Photoswitching microscopy with standard fluorophores

Linde, Sebastian van de; Kasper, Robert; Heilemann, Mike; Sauer, Markus

doi:10.1007/s00340-008-3250-9

Cited by 102 publications

(91 citation statements)

References 42 publications

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“…12, 13, and 15. Cells were prebleached for approximately 5 s to push a large proportion of fluorophores into the dark state (13,15) using only the red laser. Image sequences were then acquired, streamed to disk, and analyzed in real time using custom software.…”

Section: Methodsmentioning

confidence: 99%

“…The development of imaging techniques based on the time-multiplexed observation of single fluorescent molecules allows high-contrast fluorescence imaging with a resolution limited only by signal to noise (10)(11)(12). Using standard fluorochromes (13)(14)(15) we have used this approach to investigate the distribution of peripheral RyRs with a resolution of approximately 30 nm. Quantitative analysis of our data shows that the size and morphology of RyR clusters in peripheral couplings is quite different from that deduced by previous studies.…”

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confidence: 99%

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Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes

Baddeley

Jayasinghe

Lam

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

264

347

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We have applied an optical super-resolution technique based on single-molecule localization to examine the peripheral distribution of a cardiac signaling protein, the ryanodine receptor (RyR), in rat ventricular myocytes. RyRs form clusters with a mean size of approximately 14 RyRs per cluster, which is almost an order of magnitude smaller than previously estimated. Clusters were typically not circular (as previously assumed) but elongated with an average aspect ratio of 1.9. Edge-to-edge distances between adjacent RyR clusters were often <50 nm, suggesting that peripheral RyR clusters may exhibit strong intercluster signaling. The wide variation of cluster size, which follows a near-exponential distribution, is compatible with a stochastic cluster assembly process. We suggest that calcium sparks may be the result of the concerted activation of several RyR clusters forming a functional ''supercluster'' whose gating is controlled by both cytosolic and sarcoplasmic reticulum luminal calcium levels.excitation-contraction coupling ͉ fluorescence ͉ heart ͉ single molecule ͉ super-resolution

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes

Baddeley

Jayasinghe

Lam

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

264

347

View full text Add to dashboard Cite

show abstract

“…Thus, it is required that at any moment, most molecules are prepared in a dark state. Recently, it has been demonstrated that simply blinking of molecules, e.g., because of triplet formation, can be used, which drastically expands the range of usable fluorescent labels (24,25,38). Thus far, however, this blinking required oxygen removal (24,25,39), and the on-counts and off-times were poorly defined, although they determine the obtainable temporal and spatial resolution (25).…”

Section: [1]mentioning

confidence: 99%

Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy

Vogelsang

Cordes

Forthmann

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

253

331

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Fluorescent molecular switches have widespread potential for use as sensors, material applications in electro-optical data storages and displays, and superresolution fluorescence microscopy. We demonstrate that adjustment of fluorophore properties and environmental conditions allows the use of ordinary fluorescent dyes as efficient single-molecule switches that report sensitively on their local redox condition. Adding or removing reductant or oxidant, switches the fluorescence of oxazine dyes between stable fluorescent and nonfluorescent states. At low oxygen concentrations, the off-state that we ascribe to a radical anion is thermally stable with a lifetime in the minutes range. The molecular switches show a remarkable reliability with intriguing fatigue resistance at the single-molecule level: Depending on the switching rate, between 400 and 3,000 switching cycles are observed before irreversible photodestruction occurs. A detailed picture of the underlying photoinduced and redox reactions is elaborated. In the presence of both reductant and oxidant, continuous switching is manifested by ''blinking'' with independently controllable on-and off-state lifetimes in both deoxygenated and oxygenated environments. This ''continuous switching mode'' is advantageously used for imaging actin filament and actin filament bundles in fixed cells with subdiffraction-limited resolution.electron transfer ͉ molecular switch ͉ sensor ͉ single-molecule spectroscopy M olecular switches are single-molecule devices, and hence they are key building-blocks for future, bottom-up nanotechnological devices in computers, data storages, (bio-) sensors, and displays. Furthermore, they are exciting molecules for triggered drug-release or for superresolution imaging (1-3). Molecular switches possess at least 2 stable states and can be converted between these states by external stimuli. These external stimuli can essentially be any kind of physical or chemical trigger, such as light, electricity, or certain chemical reactions. Fluorescence is a preferred transduction mechanism because of its ease of noninvasive detection with ultimate sensitivity (i.e., single-molecule sensitivity). For future devices and for superresolution microscopy, it is also important that the molecular switches can be operated at the level of single molecules. This requirement imposes further demands with respect to reliability, reproducibility, response time, and fatigue resistance. Although the single-molecule approach provides detailed information about the properties and mechanisms, only a few examples of fluorescent switches have been demonstrated (4, 5). Among the fluorescent switches, there are systems that use 2 different wavelengths for switching (photoswitchable molecules) (6-13), those that switch the efficiency of photoinduced electron transfer (14, 15) and those that respond to pH changes (15,16).Here, we show that based on a detailed understanding of their photophysics, ordinary, fluorescent dyes can act as single-molecule switches and sensors. By adapting...

