Subcellular Mass Spectrometry Imaging and Absolute Quantitative Analysis across Organelles

Mass spectrometry imaging is a field that promises to become a mainstream bioanalysis technology by allowing the combination of single-cell imaging and subcellular quantitative analysis. The frontier of single-cell imaging has advanced to the point where it is now possible to compare the chemical contents of individual organelles in terms of raw or normalized ion signal. However, to realize the full potential of this technology, it is necessary to move beyond this concept of relative quantification. Here we present a nanoSIMS imaging method that directly measures the absolute concentration of an organelle-associated, isotopically labeled, pro-drug directly from a mass spectrometry image. This is validated with a recently developed nanoelectrochemistry method for single organelles. We establish a limit of detection based on the number of isotopic labels used and the volume of the organelle of interest, also offering this calculation as a web application. This approach allows subcellular quantification of drugs and metabolites, an overarching and previously unmet goal in cell science and pharmaceutical development

Electrochemical quantification of transmitter concentration in single nanoscale vesicles isolated from PC12 cells

We use an electrochemical platform, nanoparticle tracking analysis, and differential centrifugation of single catecholamine vesicles to study the properties of nanometer transmitter vesicles, including the number of molecules, size, and catecholamine concentration inside. Vesicle impact electrochemical cytometry (VIEC) was used to quantify the catecholamine content of single vesicles in different batches isolated from pheochromocytoma (PC12) cells with different ultracentrifugation speeds. We show that, vesicles containing less catecholamine are obtained at subsequent centrifugation steps with higher speed (force). Important to quantification, the cumulative content after subsequent centrifugation steps is equivalent to that of one-step centrifugation at the highest speed, 70,000g. Moreover, as we count molecules in the vesicles, we compared molecular numbers from VIEC, flow VIEC, and intracellular VIEC to corresponding vesicle size measured by nanoparticle tracking analysis to evaluate catecholamine concentration in vesicles. The data suggest that vesicular catecholamine concentration is relatively constant and independent of the vesicular size, indicating vesicular transmitter content as a main factor regulating the vesicle size

Quantitative Chemical Measurements of Vesicular Transmitters with Electrochemical Cytometry

Electrochemical cytometry adds a new dimension to our ability to study the chemistry and chemical storage of transmitter molecules stored in nanometer vesicles. The approach involves the adsorption and subsequent rupture of vesicles on an electrode surface during which the electroactive contents are quantitatively oxidized (or reduced). The measured current allows us to count the number of molecules in the vesicles using Faraday\u27s law and to correlate this-to the amount of molecules released when single exocytosis events take place at communicating cells. The original format for this method involved a capillary electrophoresis separation step to singly address each vesicle, but we have more recently discovered that cellular vesicles tend to adsorb to carbon electrodes and spontaneously as well as stochastically rupture to give mostly single vesicle events. This approach, called impact electrochemical cytometry, even though the impact is perhaps not the important part of this process, has been studied and the vesicle rupture appears to be at the interface between the vesicle and the electrode and is probably driven by electroporation. The pore size and rate of content electrolysis are a function of the pore diameter and the presence of a protein core in the vesicles. In model liposomes with no protein, events appear extremely rapidly as the soft nanoparticles impact the electrode and the contents are oxidized. It appears that the proteins decorating the surface of the vesicle are important in maintaining a gap from the electrode and when this gap is closed electroporation takes place. Models of the event response times suggest the pores formed are small enough so we can carry out these measurements at nanotip electrodes and we have used this to quantify the vesicle content in living cells in a mode we call intracellular impact electrochemical cytometry. The development of electrochemical cytometry allows comparison between vesicle content and vesicular release and we have found that only part of the vesicle content is released in typical exocytotic cases measured by amperometry. This has led to the novel hypothesis that most exocytosis from dense core vesicles is via mechanism where vesicles fuse with the cell membrane, some content is released and then close again to be reloaded and reused. It leaves open the possibility that cells regulate release during individual events. This might be important in learning and memory and be a nonreceptor pharmaceutical target for brain related disorders. Indeed, the concept of the chemo-brain observed in cisplatin-treated cancer patients appears to be at least in part the result of changing the fraction of transmitter released and we have been able to show this by using the combined amperometric measurement of release and electrochemical cytometry at model cells

Measuring synaptic vesicles using cellular electrochemistry and nanoscale molecular imaging

