Stability Criterion for the Assembly of Core-Shell Lipid-Polymer-Nucleic Acid Nanoparticles.

Hybrid core-shell lipid-polycation-nucleic acid nanoparticles (LPNPs) provide unique delivery strategies for nonviral gene therapeutics. Since LPNPs consist of multiple components, involving different pairwise interactions between them, they are challenging to characterize and understand. Here, we propose a method based on fluorescence cross-correlation spectroscopy to elucidate the association between the three LPNP components. Through this lens, we demonstrate that cationic lipid shells (liposomes) do not displace polycations or DNA from the polycation-DNA cores (polyplexes). Hence, polyplexes and liposomes must be oppositely charged to associate into LPNPs. Furthermore, we identify the liposome:polyplex number ratio (ρN), which was hitherto an intangible quantity, as the primary parameter predicting stable LPNPs. We establish that ρN ≥ 1 ensures that every polyplex is enveloped by a liposome, thus avoiding coexisting oppositely charged species prone to aggregation.

Atmospheric Photoionization Detector with Improved Photon Efficiency: A Proof of Concept for Application of a Nanolayer Thin-Film Electrode.

Atmospheric photoionization is a widely applied soft ionization mechanism in gas sensing devices for the detection of volatile organic compounds in ambient air. Photoionization is typically induced by low-pressure Vacuum Ultraviolet (VUV) lamps with MgF2 or LiF lamp surface windows depending on the gas fill and the required wavelength transmission window. These lamps are known to exhibit gradually reduced VUV transmission due to hydrocarbon contamination. LiF surface windows are known to be especially problematic due to their hygroscopic nature, reducing VUV lamp lifetime to a mere 100 h, approximately. Here, we present a new design for the electrode of a photoionization detector based on thin-film technology. By replacing the commonplace metal grid electrode's VUV lamp surface window with a chromium/gold thin film we obtain a doubling of photon efficiency for photoionization. Replacing the hygroscopic LiF lamp window surface with a metallic layer additionally offers the possibility to vastly increase operational lifetime of low-pressure Argon VUV lamps.

How did correlative atomic force microscopy and super-resolution microscopy evolve in the quest for unravelling enigmas in biology?

With the invention of the Atomic Force Microscope (AFM) in 1986 and the subsequent developments in liquid imaging and cellular imaging it became possible to study the topography of cellular specimens under nearly physiological conditions with nanometric resolution. The application of AFM to biological research was further expanded with the technological advances in imaging modes where topographical data can be combined with nanomechanical measurements, offering the possibility to retrieve the biophysical properties of tissues, cells, fibrous components and biomolecules. Meanwhile, the quest for breaking the Abbe diffraction limit restricting microscopic resolution led to the development of super-resolution fluorescence microscopy techniques that brought the resolution of the light microscope comparable to the resolution obtained by AFM. The instrumental combination of AFM and optical microscopy techniques has evolved over the last decades from integration of AFM with bright-field and phase-contrast imaging techniques at first to correlative AFM and wide-field fluorescence systems and then further to the combination of AFM and fluorescence based super-resolution microscopy modalities. Motivated by the many developments made over the last decade, we provide here a review on AFM combined with super-resolution fluorescence microscopy techniques and how they can be applied for expanding our understanding of biological processes.

Fluorescence cross-correlation spectroscopy as a valuable tool to characterize cationic liposome-DNA nanoparticle assembly.

The development of nonviral gene delivery vehicles for therapeutic applications requires methods capable of quantifying the association between the genes and their carrier counterparts. Here we investigate the potential of fluorescence cross-correlation spectroscopy (FCCS) to characterize and optimize the assembly of nonviral cationic liposome (CL)-DNA complexes based on a CL formulation consisting of the cationic lipid DOTAP and zwitterionic lipid DOPC. We use a DNA plasmid for lipoplex loading encoding the Oct4 gene, critically involved in reprogramming somatic cells into induced pluripotent stem cells. We demonstrate that FCCS is able to quantitatively determine the extent of the association between DNA and the liposomes and assess its loading capacity. We also establish that the cationic lipid fraction, being proportional to the liposome membrane charge density, as well as charge ratio between the CLs and anionic DNA play an important role in the degree of interaction between the liposomes and DNA.

Correlative fluorescence and atomic force microscopy to advance the bio-physical characterisation of co-culture of living cells.

An understanding of the cell mechanical properties involved in numerous cellular processes including cell division, cell migration/invasion, and cell morphology, is crucial in developing and informing cell physiology and function. Atomic force microscopy (AFM) offers a powerful biophysical technique that facilitates the imaging of living cells under physiological buffer conditions. However, AFM in isolation cannot discriminate between different cell types within heterogeneous samples for example in a solid biopsy. The current studies demonstrate the potential of AFM in combination with correlative fluorescence optical sectioning microscopy for live cell imaging. Furthermore, this work establishes the advantage of fluorescence-AFM imaging to distinguish and analyse single-cell bio-physical properties in mixed human cell populations, in real-time. Critically, our results show that correlative fluorescence-AFM imaging allows the simultaneous co-localised detection of fluorescence coupled with nano-mechanical mapping. The findings from this work contribute to the promotion and dissemination of correlative multimodal imaging in life sciences, providing a platform for further investigations in biological and pre-clinical research.

