Toward Understanding the Subcellular Distributions of Cholesterol and Sphingolipids Using High-Resolution NanoSIMS Imaging.

Characterizing the subcellular distributions of biomolecules of interest is a basic inquiry that helps inform on the potential roles of these molecules in biological functions. Presently, the functions of specific lipid species and cholesterol are not well understood, partially because cholesterol and lipid species of interest are difficult to image with high spatial resolution but without perturbing them. Because cholesterol and lipids are relatively small and their distributions are influenced by noncovalent interactions with other biomolecules, functionalizing them with relatively large labels that permit their detection may alter their distributions in membranes and between organelles. This challenge has been surmounted by exploiting rare stable isotopes as labels that may be metabolically incorporated into cholesterol and lipids without altering their chemical compositions, and the Cameca NanoSIMS 50 instrument's ability to image rare stable isotope labels with high spatial resolution. This Account covers the use of secondary ion mass spectrometry (SIMS) performed with a Cameca NanoSIMS 50 instrument for imaging cholesterol and sphingolipids in the membranes of mammalian cells. The NanoSIMS 50 detects monatomic and diatomic secondary ions ejected from the sample to map the elemental and isotopic composition at the surface of the sample with better than 50 nm lateral resolution and 5 nm depth resolution. Much effort has focused on using NanoSIMS imaging of rare isotope-labeled cholesterol and sphingolipids for testing the long-standing hypothesis that cholesterol and sphingolipids colocalize within distinct domains in the plasma membrane. By using a NanoSIMS 50 to image rare isotope-labeled cholesterol and sphingolipids in parallel with affinity-labeled proteins of interest, a hypothesis regarding the colocalization of specific membrane proteins with cholesterol and sphingolipids in distinct plasma membrane domains has been tested. NanoSIMS performed in a depth profiling mode has enabled imaging the intracellular distributions of cholesterol and sphingolipids. Important progress has also been made in developing a computational depth correction strategy for constructing more accurate three-dimensional (3D) NanoSIMS depth profiling images of intracellular component distribution without requiring additional measurements with complementary techniques or signal collection. This Account provides an overview of this exciting progress, focusing on the studies from our laboratory that shifted understanding of plasma membrane organization, and the development of enabling tools for visualizing intracellular lipids.

Depth Correction of 3D NanoSIMS Images Shows Intracellular Lipid and Cholesterol Distributions while Capturing the Effects of Differential Sputter Rate.

Knowledge of the distributions of drugs, metabolites, and drug carriers within cells is a prerequisite for the development of effective disease treatments. Intracellular component distribution may be imaged with high sensitivity and spatial resolution by using a NanoSIMS in the depth profiling mode. Depth correction strategies that capture the effects of differential sputtering without requiring additional measurements could enable producing accurate 3D NanoSIMS depth profiling images of intracellular component distributions. Here we describe an approach for depth correcting 3D NanoSIMS depth profiling images of cells that accounts for differential sputter rates. Our approach uses the secondary ion and secondary electron depth profiling images to reconstruct the cell's morphology at every raster plane. These cell morphology reconstructions are used to adjust the z-positions and heights of the voxels in the component-specific 3D NanoSIMS images. We validated this strategy using AFM topography data and reconstructions created from depth profiling images acquired with focused ion beam-secondary electron microscopy. Good agreement was found for the shapes and relative heights of the reconstructed morphologies. Application of this depth correction strategy to 3D NanoSIMS depth profiling images of a metabolically labeled cell better resolved the transport vesicles, organelles, and organellar membranes containing 18O-cholesterol and 15N-sphingolipids. Accurate 3D NanoSIMS images of intracellular component distributions may now be produced without requiring correlated analyses with separate instruments or the assumption of a constant sputter rate. This will allow visualization of the subcellular distributions of lipids, metabolites, drugs, and nanoparticles in 3D, information pivotal to understanding and treating disease.

