Single molecule experimentation in biological physics: exploring the living component of soft condensed matter one molecule at a time

Abstract

The soft matter of biological systems consists of mesoscopic length scale building blocks, composed of a variety of different types of biological molecules. Most single biological molecules are so small that 1 billion would fit on the full-stop at the end of this sentence, but collectively they carry out the vital activities in living cells whose length scale is at least three orders of magnitude greater. Typically, the number of molecules involved in any given cellular process at any one time is relatively small, and so real physiological events may often be dominated by stochastics and fluctuation behaviour at levels comparable to thermal noise, and are generally heterogeneous in nature. This challenging combination of heterogeneity and stochasticity is best investigated experimentally at the level of single molecules, as opposed to more conventional bulk ensemble-average techniques. In recent years, the use of such molecular experimental approaches has become significantly more widespread in research laboratories around the world. In this review we discuss recent experimental approaches in biological physics which can be applied to investigate the living component of soft condensed matter to a precision of a single molecule.

Single-molecule fluorescence microscopy review: shedding new light on old problems

“…When subpopulations are identified, the only way to determine which cells contribute to which group, hence, to separate competing signals, is to analyse the whole population cell-by-cell [6,7]. Population heterogeneity can arise due to environmental alterations affecting the soft matter of biological material [8], as well as through genetic variation that affects gene expression and can invoke fluctuations in various cellular components [9]. Differences in transcriptional regulation affect signal transduction pathways and hence responses to various stress factors, such as pH and oxidative stress.…”

Analytical tools for single-molecule fluorescence imaging in cellulo

“…In vivo singlemolecule approaches, arguably better termed 'in cellulo' approaches if one is observing single individual cells to discriminate from investigations on multicellular organisms (excepting single-celled organisms such as bacteria which can be described in both contexts), add significant insight not only into the native biochemistry of the living cell, but also the important physical chemistry and chemical physics of the cellular environment -the ionic strength, pH, viscosity, microrheological features, phase transition behaviour, osmolarity, as well as a breadth of information concerning free energy landscapes of observed molecular components. [1][2][3][4] The primary importance of single-molecule live-cell data is due to often highly heterogeneous behaviour in such physical parameters both as a function of localization in the cell and of history-dependent effects in the cell cycle. Namely, there are both spatial and temporal dependencies in the internal environment of cells, which therefore require not only a functioning cell as an experimental specimen but also a probe at the molecular length scale of the nanometre to offer a level of spatial precision sufficiently high to probe the highly local fluctuations in physical chemistry that can potentially occur on the nanoscale -even the most basic of living cells from the prokaryotic domain, such as bacteria, are far more than just static bags of chemicals, but rather are dynamic structures which have distinctly defined molecular architectures at the sub-cellular level affecting the physical chemistry of the internal cellular environment.…”

A System-level Approach to Single-Molecule Live-Cell Fluorescence Microscopy

“…A review article was written to assist the key architectural decision of selecting an appropriate single-molecule technique from the diversity that now exists (Harriman and Leake 2011).…”