Secondary Ion Mass Spectrometry Imaging Research Articles

Method depth corrects cell images without additional measurements

Pixel intensity of secondary electron images can be used to retroactively reshape cell morphology in 3D secondary ion mass spectrometry images.

4D Surface Reconstructions to Study Microscale Structures and Functions in Soil Biogeochemistry.

The development of high-resolution microscopy and spectroscopy techniques has allowed the analysis of microscopic 3D objects in fields like nanotechnology and life and soil sciences. Soils have the ability to incorporate and store large amounts of organic carbon. To study this organic matter (OM) sequestration, it is essential to analyze its association with soil minerals at the relevant microaggregate scale. This has been previously studied in 2D. However, 3D surface representations would allow a variable angle and magnification analysis, providing detailed insight on their architecture. Here we illustrate a 4D surface reconstruction workflow able to locate preferential sites for OM deposition with respect to microaggregate topography. We used Helium Ion Microscopy to acquire overlapping Secondary Electron (SE) images to reconstruct the soil topography in 3D. Then we used nanoscale Secondary Ion Mass Spectrometry imaging to chemically differentiate between the OM and mineral constituents forming the microaggregates. This image was projected onto the 3D SE model to create a 4D surface reconstruction. Our results show that organo-mineral associations mainly form at medium curvatures while flat and highly curved surfaces are avoided. This method presents an important step forward to survey the 3D physical structure and chemical composition of microscale biogeochemical systems correlatively.

Understanding Degradation Mechanisms in SrIrO3 Oxygen Evolution Electrocatalysts: Chemical and Structural Microscopy at the Nanoscale

AbstractDesigning acid‐stable oxygen evolution reaction electrocatalysts is key to developing sustainable energy technologies such as polymer electrolyte membrane electrolyzers but has proven challenging due to the high applied anodic potentials and corrosive electrolyte. This work showcases advanced nanoscale microscopy techniques supported by complementary structural and chemical characterization to develop a fundamental understanding of stability in promising SrIrO3 thin film electrocatalyst materials. Cross‐sectional high‐resolution transmission electron microscopy illustrates atomic‐scale bulk and surface structure, while secondary ion mass spectrometry imaging using a helium ion microscope provides the nanoscale lateral elemental distribution at the surface. After accelerated degradation tests under anodic potential, the SrIrO3 film thins and roughens, but the lateral distribution of Sr and Ir remains homogeneous. A layer‐wise dissolution mechanism is hypothesized, wherein anodic potential causes the IrOx‐rich surface to dissolve and be regenerated by Sr leaching. The characterization approaches utilized herein and mechanistic insights into SrIrO3 are translatable to a wide range of catalyst systems.

Viscoelastic Properties and Sulfur Distribution at the Nanoscale in Binary Elastomeric Blends: Toward Phase-Specific Cross-Link Density Estimations

Immiscible blends of elastomers present high technological interest, and the selection of the vulcanization system is important for the optimization of properties for different technical applications. In particular, the effect of the curing agents on the distribution of cross-links in each phase is key for the full comprehension of the structure–property relationships. Aiming at the understanding of the phase-specific network structure in rubber blends, this work presents an innovative strategy for the quantitative characterization of the local viscoelastic properties of immiscible rubber blends by atomic force microscopy (AFM) measurements. A systematic study on the quantitative nanomechanical characterization by AFM of unfilled single natural rubber (NR) matrixes with different degrees of cross-link densities ultimately allows for the estimation of the phase-specific cross-link density of the NR phase in NR/butadiene rubber (BR) blends, prepared with varying vulcanization systems. Complementary chemical information by high-resolution secondary ion mass spectrometry imaging is able to reveal differences in sulfur contents in each elastomeric phase of the blends.

Combining secondary ion mass spectrometry image depth profiling and single particle inductively coupled plasma mass spectrometry to investigate the uptake and biodistribution of gold nanoparticles in Caenorhabditis elegans

Analytical techniques capable of determining the spatial distribution and quantity (mass and/or particle number) of engineered nanomaterials in organisms are essential for characterizing nano-bio interactions and for nanomaterial risk assessments. Here, we combine the use of dynamic secondary ion mass spectrometry (dynamic SIMS) and single particle inductively coupled mass spectrometry (spICP-MS) techniques to determine the biodistribution and quantity of gold nanoparticles (AuNPs) ingested by Caenorhabditis elegans. We report the application of SIMS in image depth profiling mode for visualizing, identifying, and characterizing the biodistribution of AuNPs ingested by nematodes in both the lateral and z (depth) dimensions. In parallel, conventional- and sp-ICP-MS quantified the mean number of AuNPs within the nematode, ranging from 2 to 36 NPs depending on the size of AuNP. The complementary data from both SIMS image depth profiling and spICP-MS provides a complete view of the uptake, translocation, and size distribution of ingested NPs within Caenorhabditis elegans.

