Imaging Intracellular Fluorescent Proteins at Nanometer Resolution

Betzig, Eric; Patterson, George H.; Sougrat, Rachid; Lindwasser, O. Wolf; Olenych, Scott G.; Bonifacino, Juan S.; Davidson, Michael W.; Lippincott‐Schwartz, Jennifer; Hess, Harald F.

doi:10.1126/science.1127344

Cited by 7,873 publications

(6,855 citation statements)

References 44 publications

Supporting

Mentioning

6,562

Contrasting

Unclassified

Order By: Relevance

“…Objective-type TIR PALM imaging was performed by translating the laser beams away from the central axis of the objective using a set of mirrors in the optical pathway. Construction of PALM images was carried out according to what was described previously (Text S1) [24].…”

Section: Methodsmentioning

confidence: 99%

In Vivo Structure of the E. coli FtsZ-ring Revealed by Photoactivated Localization Microscopy (PALM)

et al. 2010

View full text Add to dashboard Cite

The FtsZ protein, a tubulin-like GTPase, plays a pivotal role in prokaryotic cell division. In vivo it localizes to the midcell and assembles into a ring-like structure-the Z-ring. The Z-ring serves as an essential scaffold to recruit all other division proteins and generates contractile force for cytokinesis, but its supramolecular structure remains unknown. Electron microscopy (EM) has been unsuccessful in detecting the Z-ring due to the dense cytoplasm of bacterial cells, and conventional fluorescence light microscopy (FLM) has only provided images with limited spatial resolution (200–300 nm) due to the diffraction of light. Hence, given the small sizes of bacteria cells, identifying the in vivo structure of the Z-ring presents a substantial challenge. Here, we used photoactivated localization microscopy (PALM), a single molecule-based super-resolution imaging technique, to characterize the in vivo structure of the Z-ring in E. coli. We achieved a spatial resolution of ∼35 nm and discovered that in addition to the expected ring-like conformation, the Z-ring of E. coli adopts a novel compressed helical conformation with variable helical length and pitch. We measured the thickness of the Z-ring to be ∼110 nm and the packing density of FtsZ molecules inside the Z-ring to be greater than what is expected for a single-layered flat ribbon configuration. Our results strongly suggest that the Z-ring is composed of a loose bundle of FtsZ protofilaments that randomly overlap with each other in both longitudinal and radial directions of the cell. Our results provide significant insight into the spatial organization of the Z-ring and open the door for further investigations of structure-function relationships and cell cycle-dependent regulation of the Z-ring.

show abstract

Section: Methodsmentioning

confidence: 99%

In Vivo Structure of the E. coli FtsZ-ring Revealed by Photoactivated Localization Microscopy (PALM)

et al. 2010

View full text Add to dashboard Cite

show abstract

“…Another set of super‐resolution techniques concerns single‐molecule localization imaging, such as photo‐activated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM), which are based on reconstructing the point source of fluorescence (often one molecule) from its detected image which is “blurred” by diffraction (Betzig et al, 2006). These techniques require stochastic excitation of only a small (sparsely distributed) fraction of fluorescent molecules per imaging cycle.…”

Section: Monitoring Of Astroglia On the Nanoscale: Emerging Techniquesmentioning

confidence: 99%

Morphological plasticity of astroglia: Understanding synaptic microenvironment

Heller

Rusakov

2015

Glia

132

137

View full text Add to dashboard Cite

Memory formation in the brain is thought to rely on the remodeling of synaptic connections which eventually results in neural network rewiring. This remodeling is likely to involve ultrathin astroglial protrusions which often occur in the immediate vicinity of excitatory synapses. The phenomenology, cellular mechanisms, and causal relationships of such astroglial restructuring remain, however, poorly understood. This is in large part because monitoring and probing of the underpinning molecular machinery on the scale of nanoscopic astroglial compartments remains a challenge. Here we briefly summarize the current knowledge regarding the cellular organisation of astroglia in the synaptic microenvironment and discuss molecular mechanisms potentially involved in use‐dependent astroglial morphogenesis. We also discuss recent observations concerning morphological astroglial plasticity, the respective monitoring methods, and some of the newly emerging techniques that might help with conceptual advances in the area. GLIA 2015;63:2133–2151