The synaptic vesicle, a cellular compartment tens to hundreds of nanometres in size, is a main player in the process of exocytosis for neuronal communication. Understanding the regulatory mechanism of neurotransmission and neurological disorders requires the quantification of chemicals transmitted between cells. These challenging single vesicle measurements can be performed using analytical techniques described in this Review. In vivo amperometry at living cells can be used to quantify the amount of neurotransmitter released from a vesicle. By contrast, intracellular vesicle impact electrochemical cytometry allows the amount of molecules to be determined inside single vesicles. Although the dominant mode of exocytosis from vesicles is still under debate, several experiments point to the importance of partial release modes. Making use of fluorescent or isotopically labelled probes enables super-resolution optical and mass spectrometric imaging of molecular composition and activity of single vesicles. Correlating results from these nanoscopic techniques with those from electrochemistry has proved advantageous in understanding the relationship between vesicle structure and function

Mass Spectrometry Imaging Suggests That Cisplatin Affects Exocytotic Release by Alteration of Cell Membrane Lipids

We used time-of-flight secondary ion mass spectrometry (TOFSIMS) imaging to investigate the effect of cisplatin, the first member of the platinum-based anticancer drugs, on the membrane lipid composition of model cells to see if lipid changes might be involved in the changes in exocytosis observed. Platinum-based anticancer drugs have been reported to affect neurotransmitter release resulting in what is called the "chemobrain"; however, the mechanism for the influence is not yet understood. TOF-SIMS imaging was carried out using a high energy 40 keV (CO2)(6000)(+) gas cluster ion beam with improved sensitivity for intact lipids in biological samples. Principal components analysis showed that cisplatin treatment of PC12 cells significantly affects the abundance of different lipids and their derivatives, particularly phosphatidylcholine and cholesterol, which are diminished. Treatment of cells with 2 mu M and 100 mu M cisplatin showed similar effects on induced lipid changes. Lipid content alterations caused by cisplatin treatment at the cell surface are associated with the molecular and bimolecular signaling pathways of cisplatin-induced apoptosis of cells. We suggest that lipid alterations measured by TOF-SIMS are involved, at least in part, in the regulation of exocytosis by cisplatin

Quantitative Measurement of Transmitters in Individual Vesicles in the Cytoplasm of Single Cells with Nanotip Electrodes

The quantification of vesicular transmitter content is important for studying the mechanisms of neurotransmission and malfunction in disease, and yet it is incredibly difficult to measure the tiny amounts of neurotransmitters in the attoliter volume of a single vesicle, especially in the cell environment. We introduce a novel method, intracellular vesicle electrochemical cytometry. A nanotip conical carbon-fiber microelectrode was used to electrochemically measure the total content of electroactive neurotransmitters in individual nanoscale vesicles in single PC12 cells as these vesicles lysed on the electrode inside the living cell. The results demonstrate that only a fraction of the quantal neurotransmitter content is released during exocytosis. These data support the intriguing hypothesis that the vesicle does not open all the way during the normal exocytosis process, thus resulting in incomplete expulsion of the vesicular contents

Quantitative Measurement of Transmitters in Individual Vesicles in the Cytoplasm of Single Cells with Nanotip Electrodes

The quantification of vesicular transmitter content is important for studying the mechanisms of neurotransmission and malfunction in disease, and yet it is incredibly difficult to measure the tiny amounts of neurotransmitters in the attoliter volume of a single vesicle, especially in the cell environment. We introduce a novel method, intracellular vesicle electrochemical cytometry. A nanotip conical carbon-fiber microelectrode was used to electrochemically measure the total content of electroactive neurotransmitters in individual nanoscale vesicles in single PC12 cells as these vesicles lysed on the electrode inside the living cell. The results demonstrate that only a fraction of the quantal neurotransmitter content is released during exocytosis. These data support the intriguing hypothesis that the vesicle does not open all the way during the normal exocytosis process, thus resulting in incomplete expulsion of the vesicular contents

Mass Spectrometry Imaging Suggests That Cisplatin Affects Exocytotic Release by Alteration of Cell Membrane Lipids

We used time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging to investigate the effect of cisplatin, the first member of the platinum-based anticancer drugs, on the membrane lipid composition of model cells to see if lipid changes might be involved in the changes in exocytosis observed. Platinum-based anticancer drugs have been reported to affect neurotransmitter release resulting in what is called the “chemobrain”; however, the mechanism for the influence is not yet understood. TOF-SIMS imaging was carried out using a high energy 40 keV (CO₂)₆₀₀₀⁺ gas cluster ion beam with improved sensitivity for intact lipids in biological samples. Principal components analysis showed that cisplatin treatment of PC12 cells significantly affects the abundance of different lipids and their derivatives, particularly phosphatidylcholine and cholesterol, which are diminished. Treatment of cells with 2 μM and 100 μM cisplatin showed similar effects on induced lipid changes. Lipid content alterations caused by cisplatin treatment at the cell surface are associated with the molecular and bimolecular signaling pathways of cisplatin-induced apoptosis of cells. We suggest that lipid alterations measured by TOF-SIMS are involved, at least in part, in the regulation of exocytosis by cisplatin