‘Top-Down’ Approaches for the Study of Single-Cells: Micro-Engineering and Electrical Phenotype

Single-cell analysis is a very important field of research and is currently at the frontier of physical and biological sciences. Understanding how the phenotype of a single-cell arises from its genotype is a complex topic. Currently, the prevailing paradigm to analyze cellular functions is the study of biochemical interactions using fluorescence based imaging systems. However, the elimination of the labelling process is highly desirable to improve the accuracy of the analysis. Living cells are electromagnetic units; in as much they use electric mechanisms to control and regulate dynamic processes involved in inter alia signal transduction, metabolism, proliferation and differentiation. Recent developments in microand nanofabrication technologies are offering great opportunities for the analysis of single cells; the combination of micro fluidic environments, nano electrodes/wires and ultra wide band electromagnetic engineering will soon make possible the investigation of local (submicrometer scale) dynamic processes integrating several events at different time scales. In the paper, we present recent approaches which aim at investigating singlecells with the help of MEMS and NEMS (Micro and Nano Electro Mechanical Systems) and ultra wide band (DC-THz) electromagnetic characterization techniques. Introduction To understand how cells respond to environmental stimuli, reconstruction of biochemical network models based on the different components of the cell, namely the genome, the transcriptome, the proteome and the metabolome is necessary (Fig. 1). The first step is the cataloging of all genes and proteins that are expressed in a mammalian cell under given external stimuli. The sequencing of the genome has been achieved thanks to different techniques like DNA microarrays [1] or chromatine immunoprecipitation [2]. Obtaining the proteome of a cell is a much more difficult task. Besides being present in low concentrations, proteins are constantly undergoing changes in their state by forming complexes with other proteins, undergoing covalent modifications and binding to myriad substrates in a cell. Most current efforts focus on analyzing a cellular milieu for specific proteins using immunoprecipitation and tagging methods [3]. At the next level of organization, the interactions between components in the cell form complex networks, and the determination of the network topology represents a formidable challenge. Measurements made on single, living cells are necessary to understand the molecular mechanisms involving these intracellular signalling molecules. Numerous studies have demonstrated that individual cells may seem identical but behave heterogeneously, both in timing and magnitude of Advances in Science and Technology Vol. 53 (2006) pp 97-106 online at http://www.scientific.net © (2006) Trans Tech Publications, Switzerland Online available since 2006/Oct/01 All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of the publisher: Trans Tech Publications Ltd, Switzerland, www.ttp.net. (ID: 130.203.133.33-14/04/08,11:20:59) signalling responses [4]. For instance, gene expression occurs stochastically, as a consequence of the low copy number of DNA and mRNA molecules involved [5]. Consequently, accurate measurement of most cellular properties requires monitoring from individual cells to avoid errors due to averaging across a population of cells. Monitoring the events that occur during signalling requires in vivo methods because the preparation of cellular extracts performed in traditional biochemical assays disrupts the normal compartmentalization and subcellular environment in which physiological signalling occurs. Such manipulation almost invariably allows time for chemical reactions to proceed during the extraction process, which may introduce inaccuracies in the measurement. Figure 1: a) Schematic overview of the connections between the different molecular components of a cell; b) AFM picture of a cell membrane showing proteins embedded in the lipid bilayer. Thus, on-line, real-time and non-invasive monitoring of single cells is currently the focus of the frontier of biophysical studies. Due to the ultra small size of single cell (volume fL-nL), ultratrace amount of component (zmol-fmol), ultrarapid biochemical reaction (ms), high sensitivity, high selectivity, high temporal resolution and ultrasmall sampling-volume are necessary. As a consequence, deciphering spatiotemporal variations of signalling molecules (i.e.: a receptor, a channel, an enzyme or several other functionally defined species) in a dynamic cell remains beyond the scope of current technologies. Microscopy and fluorescence are currently the best methods to study dynamically molecules interactions in living cell [6]. The rapid development of these techniques has been made possible thanks to new fluorescent probes and improvements in microscope systems and software. Confocal laser scanning microscopy, total internal reflection fluorescence microscopy, fluorescent correlation spectroscopy (FRAP, FLIP), fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging (FLIM) are now almost commonplace. Recent works using stimulated emission depletion (STED) or scanning near field optical microscopy (SNOM) has enabled a substantial increase in resolution below the diffraction limit. However, resolution is not the only criterion to select these PHENOTYPE NOISOME GENOME METABOLOME PROTEOME TRANSCRIPTOME EXTERNAL STIMULI a) b) 98 Biomedical Applications of Nano Technologies