Mass spectrometry imaging of small molecules from live cells and tissues using nanomaterials

We review recently developed methods for analyzing live cells and tissues in ambient conditions without the use of harsh chemical fixation or physical freezing and drying. The first method is based on laser ablation in atmospheric pressure assisted by atmospheric pressure plasma and nanomaterials such as nanoparticles and graphene to enhance laser ablation. The second method is based on secondary ion mass spectrometry imaging of live cells in solution capped with single-layer graphene to preserve intact and hydrated biological samples even under ultrahigh vacuum for secondary ion mass spectrometry bio-imaging in solution with subcellular spatial resolution. Mass spectrometry imaging of small molecules from live cells and tissues can provide an innovative molecular imaging methodology for several biomedical and material research applications.     

Fabrication of quantum dot-lectin conjugates as novel fluorescent probes for microscopic and flow cytometric identification of leukemia cells from normal lymphocytes

The present study describes a synthesis of QD-lectin conjugates and their application for identification of leukaemia cells from normal lymphocytes using fluorescent confocal microscopy and flow cytometry. The results are compared with commercially available FITC-lectin.     

Long-term exposure to CdTe quantum dots causes functional impairments in live cells

Several studies suggested that the cytotoxic effects of quantum dots (QDs) may be mediated by cadmium ions (Cd2+) released from the QDs cores. The objective of this work was to assess the intracellular Cd2+ concentration in human breast cancer MCF-7 cells treated with cadmium telluride (CdTe) and core/shell cadmium selenide/zinc sulfide (CdSe/ZnS) nanoparticles capped with mercaptopropionic acid (MPA), cysteamine (Cys), or N-acetylcysteine (NAC) conjugated to cysteamine. The Cd2+ concentration determined by a Cd2+-specific cellular assay was below the assay detection limit (<5 nM) in cells treated with CdSe/ZnS QDs, while in cells incubated with CdTe QDs, it ranged from approximately 30 to 150 nM, depending on the capping molecule. A cell viability assay revealed that CdSe/ZnS QDs were nontoxic, whereas the CdTe QDs were cytotoxic. However, for the various CdTe QD samples, there was no dose-dependent correlation between cell viability and intracellular [Cd2+], implying that their cytotoxicity cannot be attributed solely to the toxic effect of free Cd2+. Confocal laser scanning microscopy of CdTe QDs-treated cells imaged with organelle-specific dyes revealed significant lysosomal damage attributable to the presence of Cd2+ and of reactive oxygen species (ROS), which can be formed via Cd2+-specific cellular pathways and/or via CdTe-triggered photoxidative processes involving singlet oxygen or electron transfer from excited QDs to oxygen. In summary, CdTe QDs induce cell death via mechanisms involving both Cd2+ and ROS accompanied by lysosomal enlargement and intracellular redistribution.     

Conjugation of quantum dots with graphene for fluorescence imaging of live cells

It is difficult to achieve fluorescent graphene-quantum dots (QDs) conjugation because graphene quenches the fluorescence of the QDs. In the present study, the conjugation of graphene (reduced graphene oxide, RGO) with QDs via a bridge of bovine serum albumin (BSA) provides a novel highly fluorescent nano probe for the first time. BSA capped QDs are firmly grafted onto polyethylenimine (PEI)/poly(sodium 4-styrenesulfonate) (PSS) coated RGO (graphene-QDs) via electrostatic layer by layer assembly. The strong luminescence of the graphene-QDs provides a potential for non-invasive optical in vitro imaging. The graphene-QDs are used for in vitro imaging of live human carcinoma (Hela) cells. Graphene-QDs could be readily up-taken by Hela cells in the absence of specific targeting molecules, e.g., antibodies or folic acid, and no in vitro cytotoxicity is observed at 360 μg mL(-1) of the graphene-QDs. The results for the imaging of live cells indicated that the cell-penetrating graphene-QDs could be a promising nano probe for intracellular imaging and therapeutic applications.     

