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.

Water provides a dynamic matrix in which all biochemical processes occur in living organisms. The structure and dynamics of intracellular water constitute the cornerstone for understanding all aspects of cellular function. Fundamentally, direct visualization of subcellular solvation heterogeneity is essential but remains challenging with commonly used nuclear magnetic resonance methods due to poor spatial resolution. To explore this question, we demonstrate a vibrational-shift imaging approach by combining the spectral-focusing hyperspectral stimulated Raman scattering technique with an environmentally sensitive nitrile probe. The sensing ability of a near-infrared nitrile-containing molecule is validated in the solution phase, microscopic droplets, and cellular environments. Finally, we quantitatively measure the subcellular solvation variance between the cytoplasm (29.5%, S.E. 1.8%) and the nucleus (57.3%, S.E. 1.0%), which is in good agreement with previous studies. This work sheds light on heterogeneous solvation in live systems using coherent Raman microscopy and opens up new avenues to explore environmental variance in complex systems with high spatiotemporal resolution.

Solid lipid nanoparticles (SLNs) are a state-of-the-art lipid-based pharmaceutical drug delivery system. Advantages of SLNs include high biocompatibility, low immunogenicity, superiority in drug encapsulation capacity, and improved colloidal stability. They became widely known in late 2020, as several COVID-19 vaccines are built upon SLNs technology. Despite the increasing impact, the characterization methods of SLNs are currently very limited especially in biological environment, which hinders fundamental understanding of the delivery mechanism and contributes to relatively low success rate in clinical translations. Here, we present close-to-label-free imaging of deuterated SLNs using the emerging stimulated Raman scattering (SRS) microscopy. The introduction of deuterium to lipid structure renders bioorthogonal chemical specificity.Notably, with this approach, we have achieved ultrahigh single-particle sensitivity both in vitro and in vivo, even with particle counting ability. Our bioorthogonal chemical imaging modality by SRS microscopy can be generalized to visualize a wide spectrum of lipid-based drug carriers with high spatiotemporal resolution, chemical specificity, and ultimate sensitivity. This work opens up ways to address critical questions in SLN drug delivery and could also facilitate innovations in lipid nanotechnology and clinical translations. Key points• Direct imaging of lipid nanocarrier that is the basis of the COVID-19 vaccines.• Novel single-particle imaging technique applied to nanomedicine.

Water provides a dynamic matrix in which all biochemical processes occur in living organisms. The structure and dynamics of intracellular water constitute the cornerstone for understanding all aspects of cellular function. Fundamentally, direct visualization of subcellular solvation heterogeneity is essential but remains challenging with commonly used NMR methods due to poor spatial resolution. To explore this question, we demonstrate a vibrational-shift imaging approach by combining the spectral-focusing hyperspectral stimulated Raman scattering (hsSRS) technique with an environmentally-sensitive nitrile probe. The sensing ability of a near-infrared nitrile-containing molecule is validated in the solution phase, microscopic droplets and cellular environments. Finally, we quantitatively measure the subcellular solvation variance in the degree of free water content between the cytoplasm (29.7%, S.E. 1.1%) and the nucleus (56.4%, S.E. 1.3%), which is in good agreement with previous studies. This work sheds light on mapping the heterogenous solvation in live systems using coherent Raman microscopy and opens up new avenues to explore environmental variance in complex systems with high spatiotemporal resolution.

<p>Water provides a dynamic matrix in which all biochemical processes occur in living organisms. The structure and dynamics of intracellular water constitute the cornerstone for understanding all aspects of cellular function. Fundamentally, direct visualization of subcellular solvation heterogeneity is essential but remains challenging with commonly used NMR methods due to poor spatial resolution. To explore this question, we demonstrate a vibrational-shift imaging approach by combining the spectral-focusing hyperspectral stimulated Raman scattering (hsSRS) technique with an environmentally-sensitive nitrile probe. The sensing ability of a near-infrared nitrile-containing molecule is validated in the solution phase, microscopic droplets and cellular environments. Finally, we quantitatively measure the subcellular solvation variance between the cytoplasm (29.5%, S.E. 1.8%) and the nucleus (57.3%, S.E. 1.0%), which is in good agreement with previous studies. This work sheds light on heterogenous solvation in live systems using coherent Raman microscopy and opens up new avenues to explore environmental variance in complex systems with high spatiotemporal resolution.</p>