Enhancement of T2* Weighted MRI Imaging Sensitivity of U87MG Glioblastoma Cells Using γ-Ray Irradiated Low Molecular Weight Hyaluronic Acid-Conjugated Iron Nanoparticles.

IntroductionIt has been reported that low-molecular-weight hyaluronic acid (LMWHA) exhibits a potentially beneficial effect on cancer therapy through targeting of CD44 receptors on tumor cell surfaces. However, its applicability towards tumor detection is still unclear. In this regard, LMWHA-conjugated iron (Fe3O4) nanoparticles (LMWHA-IONPs) were prepared in order to evaluate its application for enhancing the T2* weighted MRI imaging sensitivity for tumor detection.MethodsLMWHA and Fe3O4 NPs were produced using γ-ray irradiation and chemical co-precipitation methods, respectively. First, LMWHA-conjugated FITC was prepared to confirm the ability of LMWHA to target U87MG cells using fluorescence microscopy. The hydrodynamic size distribution and dispersion of the IONPs and prepared LMWHA-IONPs were analyzed using dynamic light scattering (DLS). In addition, cell viability assays were performed to examine the biocompatibility of LMWHA and LMWHA-IONPs toward U87MG human glioblastoma and NIH3T3 fibroblast cell lines. The ability of LMWHA-IONPs to target tumor cells was confirmed by detecting iron (Fe) ion content using the thiocyanate method. Finally, time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging and in vitro magnetic resonance imaging (MRI) were performed to confirm the contrast enhancement effect of LMWHA-IONPs.ResultsFlorescence analysis results showed that LMWHA-FITC successfully targeted the surfaces of both tested cell types. The ability of LMWHA to target U87MG cells was higher than for NIH3T3 cells. Cell viability experiments showed that the fabricated LMWHA-IONPs possessed good biocompatibility for both cell lines. After co-culturing test cells with the LMWHA-IONPs, detected Fe ion content in the U87MG cells was much higher than that of the NIH3T3 cells in both thiocyanate assays and TOF-SIMs images. Finally, the addition of LMWHA-IONPs to the U87MG cells resulted in an obvious improvement in T2* weighted MR image contrast compared to control NIH3T3 cells.DiscussionOverall, the present results suggest that LMWHA-IONPs fabricated in this study provide an effective MRI contrast agent for improving the diagnosis of early stage glioblastoma in MRI examinations.

Facial Treatment with 3-O-Cetyl Ascorbic Acid for Improvement of Skin Texture: Uptake, Effectiveness, and In Vitro Carcinogenicity Assessment

Ascorbic acid (AA) is a water-soluble vitamin that is found at high concentrations in normal skin. The important and well-known benefits of using AA in skin health include the stimulation of collagen synthesis and the assistance of protection against photo-oxidative damages. To maintain stability and improve drug delivery to the active site, a variety of AA derivatives have been chemically synthesized. Among these compounds, we focus here on a lipophilic derivative, 3-O-cetyl ascorbic acid (3-CetylAA), which remains poorly characterized for cosmetic applications. Uptake analysis in three healthy human volunteers’ skin was conducted using a serial tape-stripping technique detecting 3-CetylAA (on average, 128 ± 27 pmol per µg) in the stratum corneum after a 5-h topical treatment when treated with 25 mM 3-CetylAA-containing cream for 13 days twice daily and continuously. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging of vertical cryosections of pig skin revealed the presence of 3-CetylAA in the epidermal layer after topical treatment with 3-CetylAA-containing cream. In sun-exposed human skin, 3-CetylAA improved the texture after treatment with 25 mM 3-CetylAA-containing cream for 4 weeks or more when used twice daily or continuously. An in vitro transformation assay using BALB/c 3T3 A31-1-1 cells demonstrated that 10 µM 3-CetylAA, which is the same concentration exhibited in vitro biological activities in another lipophilic AA derivative, 2-O-octadecyl ascorbic acid, was non-carcinogenic and did not potentiate the UVC-induced transformation frequency when applied for 3 days after UVC irradiation. These results demonstrate that 3-CetylAA is a promising candidate as a lipophilic derivative of AA for cosmetic purposes.