Identification of the Differentiation Stages of Living Cells from the Six Most Immature Murine Hematopoietic Cell Populations by Multivariate Analysis of Single-Cell Raman Spectra.

Efforts to expand hematopoietic stem and progenitor cells (HSPCs) in vitro are motivated by their use in the treatment of leukemias and other blood and immune system diseases. The combinations of extrinsic cues within the hematopoietic stem cell (HSC) niche that lead to HSC fate decisions remain unknown. New noninvasive and location-specific techniques are needed to enable identification of the differentiation stages of individual hematopoietic cells on biomaterial microarray screening platforms that minimize the usage of rare HSCs. Here, we show that a combination of Raman microspectroscopy and partial least-squares discriminant analysis (PLS-DA) enables the location-specific identification of individual living cells from the six most immature hematopoietic cell populations, HSC, multipotent progenitor (MPP)-1, MPP-2, MPP-3, common myeloid progenitor, and common lymphoid progenitor. Better than 90% accuracy was achieved. We show that the accuracy of this differentiation stage identification was based on spectral features associated with cell biochemistries. This work establishes that PLS-DA can capture the subtle spectral variations between as many as six closely related cell populations in the presence of potentially significant within-population spectral variation. This noninvasive approach can be used to screen HSC fate decisions elicited by extrinsic cues within biomaterial microarray screening platforms.

Depth correction of 3D NanoSIMS images using secondary electron pixel intensities.

Strategies that do not require additional characterization to be performed on the sample or the collection of additional secondary ion signals are needed to depth correct 3D SIMS images of cells. Here, we develop a depth correction strategy that uses the pixel intensities in the secondary electron images acquired during negative-ion NanoSIMS depth profiling to reconstruct the sample morphology. This morphology reconstruction was then used to depth correct the 3D SIMS images that show the components of interest in the sample. As a proof of concept, we applied this approach to NanoSIMS depth profiling data that show the 15N-enrichment and 18O-enrichment from 15N-sphingolipids and 18O-cholesterol, respectively, within a metabolically labeled Madin-Darby canine kidney cell. Comparison of the cell morphology reconstruction to the secondary electron images collected with the NanoSIMS revealed that the assumption of a constant sputter rate produced small inaccuracies in sample morphology after approximately 0.66 µm of material was sputtered from the cell. Nonetheless, the resulting 3D renderings of the lipid-specific isotope enrichments better matched the shapes and positions of the subcellular compartments that contained 15N-sphingolipids and 18O-cholesterol than the uncorrected 3D SIMS images. This depth correction of the 3D SIMS images also facilitated the detection of spherical cholesterol-rich compartments that were surrounded by membranes containing cholesterol and sphingolipids. Thus, we expect this approach will facilitate identifying the subcellular structures that are enriched with biomolecules of interest in 3D SIMS images while eliminating the need for correlated analyses or additional secondary ion signals for the depth correction of 3D NanoSIMS images.

Development of an inexpensive Raman-compatible substrate for the construction of a microarray screening platform.

Biomaterial microarrays are being developed to facilitate identifying the extrinsic cues that elicit stem cell fate decisions to self-renew, differentiate and remain quiescent. Raman microspectroscopy, often combined with multivariate analysis techniques such as partial least square-discriminant analysis (PLS-DA), could enable the non-invasive identification of stem cell fate decisions made in response to extrinsic cues presented at specific locations on these microarrays. Because existing biomaterial microarrays are not compatible with Raman microspectroscopy, here, we develop an inexpensive substrate that is compatible with both single-cell Raman spectroscopy and the chemistries that are often used for biomaterial microarray fabrication. Standard deposition techniques were used to fabricate a custom Raman-compatible substrate that supports microarray construction. We validated that spectra from living cells on functionalized polyacrylamide (PA) gels attached to the custom Raman-compatible substrate are comparable to spectra acquired from a more expensive commercially available substrate. We also showed that the spectra acquired from individual living cells on functionalized PA gels attached to our custom substrates were of sufficient quality to enable accurate identification of cell phenotypes using PLS-DA models of the cell spectra. We demonstrated this by using cells from laboratory lines (CHO and transfected CHO cells) as well as adult stem cells that were freshly isolated from mice (long-term and short-term hematopoietic stem cells). The custom Raman-compatible substrate reported herein may be used as an inexpensive substrate for constructing biomaterial microarrays that enable the use of Raman microspectroscopy to non-invasively identify the fate decisions of stem cells in response to extrinsic cues.

