Nanoscopic Cellular Imaging: Confinement Broadens Understanding

Lee, Stephen A.; Ponjavic, Aleks; Siv, Chanrith; Lee, Steven F.; Biteen, Julie S.

doi:10.1021/acsnano.6b02863

Cited by 13 publications

(15 citation statements)

References 144 publications

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“…Therefore, samples with a low signal/noise ratio can be more difficult to image, requiring careful experimental optimization and design. Fluorescence-background-reduction techniques such as selective plane illumination could be employed to increase signal/noise ratios (66) and offset this effect. The large depth of field of DHPSF is ideally suited to implementing simple light-sheet systems that typically have a thickness of a few microns (67).…”

Section: Advantages and Disadvantages Of The Dhpsfmentioning

confidence: 99%

Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function

Carr

Ponjavic

Basu

et al. 2017

Biophysical Journal

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Single-molecule localization microscopy, typically based on total internal reflection illumination, has taken our understanding of protein organization and dynamics in cells beyond the diffraction limit. However, biological systems exist in a complicated three-dimensional environment, which has required the development of new techniques, including the double-helix point spread function (DHPSF), to accurately visualize biological processes. The application of the DHPSF approach has so far been limited to the study of relatively small prokaryotic cells. By matching the refractive index of the objective lens immersion liquid to that of the sample media, we demonstrate DHPSF imaging of up to 15-μm-thick whole eukaryotic cell volumes in three to five imaging planes. We illustrate the capabilities of the DHPSF by exploring large-scale membrane reorganization in human T cells after receptor triggering, and by using single-particle tracking to image several mammalian proteins, including membrane, cytoplasmic, and nuclear proteins in T cells and embryonic stem cells.

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Section: Advantages and Disadvantages Of The Dhpsfmentioning

confidence: 99%

Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function

Carr

Ponjavic

Basu

et al. 2017

Biophysical Journal

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Section: Plasmonic Nanoprobes For Stimulated Emission Depletion Nanosmentioning

confidence: 99%

“…This light is then concentrated in the vicinity of the nanoparticle and can be used, for example, to deplete excited fluorescent molecules, among others. 20,21,22,23 Thus, if the LSPR of the metal NP is centred at the depletion-laser wavelength, the input depletion-power requirements for STED microscopy can be met at much lower intensities than normally used.Our recent demonstration of the proof of concept of nanoparticle-assisted STED (NP-STED) nanoscopy utilized 160 nm core@shell (silica@Au) spherical particles, with a plasmon resonance at ~800 nm and fluorescent dyes embedded in the silica core. 17 However, such large NPs are not practically useful for most studies in biology.…”

mentioning

confidence: 99%

Plasmonic Nanoprobes for Stimulated Emission Depletion Nanoscopy

Cortés¹,

Huidobro²,

Sinclair³

et al. 2016

ACS Nano

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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 ...

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“…In recent years, new techniques have been developed that can image individual membrane proteins away from the coverslip interface (28). For example, single-molecule light-sheet microscopy (smLSM) has been used to monitor the reorganization of the TCR during T cell activation at subdiffraction resolution (29).…”

Section: Introductionmentioning

confidence: 99%

Single-Molecule Light-Sheet Imaging of Suspended T Cells

Ponjavic

Carr

Santos

et al. 2018

Biophysical Journal

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Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose.

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Nanoscopic Cellular Imaging: Confinement Broadens Understanding

Cited by 13 publications

References 144 publications

Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function

Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function

Plasmonic Nanoprobes for Stimulated Emission Depletion Nanoscopy

Single-Molecule Light-Sheet Imaging of Suspended T Cells

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