In this study, peptide–gold nanoclusters with tunable fluorescence were prepared by a simple “one-pot” method, which were used for gene localization and delivery in vivo to achieve efficient intracellular colocalization, uptake, and transfection. The efficiency of pDNA transfection was up to 70.6%, and there was no obvious cytotoxicity. This study proves that the simple-composition and bio-friendly peptide–gold nanoclusters are promising gene delivery carriers and can provide a powerful theoretical and experimental basis for the application of peptide–metal nanocomplexes in gene delivery and other biomedicine fields.

To determine the molecular and/or mechanical basis of cell migration using live cell imaging tools, it is necessary to correlate multiple 3D spatiotemporal events simultaneously. Fluorescence nanoscopy and label-free nanoscale imaging can complement each other by providing both molecular specificity and structural dynamics of sub-cellular structures. A combined imaging system would permit obtaining quantitative 3D spatial temporal details of individual cellular components. In this paper, we empirically determined optimal azimuthal scanning angles of rotating beams to achieve simultaneous and label-free nanoscale and fluorescence imaging. Label-free nanoscale imaging here refers to interferometric, bright-field (BF) and dark-field (DF) rotating coherence scattering (ROCS) microscopy, while fluorescence refers to high-inclined laminated oblique (HiLO) and total internal reflection fluorescence (TIRF) imaging. The combined capabilities of interferometric, scattering, and fluorescence imaging enable (1) the identification of molecular targets (substrate or organelle), (2) quantification of 3D cell morphodynamics, and (3) tracking of intracellular organelles in 3D. This combined imaging tool was then used to characterize migrating platelets and endothelial cells, both critical to the process of infection and wound healing. The combined imaging results of more than ∼1000 platelets suggest that serum albumin (bovine) is necessary for platelets to migrate and scavenge fibrin/fibrinogen. Furthermore, we identified new asynchronous membrane fluctuations between the leading and rear edges of a migrating platelet. We further demonstrated that interferometric imaging permits the quantification of mitochondrial dynamics on human lung microvascular endothelial cells. Our data suggests that axial displacement of mitochondria is minimal when it is closer to the nucleus or the leading edge of a cell membrane. Taken together, this combined nanoscopy platform helps to quantify multiple spatial temporal events of a migrating cell that will undoubtedly open ways to new quantitative correlative nanoscale live cell imaging.

Single-objective scanning light sheet (SOLS) imaging has fueled major advances in volumetric bioimaging because it supports low phototoxic, high-resolution imaging over an extended period. The remote imaging unit in the SOLS does not use a conventional epifluorescence image detection scheme (a single tube lens). In this paper, we propose a technique called the computational SOLS (cSOLS) that achieves light sheet imaging without the remote imaging unit. Using a single microlens array after the tube lens (lightfield imaging), the cSOLS is immediately compatible with conventional epifluorescence detection. The core of cSOLS is a Fast Optical Ray (FOR) model. FOR generates 3D imaging volume (40 × 40 × 14 µm3) using 2D lightfield images taken under SOLS illumination within 0.5 s on a standard central processing unit (CPU) without multicore parallel processing. In comparison with traditional lightfield retrieval approaches, FOR reassigns fluorescence photons and removes out-of-focus light to improve optical sectioning by a factor of 2, thereby achieving a spatial resolution of 1.59 × 1.92 × 1.39 µm3. cSOLS with FOR can be tuned over a range of oblique illumination angles and directions and, therefore, paves the way for next-generation SOLS imaging. cSOLS marks an important and exciting development of SOLS imaging with computational imaging capabilities.

Current 3D microfluidic fabrication methods require hours and specialized equipment to fabricate microstructures in a single channel so as to recapitulate mixed (homogenous and heterogeneous) in vivo fluid flow. Inspired by the ancient art form of inside painting, we developed a technique for 3D fabrication of micro-patterned flow channels and mixed in vivo fluid flow in a matter of minutes. We termed this technique Multiphoton Inner Laser Lithography (MILL). We further showed that MILL is compatible with both flat and curved channel shapes. MILL recapitulated in vivo tissue topology and 3D fluid flow of the tissue microenvironment, all of which are vital for understanding of how extracellular fluid flow regulates cell function. Cells in MILL capillary tubes response to a variety of in vivo-like laminar flow patterns (homogenous and heterogeneous). Live cells were observed to organize, translocate and adhere along different fluid shear landscapes (0 - 81 dynes/cm2) in real time. Parallel strips of MILL channels were assembled for platelet function tests (~2000 microthrombi per test). The MILL technique heralds a new paradigm where dynamics of in vivo fluid flow can be readily reproduced in minutes on a standard multiphoton imaging microscope and benefit preclinical screening of drug pharmacokinetics.