Download PDF Single Cell Analysis Technologies and Applications by Dario Anselmetti

Sinopsis

Many problems in single cell analysis involve very basic questions: where do proteins or larger molecular assemblies go?How do they move: by Brownian motion, freely or in some restricted manner, or by active transport along one of the cell’s filament systems? Where are they captured, where do they bind and for how long? If binding, what are the interaction partners? Which factors are present at locations in large organelles, which are transiently bound, what is the sequence of molecular interactions? Especially the latter question is of great significance considering the functional role of large molecular complexes, which perform tasks like signal transduction, energy production, information processing, or protein formation and degradation. To approach these questions means developing an “intracellular biophysical chemistry.” Currently there are only a small number of techniques available to approach these questions of intracellular protein dynamics. Light microscopy and in particular fluorescence microscopy is certainly one of the methods of choice; and it has reached a high level of maturation and sophistication. Especially the past 20 years have seen a storm of developments in quantitative fluorescence microscopic techniques, which was triggered by the perfection of microscope optics and light detectors, the widespread use of continuous wave and pulsed lasers as excitation sources, the introduction of elegant optical concepts, the availability of massive computing power to resolve complex image processing tasks and, last but not least, the introduction of genetically engineered autofluorescent protein conjugates. In the past few years tremendous progress has been made with regard to bringing optical microscope resolution almost to the ultimate level of molecular sizes with the introduction of stimulated emission depletion microscopy [1] and nonlinear structured illumination microscopy [2, 3]. But high-resolution methods are often not applicable or optimally suited to examine dynamical processes. However for such problems fluorescence techniques appear to be almost ideal. Probably the most well known is fluorescence recovery after photobleaching, abbreviated FRAP [4]. Afurther technique, nowadays almost classic but nevertheless still rapidly expanding, is fluorescence correlation spectroscopy, FCS [5, 6]. A most recent and extremely powerful technique is single molecule tracking within cells [7]. Remarkably, monitoring single fluorescent molecules with a sufficiently high time resolution can provide real-time molecular views on biochemical processes within cells even in vivo. It is extremely fascinating and instructive to directly observe the motions and interactions of single protein molecules, ribonucleoprotein particles or oligonucleotides by state of the art light microscopy.

Content

1. Single Molecule Fluorescence Monitoring in Eukaryotic Cells: Intranuclear Dynamics of Splicing Factors
2. Gene Classification and Quantitative Analysis of Gene Regulation in Bacteria using Single Cell Atomic Force Microscopy and Single Molecule Force Spectroscopy
3. Cellular Cryo-Electron Tomography (CET): Towards a Voyage to the Inner Space of Cells
4. Single Cell Analysis: Technologies
5. Single Cell Proteomics
6. Protein Analysis of Single Cells in Microfluidic Format
7. Single Cell Mass Spectrometry
8. Single Cell Analysis for Quantitative Systems Biology
9. Optical Stretcher for Single Cells
10. Single Cell Analysis: Applications
11. Single Cell Immunology
12. Molecular Characterization of Rare Single Tumor Cells
13. Single Cell Heterogeneity
14. Genome and Transcriptome Analysis of Single Tumor Cells

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