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In JoVE (1)
Other Publications (7)
Articles by Thomas R. Huser in JoVE
JoVE General
Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)
1Center for Biophotonics, University of California, Davis, 2Department of Internal Medicine, University of California, Davis
A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.
Other articles by Thomas R. Huser on PubMed
Surface-enhanced Raman Scattering from Individual Au Nanoparticles and Nanoparticle Dimer Substrates
Surface-enhanced Raman scattering (SERS) intensities for individual Au nanospheres, nanoshells, and nanosphere and nanoshell dimers coated with nonresonant molecules are measured, where the precise nanoscale geometry of each monomer and dimer is identified through in situ atomic force microscopy. The observed intensities correlate with the integrated quartic local electromagnetic field calculated for each specific nanostructure geometry. In this study, we find that suitably fabricated nanoshells can provide SERS enhancements comparable to nanosphere dimers.
Growth, Differentiation, and Biochemical Signatures of Rhesus Monkey Mesenchymal Stem Cells
The goal of this study was to compare the growth and differentiation potential of rhesus monkey mesenchymal stem cells (rhMSCs) from different age groups (fetal, newborn, infant, juvenile), and to use confocal micro-Raman spectroscopy to assess the intrinsic biomolecular profiles of individual rhMSCs. Results indicated that fetal cells had significantly shorter population doubling times during the log growth phase (23.3 +/- 1.3 h) and greater population doubling times (66.5 +/- 6.5) when compared to other age groups (newborn 51.9 +/- 2.3, infant 38.2 +/- 3.1, juvenile 40.7 +/- 4.1). Fetal rhMSCs also differentiated toward osteogenic and adipogenic lineages at a faster rate when compared to cells from older animals. The Raman spectral analysis showed greater DNA and lower protein concentration in fetal compared to juvenile rhMSCs, although the spectra from different age groups shared many similar features. Additionally, principal component analysis (PCA), which is used to discriminate between rhMSCs, supported prior findings that suggested that cultured rhMSCs consist of a heterogeneous cell population. Although the growth potential of rhMSCs from the younger age groups was confirmed, further studies will be necessary to fully explore the potential usefulness of Raman micro-spectroscopy to characterize stem and progenitor cells such as rhMSCs.
Physico-chemical Characterization of Polylipid Nanoparticles for Gene Delivery to the Liver
Polylipid nanoparticles (PLNP) have been shown to be very effective in delivering antioxidative genes in the treatment of liver injury in mice. To build on our previous studies and to further characterize PLNP formulated from polycationic lipid (PCL) and cholesterol, we report here the synthesis of multigram quantities of PCL and employ analytical tools, such as Raman spectroscopy of single PLNP and live-cell imaging of lipofection, for the physicochemical characterization of PCL, PLNP, and the transfection process. Mass spectrometry demonstrates the characteristics of polymeric lipids. Raman spectrum of PCL reveals the polymeric structure of the polymers. The presence of cholesterol in PLNP formulation did not markedly change the Raman spectrum. PLNP-derived polyplexes exhibit Raman spectra very similar to PLNP except that the C-H out-of-plane deformation mode of the polymeric lipid is significantly suppressed, indicating the interaction with plasmid DNA. Zeta potential measurement indicates a large DNA-carrying capacity of PLNP and their stability for in vivo gene delivery. The live-cell fluorescent imaging dynamically shows that PLNP exerts transfection efficiency similar to lipofectamine in leading to early reporter gene expression in live hepatic cells. In conclusion, polylipid nanoparticles possess a high DNA carrying capacity and lipofection efficiency, rendering them suitable for testing in large animals. The employment of novel state-of-the-art technologies in the study of lipofection represents the level of physicochemical and biological characterization that is needed to best understand the key elements involved in the lipofection process.
