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.

A glucose starvation response governs endocytic trafficking and eisosomal retention of surface cargoes

“…To better understand these observations, we performed Slimfield microscopy of cells expressing Mup1-GFP and Pil1-mCherry during growth in both glucose and raffinose (Figure 6F). Using single particle tracking (Figure S3e), and previously optimised step-wise photobleach protocols (Leake et al, 2006;Shashkova and Leake, 2017) from micrographs acquired every 5 milliseconds enabling a lateral localization precision of ~40nm (Ford et al, 2001;Itoh et al, 2001;S. E. Miller et al, 2015).…”

Exploring the protein–protein interaction landscape in plants

“…SMI allows the detection of a POI in vivo with a great spatio‐temporal resolution (Danial & García‐Sáez, ; Hibino, Hiroshima, Nakamura, & Sako, ; Shen et al, ). For SMI analysis, the POI is fluorescently labelled, and the emission of an individual fluorescent molecule is recorded continuously by means of an optical microscope with techniques, such as total internal reflection fluorescence microscopy, stochastic optical reconstruction microscopy, stimulated emission depletion, and variable angle excitation microscopy (Langhans & Meckel, ; Shashkova & Leake, ; Shen et al, ). By means of SMI, the protein distribution, diffusion, subunit stoichiometry, or the number of proteins in the cluster can be monitored, and thus, the dynamics of hundreds or thousands of individual molecules over time can be followed (Okamoto, Hiroshima, & Sako, ; Shashkova & Leake, ).…”

Correlating single-molecule characteristics of the yeast aquaglyceroporin Fps1 with environmental perturbations directly in living cells

“…Every population under investigation, whether it is a cell culture or a protein bulk within a unicellular organism, is a heterogeneous system. Therefore, ensemble averaging masks important effects of subpopulations [1][2][3] , such as drug resistant bacteria or cancer cells [4][5][6] . Single-molecule optical biophysics methods permit real-time visualization of key cellular processes 7 , the action of so-called biological 'nanomachines' 8 , such as signal transduction, gene expression, immune response, mapping cellular genome, etc., providing direct insights into molecular mobility, stoichiometry, copy numbers 9,10 .…”