Localized corrosion induced surface modifications of Al-Cu-Li alloy studied by ToF-SIMS 3D imaging

Three-dimensional (3D) time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging is a promising tool in the analysis of surface modifications of alloy matrix and intermetallic particles induced by localized corrosion due to its good spatial resolution and very high sensitivity to chemical identification especially involving light mass elements like Li. In this paper, ToF-SIMS 3D chemical mapping combined with scanning electron microscopy was performed to study the localized corrosion of Al-Cu-Li alloy (AA2050-T8) in a region around an Al-Cu-Fe-Mn intermetallic particles (IMPs) in 0. 1 M NaCl solution. Initiation of corrosion was locally observed on the IMPs and between IMPs and matrix substrate. Moreover, a modified oxide/hydroxide layer was observed over IMPs compared to the matrix. This new corrosion layer was enriched with chlorides and Cu hydroxides. ToF-SIMS 3D imaging revealed that Li was present over the alloy matrix, while no Li was present over the IMPs in regions where localized corrosion occurred.

Secondary Ion Mass Spectrometry Imaging Reveals Changes in the Lipid Structure of the Plasma Membranes of Hippocampal Neurons following Drugs Affecting Neuronal Activity.

The cellular functions of lipids in the neuronal plasma membranes have been increasingly acknowledged, particularly their association to neuronal processes and synaptic plasticity. However, the knowledge of their regulatory mechanisms in neuronal cells remains sparse. To address this, we investigated the lipid organization of the plasma membranes of hippocampal neurons in relation to neuronal activity using secondary ion mass spectrometry imaging. The neurons were treated with drugs, particularly tetrodotoxin (TTX) and bicuculline (BIC), to induce chronic activation and silencing. Distinct lipid organization was found in the plasma membrane of the cell body and the neurites. Moreover, significant alterations of the levels of the membrane lipids, especially ceramides, phosphatidylserines, phosphatidic acids, and triacylglycerols, were observed under the TTX and BIC treatments. We suggest that many types of membrane lipids are affected by, and may be involved in, the regulation of neuronal function.

Direct Mapping of Phospholipid Ferroptotic Death Signals in Cells and Tissues by Gas Cluster Ion Beam Secondary Ion Mass Spectrometry (GCIB-SIMS).

Peroxidized phosphatidylethanolamine (PEox) species have been identified by liquid chromatography mass spectrometry (LC-MS) as predictive biomarkers of ferroptosis, a new program of regulated cell death. However, the presence and subcellular distribution of PEox in specific cell types and tissues have not been directly detected by imaging protocols. By applying gas cluster ion beam secondary ion mass spectrometry (GCIB-SIMS) imaging with a 70 keV (H2 O)n+ (n>28 000) cluster ion beam, we were able to map PEox with 1.2 μm spatial resolution at the single cell/subcellular level in ferroptotic H9c2 cardiomyocytes and cortical/hippocampal neurons after traumatic brain injury. Application of this protocol affords visualization of physiologically relevant levels of very low abundance (20 pmol μmol-1 lipid) peroxidized lipids in subcellular compartments and their accumulation in disease conditions.

Direct Mapping of Phospholipid Ferroptotic Death Signals in Cells and Tissues by Gas Cluster Ion Beam Secondary Ion Mass Spectrometry (GCIB‐SIMS)

AbstractPeroxidized phosphatidylethanolamine (PEox) species have been identified by liquid chromatography mass spectrometry (LC‐MS) as predictive biomarkers of ferroptosis, a new program of regulated cell death. However, the presence and subcellular distribution of PEox in specific cell types and tissues have not been directly detected by imaging protocols. By applying gas cluster ion beam secondary ion mass spectrometry (GCIB‐SIMS) imaging with a 70 keV (H2O)n+ (n>28 000) cluster ion beam, we were able to map PEox with 1.2 μm spatial resolution at the single cell/subcellular level in ferroptotic H9c2 cardiomyocytes and cortical/hippocampal neurons after traumatic brain injury. Application of this protocol affords visualization of physiologically relevant levels of very low abundance (20 pmol μmol−1 lipid) peroxidized lipids in subcellular compartments and their accumulation in disease conditions.

