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The first-ever comprehensive overview of the methods used in this key technology in modern biology provides the latest working knowledge needed by every scientist entering this growing field. It covers all the current technology and application areas, from microscopy and spectroscopy to proteomics and microfluidics.
I recently spent a summer as an intern at the Lawrence Livermore National Laboratory. I worked on a project involving the real-time, reagentless, single cell detection of aerosolized pathogens using a novel mass spectrometry approach called Bio-Aerosol Mass Spectrometry (BAMS). Based upon preliminary results showing the differentiation capabilities of BAMS, I would like to explore the development and use of this novel detection system in the context of both environmental and clinical sample pathogen detection. I would also like to explore the broader public health applications that a system such as BAMS might have in terms of infectious disease prevention and control. In order to appreciate the potential of this instrument, I will demonstrate the need for better pathogen detection methods, and outline the instrumentation, data analysis and preliminary results that lead me toward a desire to explore this technology further. I will also discuss potential experiments for the future along with possible problems that may be encountered along the way.
This dissertation, "Single-cell Analysis Using Inductively Coupled Plasma Mass Spectrometry" by Koon-sing, Ho, 何觀陞, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: The technique of single-cell analysis using time-resolved inductively coupled plasma-mass spectrometry has been characterized and optimized. Determination of the metal contents of individual cells provides data on the natural metal contents of the cells and the corresponding distributions in the population. The distribution is a useful indicator of the health and the state of development of the cells. The contents of sorbed metals of individual cells over a duration of time are required to understand the dynamics of metal-cell interactions. A green alga, Chlorella vulgaris, was used as a model biological cell in this study. The criteria and procedures for proper sampling of the cells into the ICP will be discussed. Ideally, each ICP-MS spike corresponds to one cell, but cell overlapping occurs because the cells enter the ICP randomly. Selection of cell number density and sample uptake rate to minimize spike overlapping will be discussed. A cell counting method based on the frequency of the spikes has been developed. The distribution of the metal contents of cells was determined by measuring large number of spikes. The minimum number of spikes required was determined by statistical analysis. The spike intensity distribution was correlated with the size distribution of the cells. The peak maximum of the spike intensity distribution was used for the determination of the average metal content of the cells. The use of the peak maximum reduces errors due to spike overlapping in the measurement. Quantitative determination of the metal contents was achieved using standard particles for calibration. Errors in calibration using standard solution nebulization were discussed. The technique was applied in the study of metal-cell interactions. Sorption of heavy metal ions (as environmental pollutants) by Chlorella vulgaris, and uptake of biometal (as nutrient) and metallodrug (as toxin) by Helicobacter pylori were studied. The technique requires simple sample preparation of removing the culture medium by filtration or centrifugation. The health state of the cells in the presence of toxic metals was related to the change in cell number density. The ratio of the FWHM of the spike intensity distributions of the sorbed metals to the natural metal contents of the cells is identified as a possible indicator of the location of the sorbed metals. The kinetics of metal sorption by the cells can be studied using a single cell culture. The method reduces errors due to uncertainties in cell number density and metal concentration in multiple samples that are required in conventional methods. The optimal ICP-MS sampling depth of 17 elements, introduced into the ICP by conventional solution nebulization of aqueous standard solutions, has been determined. The elements were selected to represent a wide range of boiling points and ionization potentials. Boiling point of the dried residues and ionization potential of the analyte element were identified as the major factors that determine the optimal sampling position. Since dried sample solution aerosols are effectively nanoparticles, the study provides useful insight on the optimization of the operation conditions and calibration strategies for single-particle analysis using ICP-MS. DOI: 10.5353/th_b4985851 Subjects: Inductively coupled plasma mass spectrometry Cells - Analysis
This volume explores the latest advancements made in the field of mass spectrometry (MS) based single cell proteomics. The chapters in this book focus on technologies, methods, and biological applications and together cover the entire single cell proteomics analysis workflow, from sample collection and preparation through liquid chromatographic separation and mass spectrometric data acquisition to data analysis and interpretation. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and authoritative, Mass Spectrometry Based Single Cell Proteomics: Methods and Protocols is a valuable resource for all researchers interested in using these technologies and methods in their laboratories to continue making biological discoveries in this important field.
This book provides an overview of single-cell isolation, separation, injection, lysis and dynamics analysis as well as a study of their heterogeneity using different miniaturized devices. As an important part of single-cell analysis, different techniques including electroporation, microinjection, optical trapping, optoporation, rapid electrokinetic patterning and optoelectronic tweezers are described in detail. It presents different fluidic systems (e.g. continuous micro/nano-fluidic devices, microfluidic cytometry) and their integration with sensor technology, optical and hydrodynamic stretchers etc., and demonstrates the applications of single-cell analysis in systems biology, proteomics, genomics, epigenomics, cancer transcriptomics, metabolomics, biomedicine and drug delivery systems. It also discusses the future challenges for single-cell analysis, including the advantages and limitations. This book is enjoyable reading material while at the same time providing essential information to scientists in academia and professionals in industry working on different aspects of single-cell analysis. Dr. Fan-Gang Tseng is a Distinguished Professor of Engineering and System Science at the National Tsing Hua University, Taiwan. Dr. Tuhin Subhra Santra is a Research Associate at the California Nano Systems Institute, University of California at Los Angeles, USA.
This book is a printed edition of the Special Issue "Single Cell Analysis in Biotechnology and Systems Biology" that was published in IJMS