Fredrik Svedberg

Coherent Anti-Stokes Raman Scattering Microscopy of Cellular Lipid Storage

With the increasing number of studies using nonlinear microscopy in the biosciences, an awareness for the potentials of nonlinear optics has begun to emerge among a broader audience. Coherent anti-Stokes Raman scattering (CARS) microscopy is one of the most technically challenging methods in this category, forming images of molecular distributions based on their vibrations by a multiphoton interaction process. The primary strength of CARS microscopy lies in the ability of imaging lipids; the full 3-D distribution in living cells can be mapped without exogenous tags. Thus, CARS microscopy has a strong potential to become a central instrument for in vivo studies of the lipid metabolism at cellular level, improving present understanding of the mechanisms behind the many metabolism-related diseases, the impact of natural bioactive components in foods, and supporting the development of efficient pharmaceuticals as well as bioengineering processes exploiting the metabolism of microorganisms for the production of alternative energy sources. We illustrate this wide range of biological applications of CARS microscopy with a series of examples from our research.

On the importance of optical forces in surface-enhanced Raman scattering (SERS)

This contribution reports on the combination of optical tweezers with SERS spectroscopy of colloidal silver nanoparticles covered by thiophenol. The experimental design is based on two different laser beams, one used for Raman excitation (lamda = 514.5 nm) and one for optical tweezing (lamda=830 nm). For a fixed Raman excitation power, the SERS signal from thiophenol is found to increase dramatically when the trapping laser is activated. This result is interpreted as a combination of two effects, an accumulation of nanoparticles in the optical trap and an optically induced aggregation of these nanoparticles.

Uptake of gold nanoparticles in healthy and tumor cells visualized by nonlinear optical microscopy

Understanding the mechanism underlying the interactions between inorganic nanostructures and biological systems is crucial for several rapidly growing fields that rely on nano-bio interactions. In particular, the further development of cell-targeted drug delivery using metallic nanoparticles (NP) requires new tools for understanding the mechanisms triggered by the contact of NPs with membranes in different cells at the subcellular level. Here we present a novel concept of multimodal microscopy, enabling three-dimensional imaging of the distribution of gold NPs in living, unlabeled cells. Our approach combines multiphoton induced luminescence (MIL) with coherent anti-Stokes Raman scattering (CARS) microscopy. Comparison with transmission electron microscopy (TEM) reveals in vivo sensitivity down to the single nanostructure. By monitoring the incorporation of NPs in human healthy epidermal keratinocytes and squamous carcinoma cells (SCC), we address the feasibility of noninvasive delivery of NPs for therapeutic purposes. While neutralizing PEG coating was confirmed to prevent NP integration in SCCs, an unexpectedly efficient integration of NPs into keratinocytes was observed. These results, independently validated using TEM, demonstrate the need for advanced surface modification protocols to obtain tumor selectivity for NP delivery. The CARS/MIL microscopy platform presented here is thus a promising tool for noninvasive study of the interaction between NPs and cell.

Nonlinear microscopy of lipid storage and fibrosis in muscle and liver tissues of mice fed high-fat diets

Hallmarks of high-fat Western diet intake, such as excessive lipid accumulation in skeletal muscle and liver as well as liver fibrosis, are investigated in tissues from mice using nonlinear microscopy, second harmonic generation (SHG), and coherent anti-Stokes Raman scattering (CARS), supported by conventional analysis methods. Two aspects are presented; intake of standard chow versus Western diet, and a comparison between two high-fat Western diets of different polyunsaturated lipid content. CARS microscopy images of intramyocellular lipid droplets in muscle tissue show an increased amount for Western diet compared to standard diet samples. Even stronger diet impact is found for liver samples, where combined CARS and SHG microscopy visualize clear differences in lipid content and collagen fiber development, the latter indicating nonalcoholic fatty liver disease (NAFLD) and steatohepatitis induced at a relatively early stage for Western diet. Characteristic for NAFLD, the fibrous tissue-containing lipids accumulate in larger structures. This is also observed in CARS images of liver samples from two Western-type diets of different polyunsaturated lipid contents. In summary, nonlinear microscopy has strong potential (further promoted by technical advances toward clinical use) for detection and characterization of steatohepatitis already in its early stages.

Nonlinear microscopy with fiber laser continuum excitation

A compact high-power fiber-based femtosecond laser system is presented for coherent anti-Stokes Raman scattering/second-harmonic generation (CARS/SHG) microscopy, and quantitatively compared with a conventional picosecond optical parametric oscillator (OPO)-based system. While the broad spectral width of the femtosecond pulses results in 2.5 times lower image contrast and limited spectral selectivity, lipid stores, myosin, and collagen filaments in living cells can clearly be identified at 60 times lower excitation powers compared to the picosecond system. Visually the images contain the same information. Together with simple operation, small footprint, and low cost, the capabilities of this high-power all-fiber-based laser system promise a more general use of nonlinear microscopy within the biosciences.

Chemical release from single PMMA microparticles monitored by CARS microscopy

Microparticles loaded with antigens, proteins, DNA, fungicides, and other functional agents emerge as ideal vehicles for vaccine, drug delivery, genetic therapy, surface- and crop protection. The microscopic size of the particles and their collective large specific surface area enables highly active and localized release of the functional substance. In order to develop designs with release profiles optimized for the specific application, it is desirable to map the distribution of the active substance within the particle and how parameters such as size, material and morphology affect release rates at single particle level. Current imaging techniques are limited in resolution, sensitivity, image acquisition time, or sample treatment, excluding dynamic studies of active agents in microparticles. Here, we demonstrate that the combination of CARS and THG microscopy can successfully be used, by mapping the spatial distribution and release rates of the fungicide and food preservative IPBC from different designs of PMMA microparticles at single-particle level. By fitting a radial diffusion model to the experimental data, single particle diffusion coefficients can be determined. We show that release rates are highly dependent on the size and morphology of the particles. Hence, CARS and THG microscopy provides adequate sensitivity and spatial resolution for quantitative studies on how singleparticle properties affect the diffusion of active agents at microscopic level. This will aid the design of innovative microencapsulating systems for controlled release.

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.

CARS and Raman microscopy of Alzheimer’s Brain Tissue

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder without cure, characterized by the presence of extracellular plaques surrounded by dystrophic neuritis . Fundamental understanding of the molecular processes involved is urgently needed in order to develop strategies to inhibit steps in the AD pathogenesis. Current understanding is that the senile plaques are composed of peptide fragments (Amyloid- β peptides) formed after the cleavage of the transmembrane protein amyloid protein precursor (APP) . This knowledge relies primarily on biochemical analysis of the plaques in cell extracts together with fluorescence microscopy of tissue samples exposed to harsh preparation procedures and labeling. Thus, it is disputed to which extent the observations reported reflect the true biochemistry and morphology of AD brain tissue. By the powerful combination of Raman microspectroscopy, Coherent Anti-Stokes Raman Scattering (CARS) and 2-photon fluorescence microscopy, we show that the molecular composition of AD plaques is more complex and consequently the mechanisms behind their formation.