Fatty Acids from Very Low-density Lipoprotein Lipolysis Products Induce Lipid Droplet Accumulation in Human Monocytes
One mechanism by which monocytes become activated postprandially is by exposure to triglyceride-rich lipoproteins such as very low-density lipoproteins (VLDL). VLDL are hydrolyzed by lipoprotein lipase at the blood-endothelial cell interface, releasing free fatty acids. In this study, we examined postprandial monocyte activation in more detail, and found that lipolysis products generated from postprandial VLDL induce the formation of lipid-filled droplets within cultured THP-1 monocytes, characterized by coherent antistokes Raman spectroscopy. Organelle-specific stains revealed an association of lipid droplets with the endoplasmic reticulum, confirmed by electron microscopy. Lipid droplet formation was reduced when lipoprotein lipase-released fatty acids were bound by BSA, which also reduced cellular inflammation. Furthermore, saturated fatty acids induced more lipid droplet formation in monocytes compared with mono- and polyunsaturated fatty acids. Monocytes treated with postprandial VLDL lipolysis products contained lipid droplets with more intense saturated Raman spectroscopic signals than monocytes treated with fasting VLDL lipolysis products. In addition, we found that human monocytes isolated during the peak postprandial period contain more lipid droplets compared with those from the fasting state, signifying that their development is not limited to cultured cells but also occurs in vivo. In summary, circulating free fatty acids can mediate lipid droplet formation in monocytes and potentially be used as a biomarker to assess an individual's risk of developing atherosclerotic cardiovascular disease.
Three-dimensional Structured Illumination Microscopy of Liver Sinusoidal Endothelial Cell Fenestrations
Fenestrations are pores in liver sinusoidal endothelial cells that filter substrates and debris between the blood and hepatocytes. Fenestrations have significant roles in aging and the regulation of lipoproteins. However their small size (<200 nm) has prohibited any functional analysis by light microscopy. We employed structured illumination light microscopy to observe fenestrations in isolated rat liver sinusoidal endothelial cells with great clarity and spatial resolution. With this method, the three-dimensional structure of fenestrations (diameter 123+/-24 nm) and sieve plates was elucidated and it was shown that fenestrations occur in areas of abrupt cytoplasmic thinning (165+/-54 nm vs. 292+/-103 nm in non-fenestrated regions, P<0.0001). Sieve plates were not preferentially co-localized with fluorescently labeled F-actin stress fibers and endothelial nitric oxide synthase but appeared to occur in primarily attenuated non-raft regions of the cell membrane. Labyrinthine structures were not seen and all fenestrations were short cylindrical pores. In conclusion, three-dimensional structured illumination microscopy has enabled the unlimited power of fluorescent immunostaining and co-localization to reveal new structural and functional information about fenestrations and sieve plates.
Tracking and Quantitation of Fluorescent HIV During Cell-to-cell Transmission
The green fluorescent protein (GFP) is a powerful genetic marking tool that has enabled virologists to monitor and track viral proteins during HIV infection. Expression-optimized Gag-GFP constructs have been used to study virus-like particle (VLP) assembly and localization in cell types that are easily transfected. The development of HIV-1 variants carrying GFP within the context of the viral genome has facilitated the study of infection and has been particularly useful in monitoring the transfer of virus between cells following virological synapse formation. HIV Gag-iGFP, a viral clone that contains GFP inserted between the matrix (MA) and capsid (CA) domains of Gag, is the first replication competent molecular clone that generates fluorescent infectious particles. Here, we discuss some methods that exploit HIV Gag-iGFP to quantify cell-to-cell transmission of virus by flow cytometry and to track the proteins during assembly and transmission using live-cell imaging.
Raman Scattering in Pathology
Raman scattering is the inelastic scattering of light off chemical bonds, and can therefore show molecular specificity. It can be used both in pure spectroscopy mode, and in imaging mode. While many applications of Raman spectroscopy and imaging in the biomedical field have been so far demonstrated, the use of this technology for pathology applications is still in early stages. In this paper we review some of the most important recent developments in this field, including a description of relevant technologies, applications to molecular sensing, characterization of cells and tissues of interest, and disease detection via Raman scattering.
