DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed. To address this issue, we propose the additions of ethylene carbonate (EC) to the imaging buffer, sequence repeats to the docking strand, and a spacer between the docking strand and the affinity agent. Collectively termed DNA-PAINT-ERS (E = EC, R = Repeating sequence, and S = Spacer), these strategies can be easily integrated into current DNA-PAINT workflows for both accelerated imaging speed and improved image quality through optimized DNA hybridization kinetics and efficiency. We demonstrate the general applicability of DNA-PAINT-ERS for fast, multiplexed superresolution imaging using previously validated oligonucleotide constructs with slight modifications.

Fluorophores are powerful tools for interrogating biological systems. Carbon nanotubes (CNTs) have long been attractive materials for biological imaging due to their nearinfrared excitation and bright, tunable optical properties. The difficulty in synthesizing and functionalizing these materials with precision, however, has hampered progress in this area. Carbon nanohoops, which are macrocyclic CNT substructures, are carbon nanostructures that possess ideal photophysical characteristics of nanomaterials, while maintaining the precise synthesis of small molecules. However, much work remains to advance the nanohoop class of fluorophores as biological imaging agents. Herein, we report an intracellular targeted nanohoop. This fluorescent nanostructure is noncytotoxic at concentrations up to 50 μM, and cellular uptake investigations indicate internalization through endocytic pathways. Additionally, we employ this nanohoop for two-photon fluorescence imaging, demonstrating a high two-photon absorption cross-section (65 GM) and photostability comparable to a commercial probe. This work further motivates continued investigations into carbon nanohoop photophysics and their biological imaging applications.

This work describes in detail a wide-field surface-enhanced coherent anti-Stokes Raman scattering (CARS) microscope, which enables enhanced detection of sample structures in close proximity (∼100  nm) of the substrate interface. Unlike conventional CARS microscopy, where the sample is illuminated with freely propagating light, the current implementation uses evanescent fields to drive Raman coherences across the entire object plane. By coupling the pump and Stokes excitation beams to the surface plasmon-polariton mode at the interface of a 30 nm thick gold film, we obtained strong CARS signals from cholesteryl oleate droplets adhered to the surface. The surface-enhanced CARS imaging system visualizes lipid structures with vibrational selectivity using illumination doses per unit area that are more than four orders of magnitude lower than in point-scanning CARS microscopy.

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We describe the operating principle and performance of a recently developed surface plasmon-enhanced optical sensor that utilizes two-photon excited luminescence of a planar gold film as the reporter signal. The sensor enables direct visualization of nanoscopic binding events near a sensing surface. Light is coupled to the Au/sample interface in an objective-based Kretschmann configuration to excite surface plasmon polariton (SPP) modes at a metal−dielectric interface. The gold luminescence induced by the confined optical field between the particle and the film is detected in the epi-direction by a far-field camera where individual binding events show up as diffraction limited bright spots against a dark background. We study the sensor's emission spectrum and the distance dependence between the target and substrate, which both suggest that the optical signal of the sensor originates from electron−hole pair excitations in the planar Au film. In addition, we show that the well-behaved pointspread function of the sensor enables a straightforward implementation of superresolution techniques. Finally, we demonstrate the utility of the sensor for detecting DNA binding events, underlining the sensor's usefulness for label-free imaging of nanoscopic particles and biomolecular interactions.