Dynamic surface tensiometry of tissues using Langmuir films

Langmuir monolayers are useful models of biomembranes as they allow simulation of biological conditions and rigorous thermodynamic analysis. This technique was used to characterize tissues at body temperature for the first time in our study. The organs studied include liver, kidney, stomach, testis, heart and brain from goat and certain human cancerous as well as their corresponding normal biopsies to reveal the potential of the tissue monolayer technique. Monolayers were formed on the surface of deionized water by spreading monolayer amounts of the tissue homogenates. The parameters calculated were minimum surface tension, relative lift off area, relative limiting area, compressibility and hysteresis area. Our results reveal that the parameters can differentiate between tissues obtained from different organs and were statistically significant using one-way ANOVA and Newman Keul's test (P<0.05). For example goat's stomach tissue had the lowest hysteresis area (DeltaG) value (27.6 microJ) whereas brain DeltaG value was nine folds higher than stomach value. Brain had the lowest minimum surface tension of 30.3+/-1.0 mN/m whereas stomach had a value of 40.5+/-0. 2 mN/m. Interestingly, the DeltaG values of human normal neck and esophageal tissues were 3.4 and 3.2 folds greater than that of their respective cancer tissues whereas the DeltaG values of vulval and breast cancer tissues were 4.6 and 4 folds greater than that of their respective normal tissues. While the gammamin values of neck cancer tissue showed 95% increase from normal tissue values, those of vulval and breast cancer tissues were 46 and 50% less compared to their respective normal tissue values. Though all the surface tensiometric parameters showed significant changes, minimum surface tension and hysteresis area were the most sensitive indicators of tissue types and diseased states. Further, the effects of therapeutics could also be monitored by this technique. This is evidenced by the post-radiotherapy tissue isotherms of neck and vulval cancers, where clinical radio-sensitivity was associated with a shift in the tensiometry towards their respective normal isotherms. The small sample amounts required, precision of the technique, very low within group variability, organ specificity and sensitivity to detect changes in diseased states make it a promising tool for prognostic evaluation of diseased states and monitoring effects of therapeutics. Further research is warranted in this promising and hitherto unexplored field of tissue tensiometry.     

Bioimaging of cells and tissues using accelerator-based sources

A variety of techniques exist that provide chemical information in the form of a spatially resolved image: electron microprobe analysis, nuclear microprobe analysis, synchrotron radiation microprobe analysis, secondary ion mass spectrometry, and confocal fluorescence microscopy. Linear (LINAC) and circular (synchrotrons) particle accelerators have been constructed worldwide to provide to the scientific community unprecedented analytical performances. Now, these facilities match at least one of the three analytical features required for the biological field: (1) a sufficient spatial resolution for single cell (< 1 μm) or tissue (<1 mm) analyses, (2) a temporal resolution to follow molecular dynamics, and (3) a sensitivity in the micromolar to nanomolar range, thus allowing true investigations on biological dynamics. Third-generation synchrotrons now offer the opportunity of bioanalytical measurements at nanometer resolutions with incredible sensitivity. Linear accelerators are more specialized in their physical features but may exceed synchrotron performances. All these techniques have become irreplaceable tools for developing knowledge in biology. This review highlights the pros and cons of the most popular techniques that have been implemented on accelerator-based sources to address analytical issues on biological specimens. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Efficient silicon surface and cluster modeling using quantum capping potentials

A one-electron, silicon quantum capping potential for use in capping the dangling bonds formed by artificially limiting silicon clusters or surfaces is developed. The quantum capping potentials are general and can be used directly in any computational package that can handle effective core potentials. For silicon clusters and silicon surface models, we compared the results of traditional hydrogen atom capping with those obtained from capping with quantum capping potentials. The results clearly show that cluster and surface models capped with quantum capping potentials have ionization potentials, electron affinities, and highest occupied molecular orbital-lowest unoccupied molecular orbital gaps that are in very good agreement with those of larger systems. The silicon quantum capping potentials should be applied in cases where one wishes to model processes involving charges or low-energy excitations in silicon clusters and surfaces consisting of more than ca. 150 atoms.     

Selective labeling of the endoplasmic reticulum in live cells with silicon quantum dots

A simple and novel approach was developed to obtain water-dispersible silicon quantum dots (Si-QDs) of low toxicity that were able to selectively label the endoplasmic reticulum (ER) in live cells. A block copolymer (Pluronic F127) was used to coat the surface of Si-QDs. Si-QDs form aggregates with diameters of 20-40 nm and show an outstanding optical stability upon UV irradiation. Our F127-treated Si-QDs would be a powerful tool for long-term real-time observation of the ER in live cells.     

