Kyu Young Han

Three-Dimensional Stimulated Emission Depletion Microscopy of Nitrogen-Vacancy Centers in Diamond Using Continuous-Wave Light

Charged nitrogen-vacancy (NV) color centers in diamond are excellent luminescence sources for far-field fluorescence nanoscopy by stimulated emission depletion (STED). Here we show that these photostable color centers can be visualized by STED using simple continuous-wave or high repetition pulsed lasers (76 MHz) at wavelengths >700 nm for STED. Furthermore, we show that NV centers can be imaged in three dimensions (3D) inside the diamond crystal and present single-photon signatures of single color centers recorded in high density samples, demonstrating a new recording scheme for STED and related far-field nanoscopy approaches. Finally, we exemplify the potential of using nanodiamonds containing NV centers as luminescence tags in STED microscopy. Our results offer new experimental avenues in nanooptics, nanotechnology, and the life sciences.

STED nanoscopy with time-gated detection: theoretical and experimental aspects.

In a stimulated emission depletion (STED) microscope the region in which fluorescence markers can emit spontaneously shrinks with continued STED beam action after a singular excitation event. This fact has been recently used to substantially improve the effective spatial resolution in STED nanoscopy using time-gated detection, pulsed excitation and continuous wave (CW) STED beams. We present a theoretical framework and experimental data that characterize the time evolution of the effective point-spread-function of a STED microscope and illustrate the physical basis, the benefits, and the limitations of time-gated detection both for CW and pulsed STED lasers. While gating hardly improves the effective resolution in the all-pulsed modality, in the CW-STED modality gating strongly suppresses low spatial frequencies in the image. Gated CW-STED nanoscopy is in essence limited (only) by the reduction of the signal that is associated with gating. Time-gated detection also reduces/suppresses the influence of local variations of the fluorescence lifetime on STED microscopy resolution.

Metastable dark states enable ground state depletion microscopy of nitrogen vacancy centers in diamond with diffraction-unlimited resolution.

Current far-field optical nanoscopy schemes overcome the diffraction barrier by ensuring that adjacent features assume different states upon detection. Ideally, the transition between these states can be repeated endlessly and, if performed optically, with low levels of light. Here we report such optical switching, realized by pairing the luminescent triplet and a long-lived dark state of diamond color centers, enabling their imaging with a resolution >10 times beyond the diffraction barrier (<20 nm).

Understanding the Photophysics of the Spinach–DFHBI RNA Aptamer–Fluorogen Complex To Improve Live-Cell RNA Imaging

The use of aptamer-fluorogen complexes is an emerging strategy for RNA imaging. Despite its promise for cellular imaging and sensing, the low fluorescence intensity of the Spinach-DFHBI RNA aptamer-fluorogen complex hampers its utility in quantitative live-cell and high-resolution imaging applications. Here we report that illumination of the Spinach-fluorogen complex induces photoconversion and subsequently fluorogen dissociation, leading to fast fluorescence decay and fluorogen-concentration-dependent recovery. The fluorescence lifetime of Spinach-DFHBI is 4.0 ± 0.1 ns irrespective of the extent of photoconversion. We detail a low-repetition-rate illumination scheme that enables us to maximize the potential of the Spinach-DFHBI RNA imaging tag in living cells.

An improved surface passivation method for single-molecule studies

We report a surface passivation method based on dichlorodimethylsilane (DDS)–Tween-20 for in vitro single-molecule studies, which, under the conditions tested here, more efficiently prevented nonspecific binding of biomolecules than the standard poly(ethylene glycol) surface. The DDS–Tween-20 surface was simple and inexpensive to prepare and did not perturb the behavior and activities of tethered biomolecules. It can also be used for single-molecule imaging in the presence of high concentrations of labeled species in solution.

Tandem Spinach Array for mRNA Imaging in Living Bacterial Cells.

Live cell RNA imaging using genetically encoded fluorescent labels is an important tool for monitoring RNA activities. A recently reported RNA aptamer-fluorogen system, the Spinach, in which an RNA aptamer binds and induces the fluorescence of a GFP-like 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) ligand, can be readily tagged to the RNA of interest. Although the aptamer–fluorogen system is sufficient for imaging highly abundant non-coding RNAs (tRNAs, rRNAs, etc.), it performs poorly for mRNA imaging due to low brightness. In addition, whether the aptamer-fluorogen system may perturb the native RNA characteristics has not been systematically characterized at the levels of RNA transcription, translation and degradation. To increase the brightness of these aptamer-fluorogen systems, we constructed and tested tandem arrays containing multiple Spinach aptamers (8–64 aptamer repeats). Such arrays enhanced the brightness of the tagged mRNA molecules by up to ~17 fold in living cells. Strong laser excitation with pulsed illumination further increased the imaging sensitivity of Spinach array-tagged RNAs. Moreover, transcriptional fusion to the Spinach array did not affect mRNA transcription, translation or degradation, indicating that aptamer arrays might be a generalizable labeling method for high-performance and low-perturbation live cell RNA imaging.