show abstract

“…The diffraction limit has ceased to be a practical limit to resolution in far-field microscopy, following the demonstration of STED, 1,2,3 RESOLFT 4 and localisation microscopies 5,6,7 and the subsequent development of a plethora of super-resolved nanoscopy techniques. 8 In particular, stimulated emission depletion (STED) nanoscopy, which builds on the advantages of laser scanning confocal microscopy, is a powerful technique for super-resolved imaging in complex biological samples including live organisms.…”

Section: Plasmonic Nanoprobes For Stimulated Emission Depletion Nanosmentioning

confidence: 99%

Plasmonic Nanoprobes for Stimulated Emission Depletion Nanoscopy

Cortés¹,

Huidobro²,

Sinclair³

et al. 2016

ACS Nano

View full text Add to dashboard Cite

Plasmonic nanoparticles influence the absorption and emission processes of nearby emitters due to local enhancements of the illuminating radiation and the photonic density of states. Here, we use the plasmon resonance of metal nanoparticles in order to enhance the stimulated depletion of excited molecules for super-resolved nanoscopy. We demonstrate stimulated emission depletion (STED) nanoscopy with gold nanorods with a long axis of only 26 nm and a width of 8 nm that provide an enhancement of up to 50% of the resolution compared to fluorescent-only probes without plasmonic components irradiated with the same depletion power. The nanoparticle-assisted STED probes reported here represent a ~2x10 3 reduction in probe volume compared to previously used nanoparticles. Finally, we demonstrate their application toward plasmon-assisted STED cellular imaging at low-depletion powers and we also discuss their current limitations. Key-words: STED nanoscopy, nanorods, super-resolution, plasmonic nanoparticles, bio-imagingThe diffraction limit has ceased to be a practical limit to resolution in far-field microscopy, following the demonstration of STED, 1,2,3 RESOLFT 4 and localisation microscopies 5,6,7 and the subsequent development of a plethora of super-resolved nanoscopy techniques. 8 In particular, stimulated emission depletion (STED) nanoscopy, which builds on the advantages of laser scanning confocal microscopy, is a powerful technique for super-resolved imaging in complex biological samples including live organisms. 9,10 STED nanoscopy uses stimulated emission to turn off the spontaneous fluorescence emission of dye molecules, typically overlapping a focused excitation beam with a "doughnut" shaped beam that deexcites emitters to the ground state everywhere except for the area within the centre of the doughnut, thus providing theoretically diffraction-unlimited resolution in the transverse plane by reducing the fullwidth half-maximum (FWHM) of the point spread function. By increasing the power of the depletion beam the emission region can be can be drastically reduced -theoretically allowing for sub-nanometre resolution -with resolutions of less than 10 nm being demonstrated. 11 The scaling of resolution with the square root of the depletion beam power means that relatively high-power lasers are typically used for STED nanoscopy. In practice, however, the use of high-power irradiation can result in problems such as photobleaching of the fluorophores and phototoxicity, and so the achievable resolution is compromised by the need to limit the intensity of the depletion laser radiation. Furthermore, high power lasers can add cost and complexity to STED microscopes and so the requirement for high power depletion beams presents challenges for parallelizing STED measurements 12,13 in terms of the lasers required, thus, limiting the potential for faster super-resolved imaging.To some extent the issue of photobleaching can be addressed with the development of more robust fluorescent labels such as quantum dots 14 as ...

show abstract

Imaging Intracellular Fluorescent Proteins at Nanometer Resolution

Cited by 7,873 publications

References 44 publications

In Vivo Structure of the E. coli FtsZ-ring Revealed by Photoactivated Localization Microscopy (PALM)

In Vivo Structure of the E. coli FtsZ-ring Revealed by Photoactivated Localization Microscopy (PALM)

Morphological plasticity of astroglia: Understanding synaptic microenvironment

Plasmonic Nanoprobes for Stimulated Emission Depletion Nanoscopy

Contact Info

Product

Resources

About