Exploring the maturation of a monocytic cell line using self-organizing maps of single-cell Raman spectra.

Phorbol myristate acetate (PMA)-differentiated THP-1 cells are routinely used in lieu of primary macrophages to study macrophage polarization during host-pathogen interactions and disease progression. The phenotypes of THP-1 macrophages are influenced by the level and duration of PMA stimulation and possibly also by the presence of adhesion factors. Here, we use self-organizing maps (SOMs) of single-cell Raman spectra to probe the effects of PMA stimulation conditions and adhesion factors on THP-1 cell differentiation. Raman spectra encoding for biochemical composition were acquired from individual cells on substrates coated with fibronectin or poly-l-lysine before and after stimulation with 20 or 200 nM PMA for two different time intervals. SOMs constructed from these spectra showed the extent of spectral dissimilarity between different chronological cell populations. For all conditions, the SOMs indicated that the spectra acquired from cells after three-day treatment had diverged from those of untreated cells. The SOMs also showed that the higher PMA concentration produced both fully and partially differentiated cells for both adhesion factors after three days, whereas the outcome of stimulation for three days with the lower PMA concentration depended on the adhesion factor. On poly-l-lysine, treatment with 20 nM PMA for three days induced an intermediate stage of differentiation, but the same treatment produced partially and fully differentiated cells when applied to THP-1 cells on fibronectin. These results are consistent with the modulation of the transition of THP-1 monocytes into macrophage-like cells by integrin-binding interactions. Furthermore, differences in culture and stimulation conditions may confound comparison of results from separate studies.

Measurement of Absolute Concentration at the Subcellular Scale.

The concentration of a pharmaceutical drug or bioactive metabolite within the target organelle influences the effects elicited by the drug or metabolite. Although the relative concentrations of many compounds of interest within subcellular compartments have been measured, measurements of absolute concentrations in the organelle remain elusive. In this Perspective, we discuss a significant advance in using nano secondary ion mass spectrometry (nanoSIMS) to measure the absolute concentration of a 13C-labeled metabolite within secretory vesicles, as reported by Thomen et al. in the April issue of ACS Nano.

High-Resolution Secondary Ion Mass Spectrometry Analysis of Cell Membranes.

This Feature describes the use a Cameca NanoSIMS instrument for directly imaging specific lipid and protein species in the plasma membranes of mammalian cells with approximately 100 nm-lateral resolution and discusses how these analyses have already begun to transform fundamental concepts in the field of membrane biology. Secondary ion generation is discussed with emphasis on the constraints that affect the detection and identification of membrane components, and then the sample preparation methodologies and data analysis strategies that address these constraints are described.

Tracing Hematopoietic Progenitor Cell Neutrophilic Differentiation via Raman Spectroscopy.