Study on molecular interactions between proteins on live cell membranes using quantum dot-based fluorescence resonance energy transfer

Mouse anti-human CD71 monoclonal antibody (anti-CD71) was conjugated with red quantum dots (QDs; 5.3 nm, emission wavelength λ _em = 614 nm) and used to label HeLa cells successfully. Then green QD-labeled goat anti-mouse immunoglobulin G (IgG; the size of the green QDs was 2.2 nm; λ _em = 544 nm) was added to bind the red-QD-conjugated anti-CD71 on the cell surface by immunoreactions. Such interaction between anti-CD71 and IgG lasted 4 min and was observed from the fluorescence spectra: the fluorescence intensity of the “red” peak at 614 nm increased by 32%; meanwhile that of the “green” one at 544 nm decreased by 55%. The ratio of the fluorescence intensities (I _544 nm/I _614 nm) decreased from 0.5 to 0.2. The fluorescence spectra as well as cell imaging showed that fluorescence resonance energy transfer took place between these two kinds of QDs on the HeLa cells through interactions between the primary antibody and the secondary antibody.     

Profiling and semiquantitative analysis of the cell surface proteome in human mesenchymal stem cells

Mulitpotent mesenchymal stem cells (MSCs) derived from human bone marrow are promising candidates for the development of cell therapeutic strategies. MSC surface protein profiles provide novel biological knowledge concerning the proliferation and differentiation of these cells, including the potential for identifying therapeutic targets. Basic fibroblast growth factor (bFGF) affects cell surface proteins, which are associated with increased growth rate, differentiation potential, as well as morphological changes of MSCs in vitro. Cell surface proteins were isolated using a biotinylation-mediated method and identified using a combination of one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis and mass spectrometry. The resulting gel lines were cut into 20 bands and digested with trypsin. Each tryptic fragment was analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry. Proteins were identified using the Mascot search program and the International Protein Index human database. Noble MSC surface proteins (n?=?1,001) were identified from cells cultured either with (n?=?857) or without (n?=?667) bFGF-containing medium in three independent experiments. The proteins were classified using FatiGO to elucidate their function. We also confirmed the proteomics results using Western blotting and immunofluorescence microscopic analysis. The nature of the proteins identified makes it clear that MSCs express a wide variety of signaling molecules, including those related to cell differentiation. Among the latter proteins, four Ras-related Rab proteins, laminin-R, and three 14-3-3 proteins that were fractionated from MSCs cultured on bFGF-containing medium are implicated in bFGF-induced signal transduction of MSCs. Consequently, these finding provide insight into the understanding of the surface proteome of human MSCs.

Tracking single lipase molecules on a trimyristin substrate surface using quantum dots

The mobility of single lipase molecules has been analyzed using single molecule tracking on a trimyristin substrate surface. This was achieved by conjugating lipases to quantum dots and imaging on spin-coated trimyristin surfaces by means of confocal laser scanning microscopy. Image series of single lipase molecules were collected, and the diffusion coefficient was quantified by analyzing the mean square displacement of the calculated trajectories. During no-flow conditions, the lipase diffusion coefficient was (8.0+/-5.0)x10(-10) cm2/s. The trajectories had a "bead on a string" appearance, with the lipase molecule restricted in certain regions of the surface and then migrating to another region where the restricted diffusion continued. This gave rise to clusters in the trajectories. When a flow was applied to the system, the total distance and average step length between the clusters increased, but the restricted diffusion in the cluster regions was unaffected. This can be explained by the lipase operating in two different modes on the surface. In the cluster regions, the lipase is likely oriented with the active site toward the surface and hydrolyzes the substrate. Between these regions, a diffusion process is proposed where the lipase is in contact with the surface but affected by the external flow.     

Fluorogenic assay and live cell imaging of HIV-1 protease activity using acid-stable quantum dot-peptide complex

A novel QD-peptide complex for detecting HIV-1 protease activity was prepared from simple one step electrostatic interaction. Fluorescence recovery of the pre-quenched QD through fluorescence resonance energy transfer allowed for in vitro assay and live cell imaging of the protease activity in HIV-1 transfected cells, proving the potential for cell-based protease inhibitor screening.     

