Recent advances in fluorescent probe technology have improved spatial and temporal resolution, bringing us closer to the ideal of imaging individual cellular features in real time with molecular (1–5 nm) resolution. In parallel, the development of super-resolution imaging techniques has revolutionized fluorescence microscopy. In 1873, Ernst Abbe discovered that features closer than ∼200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1–5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.

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Fluorescent probes for super-resolution imaging in living cells

Carbon Nanomaterials for Biological Imaging and Nanomedicinal Therapy

Breaking the Diffraction Barrier: Super-Resolution Imaging of Cells

The isolated electronic spin system of the nitrogen-vacancy (NV) centre in diamond offers unique possibilities to be employed as a nanoscale sensor for detection and imaging of weak magnetic fields. Magnetic imaging with nanometric resolution and field detection capabilities in the nanotesla range are enabled by the atomic-size and exceptionally long spin-coherence times of this naturally occurring defect. The exciting perspectives that ensue from these characteristics have triggered vivid experimental activities in the emerging field of 'NV magnetometry'. It is the purpose of this article to review the recent progress in high-sensitivity nanoscale NV magnetometry, generate an overview of the most pertinent results of the last years and highlight perspectives for future developments. We will present the physical principles that allow for magnetic field detection with NV centres and discuss first applications of NV magnetometers that have been demonstrated in the context of nano magnetism, mesoscopic physics and the life sciences.

https://iopscience.iop.org/article/10.1088/0034-4885/77/5/056503/pdf

Magnetometry with nitrogen-vacancy defects in diamond

Processing of materials by ultrashort laser pulses has evolved significantly over the last decade and is starting to reveal its scientific, technological and industrial potential. In ultrafast laser manufacturing, optical energy of tightly focused femtosecond or picosecond laser pulses can be delivered to precisely defined positions in the bulk of materials via two-/multi-photon excitation on a timescale much faster than thermal energy exchange between photoexcited electrons and lattice ions. Control of photo-ionization and thermal processes with the highest precision, inducing local photomodification in sub-100-nm-sized regions has been achieved. State-of-the-art ultrashort laser processing techniques exploit high 0.1–1 μm spatial resolution and almost unrestricted three-dimensional structuring capability. Adjustable pulse duration, spatiotemporal chirp, phase front tilt and polarization allow control of photomodification via uniquely wide parameter space. Mature opto-electrical/mechanical technologies have enabled laser processing speeds approaching meters-per-second, leading to a fast lab-to-fab transfer. The key aspects and latest achievements are reviewed with an emphasis on the fundamental relation between spatial resolution and total fabrication throughput. Emerging biomedical applications implementing micrometer feature precision over centimeter-scale scaffolds and photonic wire bonding in telecommunications are highlighted.

/pdf/ultrafast-laser-processing-of-materials-from-science-to-rducz2yjvs.pdf

Ultrafast laser processing of materials: from science to industry

Based on a far-field fluorescence-based optical super-resolution scheme – stimulated emission depletion microscopy – scientists resolve densely packed individual fluorescent colour centres inside crystals with a far-field spatial resolution of 5.8 nm without photobleaching. The approach will support future studies of solid-state single-photon sources and quantum optics.

/pdf/sted-microscopy-reveals-crystal-colour-centres-with-2j8r280w7h.pdf

STED microscopy reveals crystal colour centres with nanometric resolution.

Quantum dot (QD) nanocrystals remain at the forefront of fluorescence microscopy as they have the advantages of enhanced photostability, high quantum yield, and macromolecular size. Furthermore, the ability to tune the QD fluorescence, either by changing their size or by doping, allows for multiplexed imaging. The range of applications extends well beyond the realm of microscopy: QDs may also play a major role in developing novel photonic devices including lasers, light-emitting diodes, and displays. Despite significant advancements in nanocrystal research, the inability to directly modulate the fluorescence from QDs has precluded their implementation in several areas. In particular, emerging far-field diffraction-unlimited microscopy techniques uniquely benefit from the capability to reversibly modulate/switch fluorescent ensembles from a bright “on” state to a dark “off” state. This activation must occur as a response to optical stimuli which do not contain spectral components within the excitation kernel of the fluorescent markers. With the need for optical control over QD fluorescence, indirect methods have been conceived by using hybrid QD structures that incorporate a photochromic activator/quencher. Although the concept has been clearly established, hybrid QD structures suffer from inherent drawbacks, such as inadequate photostability, limited fluorescence quenching, and sensitivity to local environment/ solvent. Herein we report on the direct light-driven modulation of QD fluorescence. The mechanism for the fluorescence modulation relies only on internal electronic transitions within Mn-doped ZnSe quantum dots (Mn-QDs). It is demonstrated that the fluorescence of the QD can be reversibly depleted with efficiencies of over 90% by using continuous-wave optical intensities of approximately 1.9 MWcm . Time-domain measurements during the modulation indicate that the number of fluorescent on–off cycles exceeds 10 before a significant reduction in the fluorescence quantum efficiency occurs. Such robust nanometric probes having remotely controllable optical transitions are useful in many areas of research, particularly in far-field nanoscopy based on reversible saturable or switchable optical fluorescence transitions (RESOLFT). Consequently, we show that implementation of Mn-QDs for imaging leads to an increase in the resolution by a factor of 4.4 over that of confocal microscopy. A schematic diagram of the electronic transitions involved in light-modulated fluorescence from Mn-QDs is shown in Figure 1a. Initially, electrons are photoexcited from the

Scott E. Irvine

Papers

STED microscopy reveals crystal colour centres with nanometric resolution.

Direct light-driven modulation of luminescence from Mn-doped ZnSe quantum dots.

Innentitelbild: Direct Light‐Driven Modulation of Luminescence from Mn‐Doped ZnSe Quantum Dots (Angew. Chem. 14/2008)

Optimizing Fluorophores For Super-resolution Fluorescence STED Microscopy

Inside Cover: Direct Light-Driven Modulation of Luminescence from Mn-Doped ZnSe Quantum Dots (Angew. Chem. Int. Ed. 14/2008)