Do-it-yourself guide: how to use the modern single-molecule toolkit

Walter, Nils G.; Huang, Chunjuan; Manzo, Anthony J.; Sobhy, Mohamed A.

doi:10.1038/nmeth.1215

Cited by 302 publications

(289 citation statements)

References 137 publications

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“…In these cases, less than a hundred molecules are turned on at any time, but they are replaced during later acquisition stages by another random set of molecules. The image is then reconstructed using all single-molecule coordinates [113][114][115]. SNR and SBR considerations in wide field microscopy are slightly different from those discussed in the point-like geometry situation.…”

Section: (B) Single-molecule Detection and Analysis In Wide Field Geomentioning

confidence: 99%

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Michalet

Colyer

Scalia

et al. 2013

Phil. Trans. R. Soc. B

104

121

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Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level

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Section: (B) Single-molecule Detection and Analysis In Wide Field Geomentioning

confidence: 99%

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Michalet

Colyer

Scalia

et al. 2013

Phil. Trans. R. Soc. B

104

121

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“…Technological advances will be instrumental for creating new methods and improving current methods. For example, it is important to increase spatial and temporal resolution and sensitivity of microscopes, while minimizing phototoxicity (Hoebe et al 2007), to reveal details on dynamic organization of signalling complexes (Hell 2007;Walter et al 2008). Probe development is another very important and active area.…”

Section: Futurementioning

confidence: 99%

Fluorescence resonance energy transfer imaging of PKC signalling in living cells using genetically encoded fluorescent probes

Goedhart

Gadella

2008

J. R. Soc. Interface.

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Perception of ligands in the extracellular space by transmembrane receptors initiates signal transduction. The conformation change of the receptor induces changes of intracellular signalling components, including altered cellular concentration, altered subcellular location, altered conformation and altered interacting partners. Biochemical approaches have yielded a lot of information about these processes. However, methods that are compatible with analysis of single living cells are often preferred, since cells are highly organized and their response is usually spatially heterogeneous. In addition, the study of signalling cascades requires high temporal resolution. Fluorescence imaging approaches meet these requirements. Moreover, imaging approaches can be combined with genetically encoded green fluorescent protein-based probes that have a high selectivity and sensitivity for the process/molecule of interest. Nowadays, many genetically encoded probes are available for visualizing signalling in living cells. This review is centred on a key regulator of cellular signalling, protein kinase C (PKC). We will discuss imaging approaches that are used for analysing the molecules involved in activation of PKC, visualizing the dynamics of the location of PKC, measuring the conformation of PKC and quantifying the activity of PKC. These approaches are of general interest since they can be applied to study the dynamics, conformation and activity of any protein in living cells.

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“…2 To measure fluorescence from a single dye molecule is not an easy task because the signal is weak and the dye has a finite fluorescence lifetime. 3 When a dye molecule is excited by an excitation light source such as a laser, it becomes more vulnerable to oxidation.…”

Section: Introductionmentioning

confidence: 99%

The Effect of the Oxygen Scavenging System on the pH of Buffered Sample Solutions: in the Context of Single-molecule Fluorescence Measurements

Kim¹,

Lee²,

Hong³

2012

Bulletin of the Korean Chemical Society

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In single-molecule fluorescence experiment, the oxygen scavenging system is indispensable for avoiding photo-bleaching of fluorescent dyes. Here we report that the gloxy-based oxygen scavenging system commonly used in single molecule fluorescence experiments can disturb the solution pH considerably. To track in situ pH change, we utilized the pH-sensitive conformational transition of i-motif and examined the transition with ensemble and single-molecule FRET measurements. Based on our results, we also suggested several practical remedies for the stability of the solution pH.

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Do-it-yourself guide: how to use the modern single-molecule toolkit

Cited by 302 publications

References 137 publications

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Fluorescence resonance energy transfer imaging of PKC signalling in living cells using genetically encoded fluorescent probes

The Effect of the Oxygen Scavenging System on the pH of Buffered Sample Solutions: in the Context of Single-molecule Fluorescence Measurements

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