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

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.

Keywords: fluorescence; microscopy; single-molecule; super-resolution.

Figure 1. Jablonski diagram

An electron of a fluorophore at the ground state (S₀) receives energy from the absorption of a single photon of light which results in an excitation transition to a higher energy state (absorption). When the excited electron relaxes to the ground state, following vibrational losses, energy, lower than the incident photon and thus with a higher wavelength, is emitted as a single photon which causes fluorescence.

Figure 2. Schematic representation

Of: (A) Slimfield imaging; (B) a confocal microscope; (C), TIRF showing the illumination of fluorophores close to the glass coverslip surface (detailed explanation is provided in the text); (D) HILO microscopy.

Figure 3. Examples of methods used in single-molecule studies

(A) FRET principle based on the non-radiative energy transfer which occurs when donor and acceptor dye pairs (often, but not exclusively, fluorophores) are positioned within typically less than approximately 10 nm of each other (explanation is provided in the text); (B) FRAP illustrating photobleaching of fluorophores in a delimited region of a biological sample, here shown with a single budding yeast cell in which fluorescently labelled material in the nucleus is photobleached, followed by the measurement of fluorescence recovery over time; (C) SMLM techniques (here exemplified with PALM) illustrating selective activation of fluorophores and the final image after multiple photoactivation cycle repeats; and (D) STED showing excitation and depletion laser beams, and the effective fluorescence profile following stimulated depletion. Abbreviations: PALM, photoactivated localization microscopy; SMLM, single molecule localization microscopy; STED, stimulated emission depletion.