Facile synthesis of an aminopropylsilane layer on Si/SiO2 substrates using ethanol as APTES solvent.

(3-aminopropyl)triethoxysilane (APTES) is a commonly used organosilane on surface functionalization of silicon oxide surfaces. However, its deposition process from solution-phase usually involves the use of toluene, which has often been identified as crucial for the formation of an aminopropylsilane monolayer. Toluene is ranked as a problematic solvent in the guide developed by a group referred to as the solvent sub-team of CHEM21. In this work, we propose a facile synthetic route for functionalizing a silicon substrate with APTES via solution-phase approach using only solvents that are classified as recommended. The influence of the APTES concentration, reaction times and different post-deposition conditions using acetic acid and methanol were studied in order to evaluate the quality and thickness of the organosilane layers.•The method uses ethanol as APTES solvent for functionalizing silicon dioxide surfaces and only uses solvents classified as recommended.•The method uses a solution phase approach, does not require complicated equipment and can be prepared at room temperature.

Simultaneous co-localized super-resolution fluorescence microscopy and atomic force microscopy: combined SIM and AFM platform for the life sciences.

Correlating data from different microscopy techniques holds the potential to discover new facets of signaling events in cellular biology. Here we report for the first time a hardware set-up capable of achieving simultaneous co-localized imaging of spatially correlated far-field super-resolution fluorescence microscopy and atomic force microscopy, a feat only obtained until now by fluorescence microscopy set-ups with spatial resolution restricted by the Abbe diffraction limit. We detail system integration and demonstrate system performance using sub-resolution fluorescent beads and applied to a test sample consisting of human bone osteosarcoma epithelial cells, with plasma membrane transporter 1 (MCT1) tagged with an enhanced green fluorescent protein (EGFP) at the N-terminal.

Microscopic thin film optical anisotropy imaging at the solid-liquid interface.

Optical anisotropy of thin films has been widely investigated through ellipsometry, whereby typically an optical signal is averaged over a ∼1 cm(2) elliptical area that extends with increasing angle-of-incidence (AOI). Here, we report on spectroscopic imaging ellipsometry at the solid-liquid interface applied to a supported lipid bilayer (SLB). We detail how a differential spectrally resolved ellipsometry measurement, between samples with and without optically anisotropic thin film on an absorbing substrate, can be applied to recover in and out of plane refractive indices of the thin film with known film thickness, hence determining the thin film optical anisotropy. We also present how optimal wavelength and AOI settings can be determined ensuring low parameter cross correlation between the refractive indices to be determined from a differential measurement in Δ ellipsometry angle. Furthermore, we detail a Monte Carlo type analysis that allows one to determine the minimal required optical ellipsometry resolution to recover a given thin film anisotropy. We conclude by presenting a new setup for a spectroscopic imaging ellipsometry based on fiber supercontinuum laser technology, multi-wavelength diode system, and an improved liquid cell design, delivering a 5 ×-10 × ellipsometric noise reduction over state-of-the-art. We attribute this improvement to increased ellipsometer illumination power and a reduced light path in liquid through the use of a water dipping objective.

Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy.

Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discuss sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate.

Surface scattering of core-shell particles with anisotropic shell.

The Bobbert-Vlieger solution to light scattering of a spherical particle suspended above a surface is extended to model the scattering of core-shell structures with anisotropic shell. Numerical modeling demonstrates that ellipsometry has potential to resolve particle shell anisotropy down to 1.8×10(-4) for SiO(2)@Au core-shell particles in air with 50 nm core diameter and 10 nm shell thickness deposited on a silicon Si [100] substrate with a density of 1 µm(-2). Application of the Ibrahim and Bashara criterion for ellipsometer parameter cross correlation identifies variable-angle ellipsometry as a viable experimental approach to separate particle core radius and shell thickness from the shell anisotropy. Ellipsometry is also identified as an alternative technique for determination of liposome anisotropy and for the study of liposome fusion with a substrate in the formation process of supported lipid bilayers.

In vivo measurements of diffuse reflectance and time-resolved autofluorescence emission spectra of basal cell carcinomas.

We present a clinical investigation of diffuse reflectance and time-resolved autofluorescence spectra of skin cancer with an emphasis on basal cell carcinoma. A total of 25 patients were measured using a compact steady-state diffuse reflectance/fluorescence spectrometer and a fibre-optic-coupled multispectral time-resolved spectrofluorometer. Measurements were performed in vivo prior to surgical excision of the investigated region. Singular value decomposition was used to reduce the dimensionality of steady state diffuse reflectance and fluorescence spectra. Linear discriminant analysis was then applied to the measurements of basal cell carcinomas (BCCs) and used to predict the tissue disease state with a leave-one-out methodology. This approach was able to correctly diagnose 87% of the BCCs. With 445 nm excitation a decrease in the spectrally averaged fluorescence lifetime was observed between normal tissue and BCC lesions with a mean value of 886 ps. Furthermore, the fluorescence lifetime for BCCs was lower than that of the surrounding healthy tissue in all cases and statistical analysis of the data revealed that this decrease was significant (p = 0.002).

Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source.

We present a time-gated, optically sectioned, hyperspectral fluorescence lifetime imaging (FLIM) microscope incorporating a tunable supercontinuum excitation source extending into the UV. The system is capable of resolving the excitation spectrum, emission spectrum, and fluorescence decays in an optically sectioned image.