A major challenge to experimental studies and therapeutic uses of hematopoietic stem cells (HSC) is the limited options for analytical tools that can reliably resolve functional differences in heterogeneous HSC subpopulations at the single cell level. Currently available methods require the use of external labels and/or separate clonogenic and transplantation assays to identify bona fide stem cells, necessitating the harvest of bulk cell populations and long incubation times that obscure how individual HSCs dynamically respond to exogenous and endogenous stimuli. In this study, we employ Raman spectroscopy to noninvasively resolve the dynamics of individual differentiating hematopoietic progenitor cells during the course of neutrophilic differentiation. We collected Raman peaks of individual cells daily over the course of 14-day neutrophilic differentiation. Principal component analysis (PCA) of the Raman peaks revealed spectral differences between individual cells during differentiation that were strongly correlated with changes in the nucleus shape and surface antigen expression, the primary traditional means of monitoring neutrophilic differentiation. Additionally, our results were consistently reproducible in independent rounds of neutrophilic differentiation, as demonstrated by our partial least-squares discriminant analysis (PLS-DA) of the Raman spectral information that predicted the degree of neutrophilic differentiation with high sensitivity and specificity. Our findings highlight the utility and reliability of Raman spectroscopy as a robust molecular imaging tool to monitor the kinetics of HSC differentiation patterns.

Visualizing Intrapopulation Hematopoietic Cell Heterogeneity with Self-Organizing Maps of SIMS Data.

Characterization of the heterogeneity within stem cell populations, which affects their differentiation potential, is necessary for the design of artificial cultures for stem cell expansion. In this study, we assessed whether self-organizing maps (SOMs) of single-cell time-of-flight secondary ion mass spectrometry (TOF-SIMS) data provide insight into the spectral, and thus the related functional heterogeneity between and within three hematopoietic cell populations. SOMs were created of TOF-SIMS data from individual hematopoietic stem and progenitor cells (HSPCs), lineage-committed common lymphoid progenitors (CLPs), and fully differentiated B cells that had been isolated from murine bone marrow via conventional flow cytometry. The positions of these cells on the SOMs and the spectral variation between adjacent map units, shown on the corresponding unified distance matrix (U-matrix), indicated the CLPs exhibited the highest intrapopulation spectral variation, regardless of the age of the donor mice. SOMs of HSPCs, CLPs, and B cells isolated from young and old mice using the same surface antigen profiles revealed the HSPCs exhibited the most age-related spectral variation, whereas B cells exhibited the least. These results demonstrate that SOMs of single-cell spectra enable characterizing the heterogeneity between and within cell populations that lie along distinct differentiation pathways.

Cholesterol is enriched in the sphingolipid patches on the substrate near nonpolarized MDCK cells, but not in the sphingolipid domains in their plasma membranes.

Information about the distributions of cholesterol and sphingolipids within the plasma membranes of mammalian cells provides insight into the roles of these molecules in membrane function. In this report, high-resolution secondary ion mass spectrometry was used to image the distributions of metabolically incorporated rare isotope-labeled sphingolipids and cholesterol on the surfaces of nonpolarized epithelial cells. Sphingolipid domains that were not enriched with cholesterol were detected in the plasma membranes of subconfluent Madin-Darby canine kidney cells. Surprisingly, cholesterol-enriched sphingolipid patches were observed on the substrate adjacent to these cells. Based on the shapes of these cholesterol-enriched sphingolipid patches on the substrate and their proximity to cellular projections, we hypothesize that they are deposits of membranous particles released by the cell.

Observation of endoplasmic reticulum tubules via TOF-SIMS tandem mass spectrometry imaging of transfected cells.

Advances in three-dimensional secondary ion mass spectrometry (SIMS) imaging have enabled visualizing the subcellular distributions of various lipid species within individual cells. However, the difficulty of locating organelles using SIMS limits efforts to study their lipid compositions. Here, the authors have assessed whether endoplasmic reticulum (ER)-Tracker Blue White DPX®, which is a commercially available stain for visualizing the endoplasmic reticulum using fluorescence microscopy, produces distinctive ions that can be used to locate the endoplasmic reticulum using SIMS. Time-of-flight-SIMS tandem mass spectrometry (MS2) imaging was used to identify positively and negatively charged ions produced by the ER-Tracker stain. Then, these ions were used to localize the stain and thus the endoplasmic reticulum, within individual human embryonic kidney cells that contained higher numbers of endoplasmic reticulum-plasma membrane junctions on their surfaces. By performing MS2 imaging of selected ions in parallel with the precursor ion (MS1) imaging, the authors detected a chemical interference native to the cell at the same nominal mass as the pentafluorophenyl fragment from the ER-Tracker stain. Nonetheless, the fluorine secondary ions produced by the ER-Tracker stain provided a distinctive signal that enabled locating the endoplasmic reticulum using SIMS. This simple strategy for visualizing the endoplasmic reticulum in individual cells using SIMS could be combined with existing SIMS methodologies for imaging intracellular lipid distribution and to study the lipid composition within the endoplasmic reticulum.