Preparation of non-aggregated fluorescent nanodiamonds (FNDs) by non-covalent coating with a block copolymer and proteins for enhancement of intracellular uptake

Fluorescent nanodiamonds (FNDs) are very promising fluorophores for use in biosystems due to their high biocompatibility and photostability. To overcome their tendency to aggregate in physiological solutions, which severely limits the biological applications of FNDs, we developed a new non-covalent coating method using a block copolymer, PEG-b-P(DMAEMA-co-BMA), or proteins such as BSA and HSA. By simple mixing of the block copolymer with FNDs, the cationic DMAEMA and hydrophobic BMA moieties can strongly interact with the anionic and hydrophobic moieties on the FND surface, while the PEG block can form a shell to prevent the direct contact between FNDs. The polymer-coated FNDs, along with BSA- and HSA-coated FNDs, showed non-aggregation characteristics and maintained their size at the physiological salt concentration. The well-dispersed, polymer- or protein-coated FNDs in physiological solutions showed enhanced intracellular uptake, which was confirmed by CLSM. In addition, the biocompatibility of the coated FNDs was expressly supported by a cytotoxicity assay. Our simple non-covalent coating with the block copolymer, which can be easily modified by various chemical methods, projects a very promising outlook for future biomedical applications, especially in comparison with covalent coating or protein-based coating.

Effect of single-base mutation on activity and folding of 10-23 deoxyribozyme studied by three-color single-molecule ALEX FRET.

We investigated the effect of single-base mutation on the RNA-cleaving activity and ion-induced folding of 10-23 deoxyribozyme at the single-molecule level by 3-color ALEX FRET (alternating laser excitation fluorescence resonance energy transfer). We found that substitution or deletion of a single base in the active region of the enzyme leads to a different folding pathway and enzymatic activity for all three mutants studied, but the severity of the effect was dependent on the type of mutation and the mutation site. We suggest that mutation of even a single base may result in a considerably different ionic and hydrogen-bonding interactions. Structural changes of 10-23 deoxyribozyme as it successively binds with Mg(2+) and the substrate were also unambiguously identified by the current single-molecule-detection method.

RESOLFT nanoscopy with photoswitchable organic fluorophores.

Far-field optical nanoscopy has been widely used to image small objects with sub-diffraction-limit spatial resolution. Particularly, reversible saturable optical fluorescence transition (RESOLFT) nanoscopy with photoswitchable fluorescent proteins is a powerful method for super-resolution imaging of living cells with low light intensity. Here we demonstrate for the first time the implementation of RESOLFT nanoscopy for a biological system using organic fluorophores, which are smaller in size and easier to be chemically modified. With a covalently-linked dye pair of Cy3 and Alexa647 to label subcellular structures in fixed cells and by optimizing the imaging buffer and optical parameters, our RESOLFT nanoscopy achieved a spatial resolution of ~74 nm in the focal plane. This method provides a powerful alternative for low light intensity RESOLFT nanoscopy, which enables biological imaging with small organic probes at nanoscale resolution.

Dark state photophysics of nitrogen-vacancy centres in diamond

Nitrogen-vacancy (NV) colour centres in diamond are attractive fluorescence emitters owing to their unprecedented photostability and superior applicability to spin manipulation and sub-diffraction far-field optical microscopy. However, some applications are limited by the co-occurrence of dark state population and optical excitation. In this paper, we use fluorescence microscopy and correlation spectroscopy on single negatively charged NV centres in type IIa bulk diamond to unravel the population kinetics of a >100s long-lived dark state. The bright-dark state interconversion rates show a quadratic dependence on the applied laser intensity, which implies that higher excited states are involved. Depopulation of the dark state becomes less effective at wavelengths above 532nm, resulting in a complete fluorescence switch-off at wavelengths >600nm. This switch is reversible by the addition of shorter wavelengths. This behaviour can be explained by a model consisting of three dark and three bright states of different excitation levels, with the most efficient interconversion via the respective higher excited states. This model accounts for