Endosomal sequestration of lipid-based nanoparticles (LNPs) remains a formidable barrier to delivery. Herein, structure-activity analysis of cholesterol analogues reveals that incorporation of C-24 alkyl phytosterols into LNPs (eLNPs) enhances gene transfection and the length of alkyl tail, flexibility of sterol ring and polarity due to-OH group is required to maintain high transfection. Cryo-TEM displays a polyhedral shape for eLNPs compared to spherical LNPs, while x-ray scattering shows little disparity in internal structure. eLNPs exhibit higher cellular uptake and retention, potentially leading to a steady release from the endosomes over time. 3D single-particle tracking shows enhanced intracellular diffusivity of eLNPs relative to LNPs, suggesting eLNP traffic to productive pathways for escape. Our findings show the importance of cholesterol in subcellular transport of LNPs carrying mRNA and emphasize the need for greater insights into surface composition and structural properties of nanoparticles, and their subcellular interactions which enable designs to improve endosomal escape.

Real-time three-dimensional (3D) single-particle tracking uses optical feedback to lock on to freely diffusing nanoscale fluorescent particles, permitting precise 3D localization and continuous spectroscopic interrogation. Here we describe a new method of real-time 3D single-particle tracking wherein a diffraction-limited laser spot is dynamically swept through the detection volume in three dimensions using a two-dimensional (2D) electro-optic deflector and a tunable acoustic gradient lens. This optimized method, called 3D dynamic photon localization tracking (3D-DyPLoT), enables high-speed real-time tracking of single silica-coated non-blinking quantum dots (∼30  nm diameter) with diffusive speeds exceeding 10  μm²/s at count rates as low as 10 kHz, as well as YFP-labeled virus-like particles. The large effective detection area (1  μm×1  μm×4  μm) allows the system to easily pick up fast-moving particles, while still demonstrating high localization precision (σ_x=6.6  nm, σ_y=8.7  nm, and σ_z=15.6  nm). Overall, 3D-DyPLoT provides a fast and robust method for real-time 3D tracking of fast and lowly emitting particles, based on a single excitation and detection pathway, paving the way to more widespread application to relevant biological problems.

To date, single molecule studies have been reliant on tethering or confinement to achieve long duration and high temporal resolution measurements. Here, we present a 3D singlemolecule active real-time tracking method (3D-SMART) which is capable of locking on to single fluorophores in solution for minutes at a time with photon limited temporal resolution. As a demonstration, 3D-SMART is applied to actively track single Atto 647 N fluorophores in 90% glycerol solution with an average duration of~16 s at count rates of~10 kHz. Active feedback tracking is further applied to single proteins and nucleic acids, directly measuring the diffusion of various lengths (99 to 1385 bp) of single DNA molecules at rates up to 10 µm 2 /s. In addition, 3D-SMART is able to quantify the occupancy of single Spinach2 RNA aptamers and capture active transcription on single freely diffusing DNA. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.

The optical absorption edge of undoped, lightly Al-doped, and heavily Al-doped Bi12SiO20 single crystals is found to be exponential and follows Urbach's rule with σ0=0.71 at room temperature. The band edge is at 3.25 eV and is broadened by excitons and perhaps by impurities or defects. At 80 °K, the band edge is found to be shifted to 3.40 eV. The broad shoulder in the optical absorption and the secondary peak in the photocurrent excitation spectrum are attributed to the presence of a silicon vacancy complex. The longitudinal photocarrier response due to pulsed uv excitation leads to a value of the electron drift mobility of μd=0.029±0.003 cm2/V sec and a value for the range of electrons (μτ)e=8.5×10−7 cm2/V. The response times of electrons and holes (or the relaxation times) are determined to be 6.5×10−3 and 4.3 ×10−3 sec, respectively. Electrons dominate the photocurrent in undoped and lightly Al-doped crystals, while holes dominate the photocurrent in the heavily Al-doped crystals. Thermally stimulated current between 80 and 360 °K shows three major electron traps with energetic depths of 0.34, 0.54, and 0.65 eV in undoped crystals, and major hole traps at 0.26, 0.31, and 0.43 eV in the heavily Al-doped crystals. A band diagram for the undoped single crystals is proposed to explain the photocurrent kinetics and the temperature dependences of the photoluminescence emission bands at 1.95 and 1.30 eV and the temperature dependence of the photocurrent.