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“…For SELM on Klarite and nanovoid surfaces, image stacks of 3000 to 10000 frames with 20 ms integration time each were acquired. Strikingly, we observed very strong fluorescence blinking of the dye in the absence of any external agent such as a 'switching buffer', which is typically used in stochastic optical reconstruction microscopy [17] . A static gold background was removed effectively with a subtraction algorithm calculating a slowly converging median for improved localisation.…”

mentioning

confidence: 91%

Visualisation of plasmonic fields at the nanoscale with single molecule localisation microscopy

et al. 2015

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Plasmonic coupling of light to free electrons on metallic surfaces allows the confinement of electric fields far below the optical diffraction limit. Scattering processes of molecules placed into these plasmonic 'hotspots' are dramatically enhanced [1] which is commonly used to increase the sensitivity of spectroscopic techniques for biological and chemical sensor applications [2,3] . Strikingly, hardly any measurement technique exists for the direct visualisation and characterisation of the underlying nanoscopic electromagnetic field distributions that either do not perturb the field [3,4] or require complex electron beam imaging [5] . In this paper we introduce surface enhanced localisation microscopy (SELM), demonstrating the direct visualisation of fields on patterned plasmonic substrates using optical super resolution microscopy [6] . The observed strong photo-blinking behaviour of single molecules in plasmonic fields is exploited in SELM to map electromagnetic field distributions at nanometer resolutions.Engineered nanostructured surfaces, which can sustain plasmon modes, are extremely important in technological applications such as enhancement of Raman scattering for detection of single molecules [7] or to obtain reproducible characteristics for quantitative diagnostics [8] . The morphological characterisation of such nanostructures is generally obtained using scanning electron microscopy techniques [9] . Recently, using surface-attached photoactivatable fluorescent proteins morphological optical imaging of metallic nanostructures has also been shown to be possible [10] . Nonetheless, only a few techniques allow measuring electromagnetic field distributions of plasmonic modes with nanometer resolution [5,11] without distortion and special or complex requirements such as photoactivable molecules. Therefore, finite and boundary element simulations are widely used to predict the field distribution on plasmonic surfaces, but this comes with significant uncertainties and limitations. Simulation results are algorithm dependent and can only predict fields for 'perfect' structures. In practice, however, structures free of imperfections do not exist [12] .We demonstrate our technique to map nanoscopic EM field patterns in exemplar plasmonic structures, such as: Nanovoids, 'dish-like' structures with variable diameter D [13] ( Figure 1A) and Klarite®, featuring a pyramidal pit structure as shown in Figure 1B. SEM images of both structures are shown in Figure 2A

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Photoswitching microscopy with standard fluorophores

Cited by 102 publications

References 42 publications

Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes

Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes

Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy

Visualisation of plasmonic fields at the nanoscale with single molecule localisation microscopy

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