In Situ Identification and Spatial Mapping of Microplastic Standards in Paramecia by Secondary-Ion Mass Spectrometry Imaging.

Microplastics (MPs) are universally present in the ecosystem and pose great threats to the environment and living organisms. Research studies have shown that small MPs (<50 μm in diameter) are especially toxic and account for more than half of all MPs collected in the Atlantic Ocean. Nevertheless, current methods for the detection and analysis of MPs are incapable of achieving rapid and in situ analysis of small MPs in the biota to ultimately enable the study of their biological effects. In this work, we report a method that allows rapid in situ identification and spatial mapping of small MPs directly from paramecia with high accuracy by acquiring chemical composition information using secondary-ion mass spectrometry (SIMS) imaging. Specifically, six types of common MPs (polymethyl methacrylate, polyvinyl chloride, polypropylene, polyethylene terephthalate, polyglycidyl methacrylate, and polyamide 6) with a diameter of 1-50 μm were simultaneously imaged with high chemical specificity at a spatial resolution of 700 nm. In situ spatial mapping of a group of MPs ingested by paramecia was performed using SIMS fragments specific to the plastic composition with no sample pretreatment, revealing the aggregation of MPs in paramecia after ingestion. Compared with existing methods, one additional advantage of the developed method is that the MPs and the organism can be analyzed in the same experimental workflow to record their fingerprint spectra, acquiring biochemical information to evaluate MP fate, toxicity, and the MP-biota interaction.

Time-of-Flight Secondary Ion Mass Spectrometry Imaging of Cross Sections from the Bacchanals Paintings of Nicolas Poussin.

The two paintings Infant Bacchanals (Museo Nazionale d'Arte Antica, Palazzo Barberini, Rome, Italy) executed by Nicolas Poussin (Les Andelys, 1594-Rome, 1665) in around 1626 are thought to have been painted "a guazzo", which means either with a glue or with an egg binding medium. To date, this has never been confirmed through analysis. Dual-beam time-of-flight secondary ion mass spectrometry (TOF-SIMS), using a bismuth cluster liquid metal ion gun and an argon gas cluster ion beam, allows the mapping of organic and inorganic matter on paintings cross sections, with the possibility to acquire submicrometer-resolution mass spectrometry images of the sample, together with high mass resolution using a delayed extraction of secondary ions. The surfaces of cross sections from both paintings were prepared beforehand either by polishing or by microtome cutting and then cleaned with the gas cluster ion beam directly inside the vacuum chamber of the instrument. The nature of the binders in the two paintings was investigated by TOF-SIMS analyses. By considering the uneven physical properties of the heterogeneous analyzed surfaces, several high-resolution images were recorded with different instrument settings. The detection of lipids seems to point toward an oil-containing medium, rather than a glue-binding medium. An emulsion made of oil and glue is another hypothesis to be explored to better understand the artist's working methods in his early career.

Editorial: Marine N2 Fixation: Recent Discoveries and Future Challenges

Unicellular nitrogen fixing cyanobacteria (UCYN) are abundant members of phytoplankton communities in a wide range of marine environments, including those with rapidly changing nitrogen (N) concentrations. We hypothesized that differences in N availability (N 2 vs. combined N) would cause UCYN to shift strategies of intracellular N and C allocation. We used transmission electron microscopy and nanoscale secondary ion mass spectrometry imaging to track assimilation and intracellular allocation of 13 C-labeled CO 2 and 15 N-labeled N 2 or NO 3 at different periods across a diel cycle in Cyanothece sp. ATCC 51142. We present new ideas on interpreting these imaging data, including the influences of pre-incubation cellular C and N contents and turnover rates of inclusion bodies. Within cultures growing diazotrophically, distinct subpopulations were detected that fixed N 2 at night or in the morning. Additional significant within-population heterogeneity was likely caused by differences in the relative amounts of N assimilated into cyanophycin from sources external and internal to the cells. Whether growing on N 2 or NO 3 , cells prioritized cyanophycin synthesis when N assimilation rates were highest. N assimilation in cells growing on NO 3 switched from cyanophycin synthesis to protein synthesis, suggesting that once a cyanophycin quota is met, it is bypassed in favor of protein synthesis. Growth on NO 3 also revealed that at night, there is a very low level of CO 2 assimilation into polysaccharides simultaneous with their catabolism for protein synthesis. This study revealed multiple, detailed mechanisms underlying C and N management in Cyanothece that facilitate its success in dynamic aquatic environments.

