Ultrahigh Resolution Imaging of Biomolecules by Fluorescence Photoactivation Localization Microscopy

Hess, Samuel T.; Gould, Travis J.; Gunewardene, Mudalige S.; Bewersdorf, Joerg; Mason, Michael D.

doi:10.1007/978-1-59745-483-4_32

Cited by 61 publications

(50 citation statements)

References 59 publications

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“…It follows that this convenient imaging technique cannot appreciate the subtle factors that govern biological processes and define biological structures at the molecular level. These stringent limitations can be overcome with the aid of photoactivatable fluorophores [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42]. Indeed, the ability to switch fluorescence on under optical control permits the separation of distinct probes in time and the sequential reconstruction of images with subdiffraction resolution.…”

Section: Isrn Physical Chemistrymentioning

confidence: 99%

“…Indeed, fluorescence photoactivation permits the monitoring of dynamic processes in real time [13][14][15][16][17][18][19][20][21][22][23][24] as well as the acquisition of images with spatial resolution at the nanometer level [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42]. As a result, significant research efforts are presently directed to the development of strategies for fluorescence photoactivation with fluorescent proteins and synthetic dyes together with their implementation in imaging applications.…”

Section: Introductionmentioning

confidence: 99%

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Photoactivatable Fluorophores

Raymo

2012

ISRN Physical Chemistry

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Photoactivatable fluorophores switch from a nonemissive to an emissive state upon illumination at an activating wavelength and then emit after irradiation at an exciting wavelength. The interplay of such activation and excitation events can be exploited to switch fluorescence on in a defined region of space at a given interval of time. In turn, the spatiotemporal control of fluorescence translates into the opportunity to implement imaging and spectroscopic schemes that are not possible with conventional fluorophores. Specifically, photoactivatable fluorophores permit the monitoring of dynamic processes in real time as well as the reconstruction of images with subdiffraction resolution. These promising applications can have a significant impact on the characterization of the structures and functions of biomolecular systems. As a result, strategies to implement mechanisms for fluorescence photoactivation with synthetic fluorophores are particularly valuable. In fact, a number of versatile operating principles have already been identified to activate the fluorescence of numerous members of the main families of synthetic dyes. These methods are based on either the irreversible cleavage of covalent bonds or the reversible opening and closing of rings. This paper overviews the fundamental mechanisms that govern the behavior of these photoresponsive systems, illustrates structural designs for fluorescence photoactivation, and provides representative examples of photoactivatable fluorophores in actions.

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Section: Isrn Physical Chemistrymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Photoactivatable Fluorophores

Raymo

2012

ISRN Physical Chemistry

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“…The measured molecule intensity signal is influenced by both fluctuations in camera shot noise (Poisson distributed) and photons coming from sources other than the molecule of interest, which is referred to as the background intensity (2). The background intensity can be affected considerably by a number of global and local effects, including inhomogeneous or fluctuating laser spot profiles and crowded molecular environments (10,11). Particularly in dense samples, photons coming from neighboring molecules may contribute substantially to the background intensity.…”

Section: Introductionmentioning

confidence: 99%

Optimal Background Estimators in Single-Molecule FRET Microscopy

Preus

Hildebrandt

Birkedal

2016

Biophysical Journal

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Single-molecule total internal reflection fluorescence (TIRF) microscopy constitutes an umbrella of powerful tools that facilitate direct observation of the biophysical properties, population heterogeneities, and interactions of single biomolecules without the need for ensemble synchronization. Due to the low signal/noise ratio in single-molecule TIRF microscopy experiments, it is important to determine the local background intensity, especially when the fluorescence intensity of the molecule is used quantitatively. Here we compare and evaluate the performance of different aperture-based background estimators used particularly in single-molecule Fö rster resonance energy transfer. We introduce the general concept of multiaperture signatures and use this technique to demonstrate how the choice of background can affect the measured fluorescence signal considerably. A new, to our knowledge, and simple background estimator is proposed, called the local statistical percentile (LSP). We show that the LSP background estimator performs as well as current background estimators at low molecular densities and significantly better in regions of high molecular densities. The LSP background estimator is thus suited for single-particle TIRF microscopy of dense biological samples in which the intensity itself is an observable of the technique.

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“…Despite the variety of methods which emerged subsequently [7][8][9][10][11][12][13][14], for years nano- …”

Section: Introductionmentioning

confidence: 99%

Comparing video‐rate STED nanoscopy and confocal microscopy of living neurons

Lauterbach

Keller

Schönle

et al. 2010

Journal of Biophotonics

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We compare the performance of video‐rate Stimulated Emission Depletion (STED) and confocal microscopy in imaging the interior of living neurons. A lateral resolution of 65 nm is observed in STED movies of 28 frames per second, which is 4‐fold higher in spatial resolution than in their confocal counterparts. STED microscopy, but not confocal microscopy, allows discrimination of single features at high spatial densities. Specific patterns of movement within the confined space of the axon are revealed in STED microscopy, while confocal imaging is limited to reporting gross motion. Further progress is to be expected, as we demonstrate that the use of continuous wave (CW) beams for excitation and STED is viable for video‐rate STED recording of living neurons. Tentatively providing a larger photon flux, CW beams should facilitate extending fast STED imaging towards imaging fainter living samples. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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Ultrahigh Resolution Imaging of Biomolecules by Fluorescence Photoactivation Localization Microscopy

Cited by 61 publications

References 59 publications

Photoactivatable Fluorophores

Photoactivatable Fluorophores

Optimal Background Estimators in Single-Molecule FRET Microscopy

Comparing video‐rate STED nanoscopy and confocal microscopy of living neurons

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