Three-dimensional imaging of cholesterol and sphingolipids within a Madin-Darby canine kidney cell.

Metabolic stable isotope incorporation and secondary ion mass spectrometry (SIMS) depth profiling performed on a Cameca NanoSIMS 50 were used to image the (18)O-cholesterol and (15)N-sphingolipid distributions within a portion of a Madin-Darby canine kidney (MDCK) cell. Three-dimensional representations of the component-specific isotope distributions show clearly defined regions of (18)O-cholesterol and (15)N-sphingolipid enrichment that seem to be separate subcellular compartments. The low levels of nitrogen-containing secondary ions detected at the (18)O-enriched regions suggest that these (18)O-cholesterol-rich structures may be lipid droplets, which have a core consisting of cholesterol esters and triacylglycerides.

The importance of selecting a proper biological milieu for protein corona analysis in vitro: Human plasma versus human serum.

Nanoparticle (NP) exposure to biological fluids in the body results in protein binding to the NP surface, which forms a protein coating that is called the "protein corona". To simplify studies of protein-NP interactions and protein corona formation, NPs are incubated with biological solutions, such as human serum or human plasma, and the effects of this exposure are characterized in vitro. Yet, how NP exposure to these two different biological milieus affects protein corona composition and cell response has not been investigated. Here, we explore the differences between the protein coronas that form when NPs are incubated in human serum versus human plasma. NP characterization indicated that NPs that were exposed to human plasma had higher amounts of proteins bound to their surfaces, and were slightly larger in size than those exposed to human serum. In addition, significant differences in corona composition were also detected with gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry, where a higher fraction of coagulation proteins and complement factors were found on the plasma-exposed NPs. Flow cytometry and confocal microscopy showed that the uptake of plasma-exposed NPs was higher than that of serum-exposed NPs by RAW 264.7 macrophage immune cells, but not by NIH 3T3 fibroblast cells. This difference is likely due to the elevated amounts of opsonins, such as fibrinogen, on the surfaces of the NPs exposed to plasma, but not serum, because these components trigger NP internalization by immune cells. As the human plasma better mimics the composition of the in vivo environment, namely blood, in vitro protein corona studies should employ human plasma, and not human serum, so the biological phenomena that is observed is more similar to that occurring in vivo.

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It.

Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with cholesterol or hemagglutinin. The alternate views of plasma membrane organization suggested by these findings are discussed.

Impact of protein pre-coating on the protein corona composition and nanoparticle cellular uptake.

Nanoparticles (NPs) are functionalized with targeting ligands to enable selectively delivering drugs to desired locations in the body. When these functionalized NPs enter the blood stream, plasma proteins bind to their surfaces, forming a protein corona that affects NP uptake and targeting efficiency. To address this problem, new strategies for directing the formation of a protein corona that has targeting capabilities are emerging. Here, we have investigated the feasibility of directing corona composition to promote targeted NP uptake by specific types of cells. We used the well-characterized process of opsonin-induced phagocytosis by macrophages as a simplified model of corona-mediated NP uptake by a desired cell type. We demonstrate that pre-coating silica NPs with gamma-globulins (γ-globulins) produced a protein corona that was enriched with opsonins, such as immunoglobulins. Although immunoglobulins are ligands that bind to receptors on macrophages and elicit phagocytois, the opsonin-rich protein corona did not increase NP uptake by macrophage RAW 264.7 cells. Immunolabeling experiments indicated that the binding of opsonins to their target cell surface receptors was impeded by other proteins in the corona. Thus, corona-mediated NP targeting strategies must optimize both the recruitment of the desired plasma proteins as well as their accessibility and orientation in the corona layer.

