combine low toxicity with high sensitivity, resolution, and stability. Toward that end, a new class of highly fluorescent core-shell silica nanoparticles with narrow size distributions and enhanced photostability, known as C dots, provide an appealing alternative to quantum dots. Here, C dots are evaluated with a particular emphasis on in-vivo applications in cancer biology. It is established that C dots are nontoxic at biologically relevant concentrations, and can be used in a broad range of imaging applications including intravital visualization of capillaries and macrophages, sentinel lymph node mapping, and peptide-mediated multicolor cell labeling for real-time imaging of tumor metastasis and tracking of injected bone marrow cells in mice. These results demonstrate that fluorescent core-shell silica nanoparticles represent a powerful novel imaging tool within the emerging field of nanomedicine.