Organelle-specific zinc detection using zinpyr-labeled fusion proteins in live cells

A protein labeling approach is employed for the localization of a zinc-responsive fluorescent probe in the mitochondria and in the Golgi apparatus of living cells. ZP1, a zinc sensor of the Zinpyr family, was functionalized with a benzylguanine moiety and thus converted into a substrate (ZP1BG) for the human DNA repair enzyme alkylguaninetransferase (AGT or SNAP-Tag). The labeling reaction of purified glutathione S-transferase tagged AGT with ZP1BG and the zinc response of the resulting protein-bound sensor were confirmed in vitro. The new detection system, which combines a protein labeling methodology with a zinc fluorescent sensor, was tested in live HeLa cells expressing AGT in specific locations. The enzyme was genetically fused to site-directing proteins that anchor the probe onto targeted organelles. Localization of the zinc sensors in the Golgi apparatus and in the mitochondria was demonstrated by fluorescence microscopy. The protein-bound fluorescence detection system is zinc-responsive in living cells.     

Imaging live cells grown on a three dimensional collagen matrix using Raman microspectroscopy

Three dimensional collagen gels have been used as matrices for the imaging of live cells by Raman spectroscopy. The study is conducted on a human lung adenocarcinoma (A549) and a spontaneously immortalized human epithelial keratinocyte (HaCaT) cell line. The lateral resolution of the system has been estimated to be <1.5 μm making it possible to access the subcellular organization. Using K-means clustering analysis, it is shown that the different subcellular compartments of individual cells can be identified and differentiated. The biochemical specificity of the information contained in the Raman spectra allows the visualization of differences in the molecular signature of the different sub-cellular structures. Furthermore, to enhance the chemical information obtained from the spectra, principal component analysis has been employed, allowing the identification of spectral windows with a high variability. The comparison between the loadings calculated and spectra from pure biochemical compounds enables the correlation of the variations observed with the molecular content of the different cellular compartments.     

Profiling and imaging of tissues by imaging ion mobility-mass spectrometry

Molecular profiling and imaging mass spectrometry (IMS) of tissues can often result in complex spectra that are difficult to interpret without additional information about specific signals. This report describes increasing data dimensionality in IMS by combining two-dimensional separations at each spatial location on the basis of imaging ion mobility-mass spectrometry (IM-MS). Analyte ions are separated on the basis of both ion-neutral collision cross section and m/z, which provides rapid separation of isobaric, but structurally distinct ions. The advantages of imaging using ion mobility prior to MS analysis are demonstrated for profiling of human glioma and selective lipid imaging from rat brain.     

Synthetic studies on cell surface glycans 3: Branching pentasaccharides of glycoprotein

Synthetic routes for the branching pentasaccharides 4 and 5 of glycoproteins are described in a regio- and stereo- controlled way.     

A metabolically engineered spin-labeling approach for studying glycans on cells

Metabolic glycan engineering (MGE) coupled with nitroxide spin-labeling (SL) was utilized to investigate the heterogeneous environment of cell surface glycans in select cancer and normal cells. This approach exploited the incorporation of azides into cell surface glycans followed by a click reaction with a new nitroxide spin label. Both sialic acid and N-acetylglucosamine (GlcNAc) were targeted for spin labelling. Although each of these moieties experiences a diverse and heterogeneous glycan environment, their EPR spectra and hence mobility are both characterized as a linear combination of two distinct spectra where one component reflects a highly mobile or uncrowded micro-environment with the second component reflecting more restricted motion, reflective of increased crowding and packing within the glycocalyx. What differs among the spectra of the targeted glycans is the relative percentage of each component, with sialic acid moieties experiencing on average an ∼80% less crowded environment, where conversely GlcNAc/GalNAz labeled sites reported on average a ∼50% more crowded environment. These distinct environments are consistent with the organization of sugar moieties within cellular glycans where some residues occur close to the cell membrane/protein backbone (i.e. more restricted) and others are more terminal in the glycan (i.e. more mobile). Strikingly, different cell lines displayed varied relative populations of these two components, suggesting distinctive glycan packing, organization, and composition of different cells. This work demonstrates the capability of SDSL EPR to be a broadly useful tool for studying glycans on cells, and interpretation of the results provides insights for distinguishing the differences and changes in the local organization and heterogeneity of the cellular glycocalyx.

Metabolic glycan engineering (MGE) coupled with nitroxide spin-labeling (SL) was utilized to investigate the heterogeneous environment of cell surface glycans in select cancer and normal cells.