Identifying States along the Hematopoietic Stem Cell Differentiation Hierarchy with Single Cell Specificity via Raman Spectroscopy.

A major challenge for expanding specific types of hematopoietic cells ex vivo for the treatment of blood cell pathologies is identifying the combinations of cellular and matrix cues that direct hematopoietic stem cells (HSC) to self-renew or differentiate into cell populations ex vivo. Microscale screening platforms enable minimizing the number of rare HSCs required to screen the effects of numerous cues on HSC fate decisions. These platforms create a strong demand for label-free methods that accurately identify the fate decisions of individual hematopoietic cells at specific locations on the platform. We demonstrate the capacity to identify discrete cells along the HSC differentiation hierarchy via multivariate analysis of Raman spectra. Notably, cell state identification is accurate for individual cells and independent of the biophysical properties of the functionalized polyacrylamide gels upon which these cells are cultured. We report partial least-squares discriminant analysis (PLS-DA) models of single cell Raman spectra enable identifying four dissimilar hematopoietic cell populations across the HSC lineage specification. Successful discrimination was obtained for a population enriched for long-term repopulating HSCs (LT-HSCs) versus their more differentiated progeny, including closely related short-term repopulating HSCs (ST-HSCs) and fully differentiated lymphoid (B cells) and myeloid (granulocytes) cells. The lineage-specific differentiation states of cells from these four subpopulations were accurately identified independent of the stiffness of the underlying biomaterial substrate, indicating subtle spectral variations that discriminated these populations were not masked by features from the culture substrate. This approach enables identifying the lineage-specific differentiation stages of hematopoietic cells on biomaterial substrates of differing composition and may facilitate correlating hematopoietic cell fate decisions with the extrinsic cues that elicited them.

Hemagglutinin clusters in the plasma membrane are not enriched with cholesterol and sphingolipids.

The clusters of the influenza envelope protein, hemagglutinin, within the plasma membrane are hypothesized to be enriched with cholesterol and sphingolipids. Here, we directly tested this hypothesis by using high-resolution secondary ion mass spectrometry to image the distributions of antibody-labeled hemagglutinin and isotope-labeled cholesterol and sphingolipids in the plasma membranes of fibroblast cells that stably express hemagglutinin. We found that the hemagglutinin clusters were neither enriched with cholesterol nor colocalized with sphingolipid domains. Thus, hemagglutinin clustering and localization in the plasma membrane is not controlled by cohesive interactions between hemagglutinin and liquid-ordered domains enriched with cholesterol and sphingolipids, or from specific binding interactions between hemagglutinin, cholesterol, and/or the majority of sphingolipid species in the plasma membrane.

Secondary ion mass spectrometry and Raman spectroscopy for tissue engineering applications.

Identifying the matrix properties that permit directing stem cell fate is crucial for expanding desired cell lineages ex vivo for disease treatment. Such efforts require knowledge of matrix surface chemistry and the cell responses they elicit. Recent progress in analyzing biomaterial composition and identifying cell phenotype with two label-free chemical imaging techniques, TOF-SIMS and Raman spectroscopy are presented. TOF-SIMS is becoming indispensable for the surface characterization of biomaterial scaffolds. Developments in TOF-SIMS data analysis enable correlating surface chemistry with biological response. Advances in the interpretation of Raman spectra permit identifying the fate decisions of individual, living cells with location specificity. Here we highlight this progress and discuss further improvements that would facilitate efforts to develop artificial scaffolds for tissue regeneration.