Mass spectrometry imaging of untreated wet cell membranes in solution using single-layer graphene

We report a means by which atomic and molecular secondary ions, including cholesterol and fatty acids, can be sputtered through single-layer graphene to enable secondary ion mass spectrometry (SIMS) imaging of untreated wet cell membranes in solution at subcellular spatial resolution. We can observe the intrinsic molecular distribution of lipids, such as cholesterol, phosphoethanolamine and various fatty acids, in untreated wet cell membranes without any labeling. We show that graphene-covered cells prepared on a wet substrate with a cell culture medium reservoir are alive and that their cellular membranes do not disintegrate during SIMS imaging in an ultra-high-vacuum environment. Ab initio molecular dynamics calculations and ion dose-dependence studies suggest that sputtering through single-layer graphene occurs through a transient hole generated in the graphene layer. Cholesterol imaging shows that methyl-β-cyclodextrin preferentially extracts cholesterol molecules from the cholesterol-enriched regions in cell membranes.

Temporal Patterns and Intra- and Inter-Cellular Variability in Carbon and Nitrogen Assimilation by the Unicellular Cyanobacterium Cyanothece sp. ATCC 51142.

Unicellular nitrogen fixing cyanobacteria (UCYN) are abundant members of phytoplankton communities in a wide range of marine environments, including those with rapidly changing nitrogen (N) concentrations. We hypothesized that differences in N availability (N2 vs. combined N) would cause UCYN to shift strategies of intracellular N and C allocation. We used transmission electron microscopy and nanoscale secondary ion mass spectrometry imaging to track assimilation and intracellular allocation of 13C-labeled CO2 and 15N-labeled N2 or NO3 at different periods across a diel cycle in Cyanothece sp. ATCC 51142. We present new ideas on interpreting these imaging data, including the influences of pre-incubation cellular C and N contents and turnover rates of inclusion bodies. Within cultures growing diazotrophically, distinct subpopulations were detected that fixed N2 at night or in the morning. Additional significant within-population heterogeneity was likely caused by differences in the relative amounts of N assimilated into cyanophycin from sources external and internal to the cells. Whether growing on N2 or NO3, cells prioritized cyanophycin synthesis when N assimilation rates were highest. N assimilation in cells growing on NO3 switched from cyanophycin synthesis to protein synthesis, suggesting that once a cyanophycin quota is met, it is bypassed in favor of protein synthesis. Growth on NO3 also revealed that at night, there is a very low level of CO2 assimilation into polysaccharides simultaneous with their catabolism for protein synthesis. This study revealed multiple, detailed mechanisms underlying C and N management in Cyanothece that facilitate its success in dynamic aquatic environments.

Nanosims Tools and Probes for Imaging Intracellular Structures

In eukaryotic cells, intracellular lipid membranes act as barriers that segregate specific chemical reactions into organelles for increased biochemical efficiency. Organelle efficiency is maintained by the regulation of cholesterol and different lipid distributions within these organelles and is tightly controlled by intracellular transport and lipid metabolism. Disturbances in intracellular lipid trafficking, biosynthesis and degradation are hallmarks of fatal diseases involving an accumulation of cholesterol or lipids in endosomes and lysosomes, such as Niemann Pick Type II disease. Efforts to elucidate the mechanisms for lipid-mediated cellular (dys)function would benefit from an imaging technique that enables assessing the relative abundances of cholesterol and lipid species of interest between organelles. Towards this aim, we developed a secondary ion mass spectrometry (SIMS) approach to image the sphingolipid and cholesterol distributions within intact cells in three dimensions with a NanoSIMS instrument. Because the components of interest must produce secondary ions that contain distinctive elements or isotopes to permit their detection with a NanoSIMS instrument, organelle-specific labels must be incorporated into the cells to enable identifying specific organelles. This work describes a strategy that utilizes small protein tags and functionalized substrates that permit identify organelles of interest with NanoSIMS, and the development of data processing tools that produce images of these organelles at the proper depth in the 3D SIMS images. We created a transfected cell line that stably expresses enzymatic proteins fused to organelle-specific proteins. These proteins covalently attach to functionalized small-molecule substrates which can be detected with NanoSIMS and fluorescence microscopy. By combining these tools for organelle detection with lipid-specific metabolic isotope incorporation, semi-quantitative information concerning the abundance of cholesterol or specific lipids within organelles of interest may be obtained.

