Self-activated photoblinking of nitrogen vacancy centers in nanodiamonds (sandSTORM): A method for rapid single molecule localization microscopy with unlimited observation time

Abstract

Stochastic optical reconstruction microscopy (STORM) is one of the most commonly used super-resolution microscopy techniques. Popular implementations of STORM utilize aromatic fluorophores and consist of a number of intrinsic limitations such the finite photostability of the dyes, the reliance upon non-physiological redox buffers and speed which is ultimately limited by the 'off'rates of the photoblinking. Self-activated nanodiamond-based STORM (sandSTORM) has been developed as an accelerated STORM protocol which harvests the rapid, high quantum-yield and sustained photoblinking of nanodiamonds (ND). Photoluminescence emanating from the stochastic charge-state interconversion of Nitrogen Vacancy (NV) centers between NV 0 and NVis localized using conventional STORM-optimized hardware and image processing protocols over an unlimited duration of imaging. This produces super-resolution images of matching resolution at ~ 3-times the speed and ~ 100 times less light exposure to the sample compared to traditional STORM. The enabling NDs have been used to map arrays of ryanodine receptor in skeletal muscle tissues via immunolabelling and directly visualize the internal spaces of living neurons via endocytosis of NDs. This paper details the physical basis of sandSTORM, factors which optimize its performance, and key characteristics which make it a powerful STORM protocol suitable for imaging nanoscale sub-cellular structures.

High-throughput nitrogen-vacancy center imaging for nanodiamond photophysical characterization and pH nanosensing

“…42,44 A better investigation of NV charge-state transition in NDs is essential for the development of ND-based biosensing or bio-imaging approaches, as recently reported. 53 The lifetime measurement methodology we introduced allows automated time-trace analysis from multiple single-NV centres. The closest report to our study of single-NV charge transition is a study by Aslam et al who investigated charge-state transitions of a single NV center in bulk diamond, and measured lifetimes Fig.…”

“…A better investigation of NV charge-state transition in NDs is essential for the development of ND-based biosensing or bio-imaging approaches, as recently reported. 53 The lifetime measurement methodology we introduced allows automated time-trace analysis from multiple single-NV centres. The closest report to our study of single-NV charge transition is a study by Aslam et al who investigated charge-state transitions of a single NV center in bulk diamond, and measured lifetimes for NV − and NV 0 (57 ms and 465 ms respectively) three orders of magnitude shorter than what we measured; however, those measurements used substantially different conditions (study in bulk diamond instead of in a ND, and use of a different excitation wavelength: 593 nm), and examined only one NV center.…”

Multifunctional nanodiamonds to enable neuritogenesis and super-resolution imaging

“…Preparation and physico-optical characterization of NDs Red uorescent NDs (brFND-100) with more than 1000 NV centers of both NV − and NV 0 per particle were purchased from FND Biotech (Taipei, Taiwan). These particles offer the advantages of photoblinking and higher uorescence intensities [36,39]. Before each experimental run, the ND powders were suspended in deionized (DI) water at 2 mg/mL, sonicated for 1 h, and vortexed for 10 min.…”

“…Immediately before entering the PMTs, the beams were passed through additional bandpass lters to maintain the wavelength range. Occasionally, a single PMT with a bandpass lter with transmission range 573-637 nm was used when the NDs were used as the only probe for imaging because the wavelength range was determined to be optimum for capturing photoblinking emissions [39].…”

“…Moreover, NV centers, an internal defect in NDs, can be utilized in superresolution imaging probes owing to their photochromic property, which is caused by the electron transition between NV − and NV 0 when illuminated by a laser. This photoionization is hypothetically related to photoblinking, which is stochastically converted to a uorescent ('on') or non-uorescent ('off') state during the detection of certain spectra [38,39]. Because of this phenomenon, NDs can be applied to the dSTORM technique without any additional manipulations, on neither the particle itself nor systemically.…”