Detectors for single-molecule fluorescence imaging and spectroscopy

Michalet, Xavier; Siegmund, Oswald H. W.; Vallerga, J. V.; Jelinsky, Patrick; Millaud, J.; Weiss, Shimon

doi:10.1080/09500340600769067

Cited by 116 publications

(106 citation statements)

References 148 publications

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“…TIR Microscopy has gained popularity partly spurred by the new wave of highly sensitive and fast frame transfer electron-multiplying charged couple device (EM-CCD) cameras 3,42 . smFRET setups usually employ EM-CCDs that have high quantum efficiency (85-95%) in the 450 -700 nm range, low effective read out noise (< 1 electron rms) even at the fastest readout speed (≥ 10 MHz), fast vertical shift speeds (≤ 1 sec/row; to achieve faster frame rates) and a low multiplication noise.…”

Section: Emission Detectionmentioning

confidence: 99%

A practical guide to single-molecule FRET

2008

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Despite the explosive growth in the biological applications of single molecule methods over the last decade, these techniques have thus far been practiced mostly by researchers who are biophysically oriented. This is partly because of the lack of commercial instruments in many cases and also because of the perceived steep learning curve and need for expensive equipments. We wish to provide a practical guide to using Förster (or Fluorescence) Resonance Energy Transfer (FRET) at the single molecule level, focusing on the study of immobilized molecules that allow measurements of single molecule reaction trajectories from about 1 millisecond to many minutes. An instrument can be built at a reasonable cost using various off-the-shelf components and operated reliably using current well-established protocols and freely available software.The future holds the promise of personalized DNA sequencing and high throughput screening for pathogens at affordable cost and viable time. These promises are riding high on a surge of single-molecule based technologies that enable us to manipulate and probe individual molecules. Using this approach, several important biological riddles that have intrigued scientists for a long time are coming under the microscope. As Feynman famously said "It is very easy to answer many of these fundamental biological questions; you just look at the thing!" 1 . Single-molecule methods are allowing us to do just that 2,3 . They may one day become an elementary tool for characterizing proteins, signaling pathways or any biological phenomenon. In the hopes of facilitating this objective, we provide a brief, but practical guide for single-molecule FRET 4 measurements (smFRET) [5][6][7] , one of the most general and adaptable single-molecule techniques. Since its humble beginning under nonaqueous conditions in 1996 8 , smFRET has rapidly developed to answer fundamental questions about replication, recombination, transcription, translation, RNA folding and catalysis, non-canonical DNA dynamics, protein folding and conformational changes, various motor proteins, membrane fusion proteins, ion channels, signal transduction, to name just a few, and the list keeps growing at a fast pace. Since it is not the purpose of this review to survey the vast literature on such studies, we refer the reader to reviews in the field and the references therein 6, 9-13 .Correspondence to: Taekjip Ha. Competing interests statement:The authors declare no competing financial interests. In FRET measurements, the extent of non-radiative energy transfer between two fluorescent dye molecules, termed donor and acceptor, reports the intervening distance which can be estimated from the ratio of acceptor to total emission intensity ( Fig. 1) 4,14,15 . This efficiency of energy transfer, E is given as E = [1 + (R/R 0 ) 6 ] −1 , where R is the inter-dye distance and R 0 is the Förster radius at which E = 0.5 (Fig. 1a). Conformational dynamics of single molecules can be observed in real-time by tracking FRET changes (Fig. 1b). The ad...

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Section: Emission Detectionmentioning

confidence: 99%

A practical guide to single-molecule FRET

2008

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“…The practical localization precision is bounded by the Cramér-Rao lower bound, which depends in a nonlinear manner on both SNR and SBR [9,[116][117][118][119]. A good approximation, provided the PSF image is sufficiently well sampled, is given by the product of the PSF size (s) and the inverse of the SNR.…”

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

confidence: 99%

“…Different sensor technologies and readout designs exist, most of which are compatible with single-molecule detection, provided they meet a few performance requirements [9]. Back-thinned CCD cameras have the best sensitivity (figure 1b) but relatively low readout rate and suffer from increasing readout noise at higher frame rate.…”

Section: (D) Detectors Used For Wide Field Single-molecule Imagingmentioning

confidence: 99%

“…If present, readout noise should be minimized, or at least compensated by additional signal gain (such as in intensified CCDs or EMCCDs). We will not discuss this latter strategy, as it is irrelevant for the photon-counting detectors discussed here, but it is worth mentioning that it comes at a cost: the gain generates additional signal variance, quantified as an excess noise factor (ENF), which translates into reduced SNR [8,9]. Finally, large dark count rate can affect some detectors, reducing the SBR and making it difficult to detect single molecules.…”

Section: Introductionmentioning

confidence: 99%

“…A simple way to increase burst size would seem to be increasing the excitation power. However, this is not always possible, and advantageous only up to a certain point: owing to emission saturation and photobleaching, background eventually increases faster than signal, reducing SBR and SNR [9].…”

Section: Introductionmentioning

confidence: 99%

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

Michalet

Colyer

Scalia

et al. 2013

Phil. Trans. R. Soc. B

Self Cite

105

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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Single‐molecule Fluorescence Imaging Techniques

Reid

Rothenberg

2015

Encyclopedia of Analytical Chemistry

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The past decade has been witnessed to exciting developments in advanced fluorescence microscopy techniques that rely on visualizing single emitting fluorophores. The proliferation of single‐molecule fluorescent imaging techniques and their application in biological research have the potential to revolutionize how research is performed and greatly increase our understanding of biological systems. Presently, these techniques are still relatively niche owing to technological barriers, but it is foreseeable that they will become an increasingly common way in which insights are sought in biology. Here, we review the basic principles of key single‐molecule techniques and their recent biological applications.

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Detectors for single-molecule fluorescence imaging and spectroscopy

Cited by 116 publications

References 148 publications

A practical guide to single-molecule FRET

A practical guide to single-molecule FRET

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

Single‐molecule Fluorescence Imaging Techniques

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