Abstract
Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called ‘green revolution’, has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called ‘super-resolution’ fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques.
Highlights
Why do we care about detecting single molecules in cells?Experimental investigations in the life sciences have traditionally been performed on a population ‘ensemble average’ level
Using a population signature as a metric for the physical or chemical status of different cellular parameters is valuable at one level, since it averages out the observations of potential minor and anomalous cells in that population, in effect smoothing out the ‘noise’
Single-molecule fluorescence microscopy approaches since have uncovered many fundamental molecular scale biological processes that were previously not studied primarily due to the limitations imposed by population methods, including studies of the bacterial flagellar motor rotation [17,18,19,20,21], protein folding, translocation and movement [11,22,23,24,25], signal transduction [26], biopolymer mechanics [27,28,29,30,31,32], DNA replication and remodelling [33,34,35,36,37], oxidative phosphorylation [38,39,40,41], as well as biomedically relevant areas such as the probing of processes relating to infection and general pathology [42,43,44], cell division mechanisms [45], mitochondrial protein dynamics [46], viral infection processes [47], endocytocis and exocytosis pathways [48], osmolarity receptor dynamics [49], cell wall synthesis [50], and structural dynamics of DNA [51]
Summary
Single-molecule fluorescence microscopy review: shedding new light on old problems. Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. We discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called ‘super-resolution’ fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. How these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques. Accepted Manuscript Online: 10 July 2017 Version of Record published: 21 July 2017
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