We demonstrate high performance coherent anti-Stokes Raman scattering (CARS) microscopy of live cells and tissues with user-variable spectral resolution and broad Raman tunability (2500 - 4100 cm(-1)), using a femtosecond Ti:Sapphire pump and photonic crystal fiber output for the broadband synchronized Stokes pulse. Spectral chirp of the fs laser pulses was a user-variable parameter for optimization in a spectral focusing implementation of multimodal CARS microscopy. High signal-to-noise, high contrast multimodal imaging of live cells and tissues was achieved with pixel dwell times of 2-8 micros and low laser powers (< 30 mW total).

The contraction of cardiac myocytes is initiated by ligand binding to adrenergic receptors contained in nanoscale multiprotein complexes called signalosomes. The composition and number of functional signalosomes within cardiac myocytes defines the molecular basis of the response to adrenergic stimuli. For the first time, we demonstrated the ability of near-field scanning optical microscopy to visualize beta-adrenergic receptors at the nanoscale in situ. On H9C2 cells, mouse neonatal and mouse embryonic cardiac myocytes, we showed that functional receptors are organized into multiprotein domains of approximately 140 nm average diameter. Colocalization experiments in primary cells at the nanometer scale showed that 15-20% of receptors were preassociated in caveolae. These nanoscale complexes were sufficient to effect changes in ligand-induced contraction rate without the requirement for substantial changes in receptor distribution in the cellular membrane. Using fluorescence intensities associated with these nanodomains, we estimated the receptor density within the observed nanometer features and established a lower limit for the number of receptors in the signalosome.

Coronaviruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the coronavirus disease 2019 (COVID-19) pandemic, present a significant threat to human health by inflicting a wide variety of health complications and even death. While conventional therapeutics often involve administering small molecules to fight viral infections, small non-coding RNA sequences, known as microRNAs (miRNAs/miR-), may present a novel antiviral strategy. We can take advantage of their ability to modulate host–virus interactions through mediating RNA degradation or translational inhibition. Investigations into miRNA and SARS-CoV-2 interactions can reveal novel therapeutic approaches against this virus. The viral genomes of SARS-CoV-2, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) were searched using the Nucleotide Basic Local Alignment Search Tool (BLASTn) for highly similar sequences, to identify potential binding sites for miRNAs hypothesized to play a role in SARS-CoV-2 infection. miRNAs that target angiotensin-converting enzyme 2 (ACE2), the receptor used by SARS-CoV-2 and SARS-CoV for host cell entry, were also predicted. Several relevant miRNAs were identified, and their potential roles in regulating SARS-CoV-2 infections were further assessed. Current treatment options for SARS-CoV-2 are limited and have not generated sufficient evidence on safety and efficacy for treating COVID-19. Therefore, by investigating the interactions between miRNAs and SARS-CoV-2, miRNA-based antiviral therapies, including miRNA mimics and inhibitors, may be developed as an alternative strategy to fight COVID-19.

Copper ions are vital to human health, and mis-trafficking of them can result in many diseases including Wilson's, Menkes', and Alzheimer's diseases. Coherent anti-Stokes Raman scattering (CARS) microscopy can be used to observe changes in lipid phenotype in a noninvasive manner and is employed here to show that copper accumulation in hepatic cells results in rapid changes in lipid storage and lipid droplet density. The increase in lipid storage is dependent on the coordination environment of the copper to which the cells are exposed and changes in toxicity, lipid phenotype, and rate of copper accumulation upon treatment vary using different Cu species.

Near-field scanning optical microscopy (NSOM) has been used to study the nanoscale distribution of voltage-gated L-type Ca2+ ion channels, which play an important role in cardiac function. NSOM fluorescence imaging of immunostained cardiac myocytes (H9C2 cells) demonstrates that the ion channel is localized in small clusters with an average diameter of 100 nm. The clusters are randomly distributed throughout the cell membrane, with some larger fluorescent patches that high-resolution images show to consist of many small closely-spaced clusters. We have imaged unstained cells to assess the contribution of topography-induced artifacts and find that the topography-induced signal is <10% of the NSOM fluorescence intensity. We have also examined the dependence of the NSOM signal intensity on the tip-sample separation to assess the contributions from fluorophores that are significantly below the cell surface. This indicates that chromophores > approximately 200 nm below the probe will have negligible contributions to the observed signal. The ability to quantitatively measure small clusters of ion channels will facilitate future studies that examine changes in protein localization in stimulated cells and during cardiac development. Our work illustrates the potential of NSOM for studying membrane domains and protein localization/colocalization on a length scale which exceeds that available with optical microscopy.

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Using an all-fiber laser system consisting of a femtosecond Er/Yb fiber oscillator as the pump and an ultra-highly nonlinear fiber for Stokes generation, we demonstrate multimodal (TPF+SHG+CARS) non-linear optical microscopy of both tissue samples and live cells. Multimodal imaging was successfully performed with pixel dwell times as short as 4 micros at low laser powers (< 40 mW total).

The first estimates of the lifetimes of the 2-propyl cation (4a), cyclobutonium ion (4b), cyclopropylethyl cation (4c), and 2-adamantyl cation (4d) in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), trifluoroethanol (TFE), and acetonitrile solvents have been determined using electrophilic aromatic addition to 1,3,5-trimethoxybenzene as a kinetic probe reaction in laser flash photolysis (LFP) experiments. The lifetimes ranged from ∼100 ps to ∼40 ns at 22 °C. Oxadiazoline precursors 1 were used to generate sec-diazoalkanes 2 by LFP which, in the presence of a proton source, give rise to sec-alkanediazonium ions 3 that lose N2 rapidly to form carbocationic intermediates 4. The cations were found to react with 1,3,5-trimethoxybenzene to form cyclohexadienyl cations. The latter were monitored by LFP. Stern−Volmer kinetics were used to determine lifetimes and rate constants for reactions of cations 4a − d. Short lifetimes of 4a − d imply that simple sec-alkanediazonium ions, from carcinogenic N-alkyl-N-nitrosamines, must be generated within contact distances of DNA through a preassociation mechanism in order to effect alkylation.