Juris Kiskis

Plaque-associated lipids in Alzheimer’s diseased brain tissue visualized by nonlinear microscopy

By simultaneous coherent anti-Stokes Raman scattering (CARS) and 2-photon fluorescence microscopy of Thioflavin-S stained Alzheimer´s diseased human brain tissues, we show evidence of lipid deposits co-localizing with fibrillar β-amyloid (Aβ) plaques. Two lipid morphologies can be observed; lamellar structures and coalescing macro-aggregates of sub-micron sizes to ~25 μm. No significant lipid deposits were observed in non-fibrillar, diffuse plaques identified by Aβ immuno-staining. CARS microscopy of unlabeled samples confirms the lamellar and macro-aggregate lipid morphologies. The composition of the plaques was analyzed by CARS microspectroscopy and Raman microscopy; vibrational signatures of lipids with long acyl chains co-localize with the β-sheet vibrations. The lipid fluidity was evaluated from the CARS spectra, illustrating that the lipid composition/organization varies throughout the plaques. Altogether this indicates close amyloid-lipid interplay in fibrillar Aβ plaques, rendering them more dynamic compositions than previously believed and, hence, potential sources of toxic oligomers.

Imaging of Lipids in Microalgae with Coherent Anti-Stokes Raman Scattering Microscopy

Lipids are accumulated as giant droplets alongside coalescing emerging droplets under excessive lipid storage, in contrast to the multiple micron-sized droplets formed at normal conditions. Microalgae have great prospects as a sustainable resource of lipids for refinement into nutraceuticals and biodiesel, which increases the need for detailed insights into their intracellular lipid synthesis/storage mechanisms. As an alternative strategy to solvent- and label-based lipid quantification techniques, we introduce time-gated coherent anti-Stokes Raman scattering (CARS) microscopy for monitoring lipid contents in living algae, despite strong autofluorescence from the chloroplasts, at approximately picogram and subcellular levels by probing inherent molecular vibrations. Intracellular lipid droplet synthesis was followed in Phaeodactylum tricornutum algae grown under (1) light/nutrient-replete (control [Ctrl]), (2) light-limited (LL), and (3) nitrogen-starved (NS) conditions. Good correlation (r2 = 0.924) was found between lipid volume data yielded by CARS microscopy and total fatty acid content obtained from gas chromatography-mass spectrometry analysis. In Ctrl and LL cells, micron-sized lipid droplets were found to increase in number throughout the growth phases, particularly in the stationary phase. During more excessive lipid accumulation, as observed in NS cells, promising commercial harvest as biofuels and nutritional lipids, several micron-sized droplets were present already initially during cultivation, which then fused into a single giant droplet toward stationary phase alongside with new droplets emerging. CARS microspectroscopy further indicated lower lipid fluidity in NS cells than in Ctrl and LL cells, potentially due to higher fatty acid saturation. This agreed with the fatty acid profiles gathered by gas chromatography-mass spectrometry. CARS microscopy could thus provide quantitative and semiqualitative data at the single-cell level along with important insights into lipid-accumulating mechanisms, here revealing two different modes for normal and excessive lipid accumulation.

The histone deacetylase inhibiting drug Entinostat induces lipid accumulation in differentiated HepaRG cells

Dietary overload of toxic, free metabolic intermediates leads to disrupted insulin signalling and fatty liver disease. However, it was recently reported that this pathway might not be universal: depletion of histone deacetylase (HDAC) enhances insulin sensitivity alongside hepatic lipid accumulation in mice, but the mechanistic role of microscopic lipid structure in this effect remains unclear. Here we study the effect of Entinostat, a synthetic HDAC inhibitor undergoing clinical trials, on hepatic lipid metabolism in the paradigmatic HepaRG liver cell line. Specifically, we statistically quantify lipid droplet morphology at single cell level utilizing label-free microscopy, coherent anti-Stokes Raman scattering, supported by gene expression. We observe Entinostat efficiently rerouting carbohydrates and free-fatty acids into lipid droplets, upregulating lipid coat protein gene Plin4, and relocating droplets nearer to the nucleus. Our results demonstrate the power of Entinostat to promote lipid synthesis and storage, allowing reduced systemic sugar levels and sequestration of toxic metabolites within protected protein-coated droplets, suggesting a potential therapeutic strategy for diseases such as diabetes and metabolic syndrome.