NanoSIMS observations of mouse retinal cells reveal strict metabolic controls on nitrogen turnover

BackgroundMost of the cells of the mammalian retina are terminally differentiated, and do not regenerate once fully developed. This implies that these cells have strict controls over their metabolic processes, including protein turnover. We report the use of metabolic labelling procedures and secondary ion mass spectrometry imaging to examine nitrogen turnover in retinal cells, with a focus on the outer nuclear layer, inner nuclear layer, and outer plexiform layer.ResultsWe find that turnover can be observed in all cells imaged using NanoSIMS. However, the rate of turnover is not constant, but varies between different cellular types and cell regions. In the inner and outer nuclear layers, turnover rate is higher in the cytosol than in the nucleus of each cell. Turnover rates are also higher in the outer plexiform layer. An examination of retinal cells from mice that were isotopically labeled very early in embryonic development shows that proteins produced during this period can be found in all cells and cell regions up to 2 months after birth, even in regions of high turnover.ConclusionsOur results indicate that turnover in retinal cells is a highly regulated process, with strict metabolic controls. We also observe that turnover is several-fold higher in the synaptic layer than in cell layers. Nevertheless, embryonic proteins can still be found in this layer 2 months after birth, suggesting that stable structures persist within the synapses, which remain to be determined.

Molecular imaging of plant-microbe interactions on the Brachypodium seed surface.

Plant growth-promoting rhizobacteria (PGPR) play a crucial role in biological control and pathogenic defense on and within plant tissues, however the mechanisms by which plants associate with PGPR to elicit such beneficial effects need further study. Here, we present time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging of Brachypodium distachyon (Brachypodium) seeds with and without exposure to two model PGPR, i.e., Gram-negative Pseudomonas fluorescens SBW25 (P.) and Gram-positive Arthrobacter chlorophenolicus A6 (A.). Delayed image extraction was used to image PGPR-treated seed sections to reveal morphological changes. ToF-SIMS spectral comparison, principal component analysis (PCA), and two-dimensional (2D) imaging show that the selected PGPR have different effects on the host seed surface, resulting in changes in chemical composition and morphology. Metabolite products and biomarkers, such as flavonoids, phenolic compounds, fatty acids, and indole-3-acetic acid (IAA), were identified on the PGPR-treated seed surfaces. These compounds have different distributions on the Brachypodium seed surface for the two PGPR, indicating that the different bacteria elicit distinct responses from the host. Our results illustrate that ToF-SIMS is an effective tool to study plant-microbe interactions and to provide insightful information with submicrometer lateral resolution of the chemical distributions associated with morphological features, potentially offering a new way to study the mechanisms underlying beneficial roles of PGPR.

Single cell imaging reveals cisplatin regulating interactions between transcription (co)factors and DNA.

Cisplatin is an extremely successful anticancer drug, and is commonly thought to target DNA. However, the way in which cisplatin-induced DNA lesions regulate interactions between transcription factors/cofactors and genomic DNA remains unclear. Herein, we developed a dual-modal microscopy imaging strategy to investigate, in situ, the formation of ternary binding complexes of the transcription cofactor HMGB1 and transcription factor Smad3 with cisplatin crosslinked DNA in single cells. We utilized confocal microscopy imaging to map EYFP-fused HMGB1 and fluorescent dye-stained DNA in single cells, followed by the visualization of cisplatin using high spatial resolution (200–350 nm) time of flight secondary ion mass spectrometry (ToF-SIMS) imaging of the same cells. The superposition of the fluorescence and the mass spectrometry (MS) signals indicate the formation of HMGB1-Pt-DNA ternary complexes in the cells. More significantly, for the first time, similar integrated imaging revealed that the cisplatin lesions at Smad-binding elements, for example GGC(GC)/(CG) and AGAC, disrupted the interactions of Smad3 with DNA, which was evidenced by the remarkable reduction in the expression of Smad-specific luciferase reporters subjected to cisplatin treatment. This finding suggests that Smad3 and its related signalling pathway are most likely involved in the intracellular response to cisplatin induced DNA damage.