Full volume super-resolution imaging of thick mitotic spindle using 3D AO STED microscope

© 2019 Optical Society of America under the terms of the OSA Open Access Publishing. Stimulated emission depletion (STED) nanoscopy is one of a suite of modern optical microscopy techniques capable of bypassing the conventional diffraction limit in fluorescent imaging. STED makes use of a spiral phase mask to enable 2D super-resolution imaging whereas to achieve full volumetric 3D super-resolution an additional bottle-beam phase mask must be applied. The resolution achieved in biological samples 10 μm or thicker is limited by aberrations induced mainly by scattering due to refractive index heterogeneity in the sample. These aberrations impact the fidelity of both types of phase mask, and have limited the application of STED to thicker biological systems. Here we apply an automated adaptive optics solution to correct the performance of both STED masks, enhancing robustness and expanding the capabilities of this nanoscopic technique. Corroboration in terms of successful high-quality imaging of the full volume of a 15μm mitotic spindle with resolution of 50nm x 50nm x 150nm achieved in all three dimensions is presented

Multicolour single molecule imaging on cells using a supercontinuum source

Multicolour single molecule fluorescence imaging enables the study of multiple proteins in the membranes of living cells. We describe the use of a supercontinuum laser as the excitation source, show its comparability with multiplexed single-wavelength lasers and demonstrate that it can be used to study membrane proteins such as the ErbB receptor family. We discuss the benefits of white-light sources for single molecule fluorescence, in particular their ease of use and the freedom to use the most appropriate dye without being constrained by available laser wavelengths

Screening and engineering of colour centres in diamond

We present a high throughput and systematic method for the screening of colour centres in diamond with the aim of searching for and reproducibly creating new optical centres down to the single defect level, potentially of interest for a wide range of diamond-based quantum applications. The screening method presented here should, moreover, help to identify some already indexed defects among hundreds in diamond (Zaitsev 2001 Optical Properties of Diamond (Berlin: Springer)) but also some promising defects of a still unknown nature, such as the recently discovered ST1 centre (Lee et al 2013 Nat. Nanotechnol. 8 487; John et al 2017 New J. Phys. 19 053008). We use ion implantation in a systematic manner to implant several chemical elements. Ion implantation has the advantage of addressing single atoms inside the bulk with defined depth and high lateral resolution, but the disadvantage of producing intrinsic defects. The implanted samples are annealed in vacuum at different temperatures (between 600 degrees C and 1600 degrees C with 200 degrees C steps) and fully characterised at each step in order to follow the evolution of the defects: formation, dissociation, diffusion, re-formation and charge state, at the ensemble level and, if possible, at the single centre level. We review the unavoidable ion implantation defects (such as the GR1 and 3H centres), discuss ion channeling and thermal annealing and estimate the diffusion of the vacancies, nitrogen and hydrogen. We use different characterisation methods best suited for our study (from widefield fluorescence down to subdiffraction optical imaging of single centres) and discuss reproducibility issues due to diamond and defect inhomogeneities. Nitrogen is also implanted for reference, taking advantage of the considerable knowledge on NV centres as a versatile sensor in order to retrieve or deduce the conditions and local environment in which the different implanted chemical elements are embedded. We show here the preliminary promising results of a long-term study and focus on the elements O, Mg, Ca, F and P from which fluorescent centres were found.Peer reviewe

Fourier Magnetic Imaging with Nanoscale Resolution and Compressed Sensing Speed-up using Electronic Spins in Diamond

Optically-detected magnetic resonance using Nitrogen Vacancy (NV) color centres in diamond is a leading modality for nanoscale magnetic field imaging, as it provides single electron spin sensitivity, three-dimensional resolution better than 1 nm, and applicability to a wide range of physical and biological samples under ambient conditions. To date, however, NV-diamond magnetic imaging has been performed using real space techniques, which are either limited by optical diffraction to 250 nm resolution or require slow, point-by-point scanning for nanoscale resolution, e.g., using an atomic force microscope, magnetic tip, or super-resolution optical imaging. Here we introduce an alternative technique of Fourier magnetic imaging using NV-diamond. In analogy with conventional magnetic resonance imaging (MRI), we employ pulsed magnetic field gradients to phase-encode spatial information on NV electronic spins in wavenumber or k-space followed by a fast Fourier transform to yield real-space images with nanoscale resolution, wide field-of-view (FOV), and compressed sensing speed-up.Comment: 31 pages, 10 figure

Power dependence of supercontinuum noise in uniform and tapered PCFs

An error was made in the calculation of the relative intensity noise (RIN) because of an incorrectly specified value of the photodetector DC transimpedance gain

Formation of a Bazooka–Stardust complex is essential for plasma membrane polarity in epithelia

Recruitment of the Crumbs–Stardust polarity complex depends on interactions between Bazooka and the Stardust PDZ domain and is regulated by aPKC-mediated phosphorylation

Subdiffraction, Luminescence-Depletion Imaging of Isolated, Giant, CdSe/CdS Nanocrystal Quantum Dots

Subdiffraction spatial resolution luminescence depletion imaging was performed with giant CdSe/14CdS nanocrystal quantum dots (g-NQDs) dispersed on a glass slide. Luminescence depletion imaging used a Gaussian shaped excitation laser pulse overlapped with a depletion pulse, shaped into a doughnut profile, with zero intensity in the center. Luminescence from a subdiffraction volume is collected from the central portion of the excitation spot, where no depletion takes place. Up to 92% depletion of the luminescence signal was achieved. An average full width at half-maximum of 40 ± 10 nm was measured in the lateral direction for isolated g-NQDs at an air interface using luminescence depletion imaging, whereas the average full width at half-maximum was 450 ± 90 nm using diffraction-limited, confocal luminescence imaging. Time-gating of the luminescence depletion data was required to achieve the stated spatial resolution. No observable photobleaching of the g-NQDs was present in the measurements, which allowed imaging with a dwell time of 250 ms per pixel to obtain images with a high signal-to-noise ratio. The mechanism for luminescence depletion is likely stimulated emission, stimulated absorption, or a combination of the two. The g-NQDs fulfill a need for versatile, photostable tags for subdiffraction imaging schemes where high laser powers or long exposure times are used