Plasmon-enhanced four-wave mixing by nanoholes in thin gold films

Nonlinear plasmonics opens up for wavelength conversion, reduced interaction/emission volumes, and nonlinear enhancement effects at the nanoscale with many compelling nanophotonic applications foreseen. We investigate nonlinear plasmonic responses of nanoholes in thin gold films by exciting the holes individually with tightly focused laser beams, employing a degenerated pump/probe and Stokes excitation scheme. Excitation of the holes results in efficient generation of both narrowband four-wave mixing (FWM) and broadband multiphoton excited luminescence, blueshifted relative to the excitation beams. Clear enhancements were observed when matching the pump/probe wavelength with the hole plasmon resonance. These observations show that the FWM generation is locally excited by nanoholes and has a resonant behavior primarily governed by the dimensions of the individual holes.

Visualization of structural changes in cellulosic substrates during enzymatic hydrolysis using multimodal nonlinear microscopy

Enzymatic hydrolysis of cellulose provides a renewable source of monosaccharides for production of variety of biochemicals and biopolymers. Unfortunately, the enzymatic hydrolysis of cellulose is often incomplete, and the reasons are not fully understood. We have monitored enzymatic hydrolysis in terms of molecular density, ordering and autofluorescence of cellulose structures in real time using simultaneous CARS, SHG and MPEF microscopy with the aim of contributing to the understanding and optimization of the enzymatic hydrolysis of cellulose. Three cellulose-rich substrates with different supramolecular structures, pulp fibre, acid-treated pulp fibre and Avicel, were studied at microscopic level. The microscopy studies revealed that before enzymatic hydrolysis Avicel had the greatest carbon-hydrogen density, while pulp fibre and acid-treated fibre had similar density. Monitoring of the substrates during enzymatic hydrolysis revealed the double exponential SHG decay for pulp fibre and acid-treated fibre indicating two phases of the process. Acid-treated fibre was hydrolysed most rapidly and the hydrolysis of pulp fibre was spatially non-uniform leading to fractioning of the particles, while the hydrolysis of Avicel was more than an order of magnitude slower than that of both fibres.

Nonlinear nearfield microscopy

Higher-order nonlinearity of light-matter interactions, such as second and third harmonic generation (SHG & THG) and Coherent anti-Stokes Raman Scattering (CARS) can be used for improving spatial resolution in microscopy as a consequence of the confinement of the nonlinear polarization to the high-intensity region of the focal volume. However, the resolution is limited to similar to 300 nm, not sufficient to resolve macromolecules or nanostructures of interest in the bio-, life- and nano-sciences. In the strive to push the resolution beyond the diffraction limit, allowing for nanoscale imaging, we have equipped a nonlinear optical microscope with a scanning-probe setup operated in tapping-mode feedback. A tapered, gold-coated, open-aperture tip with an aperture diameter of similar to 150 nm is scanned over the sample, probing the nonlinear nearfield generated by free-beam excitation. First nonlinear coherent Raman nearfield images of biological macromolecules and metallic nanostructures are shown. Limitations and future challenges with nonlinear nearfield microscopy are discussed.Higher-order nonlinearity of light–matter interactions, such as second and third harmonic generation (SHG & THG) and Coherent anti-Stokes Raman Scattering (CARS) can be used for improving spatial resolution in microscopy as a consequence of the confinement of the nonlinear polarization to the high-intensity region of the focal volume. However, the resolution is limited to ~300 nm, not sufficient to resolve macromolecules or nanostructures of interest in the bio-, lifeand nano-sciences. In the strive to push the resolution beyond the diffraction limit, allowing for nanoscale imaging, we have equipped a nonlinear optical microscope with a scanning-probe setup operated in tapping-mode feedback. A tapered, gold-coated, open-aperture tip with an aperture diameter of ~150 nm is scanned over the sample, probing the nonlinear nearfield generated by free-beam excitation. First nonlinear coherent Raman nearfield images of biological macromolecules and metallic nanostructures are shown. Limitations and future challenges with nonlinear nearfield microscopy are discussed.

CARS microscopy of Alzheimer's diseased brain tissue

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder currently without cure, characterized by the presence of extracellular plaques surrounded by dystrophic neurites. In an effort to understand the underlying mechanisms, biochemical analysis (protein immunoblot) of plaque extracts reveals that they consist of amyloid-beta (Aβ) peptides assembled as oligomers, protofibrils and aggregates. Their spatial distribution has been confirmed by Thioflavin-S or immuno-staining with fluorescence microscopy. However, it is increasingly understood that the protein aggregation is only one of several mechanism that causes neuronal dysfunction and death. This raises the need for a more complete biochemical analysis. In this study, we have complemented 2-photon fluorescence microscopy of Thioflavin-S and Aβ immuno-stained human AD plaques with CARS microscopy. We show that the chemical build-up of AD plaques is more complex and that Aβ staining does not provide the complete picture of the spatial distribution or the molecular composition of AD plaques. CARS images provide important complementary information to that obtained by fluorescence microscopy, motivating a broader introduction of CARS microscopy in the AD research field.