Bernhard von Vacano

Highly sensitive single-beam heterodyne coherent anti-Stokes Raman scattering

Single-beam coherent anti-Stokes Raman-scattering (CARS) microspectroscopy achieves a complete CARS scheme with a femtosecond laser. Here, we introduce heterodyne detection in a simple experimental extension: the optical fields driving the CARS process and the local oscillator used for heterodyning are derived from a single beam of ultrashort laser pulses by pulse shaping. The heterodyne signal is amplified by more than 3 orders of magnitude and is linearly dependent on the concentration of Raman scatterers. This dramatically increases the sensitivity of chemically selective detection at microscopic resolution while maintaining the simplicity of the single-beam setup.

Actively shaped supercontinuum from a photonic crystal fiber for nonlinear coherent microspectroscopy

The combination of broadband pulses from a photonic crystal fiber (PCF) pumped by a standard 100 fs oscillator and pulse shaping is successfully employed for coherently controlled nonlinear spectroscopy. The pulse shaper manages not only to compress the PCF supercontinuum in a closed-loop optimization scheme but also to manipulate the phase at the same time for quantum control applications. This approach is demonstrated by single-beam coherent anti-Stokes Raman microspectroscopy and should be, due to its simplicity, well suited for general applications in nonlinear microscopy.

Shaper-assisted collinear SPIDER: fast and simple broadband pulse compression in nonlinear microscopy

In situ characterization and control of the phase of broadband femtosecond pulses in microscopy can be achieved with a novel simplified scheme based on spectral shear interferometry for direct electric field reconstruction (SPIDER): the use of a femtosecond pulse shaper eliminates the need for an interferometer setup, allows dispersion-free SPIDER operation and at the same time compression even of complex pulses. Beyond compression, the scheme allows precise phase control at the site of the microscopic experiment. We present the underlying principles, design considerations, and details of the experimental implementation, and show the successful operation of the shaper-assisted collinear (SAC) SPIDER to characterize, compress, and tailor broadband femtosecond pulses in situ. The reliability is demonstrated by comparison with independent cross-frequency-resolved optical gating measurement, and improved multiphoton imaging with SAC-SPIDER-compressed pulses is shown. Its simplicity and versatility make SAC-SPIDER an extremely useful tool for next-generation broadband nonlinear microscopy.

In situ broadband pulse compression for multiphoton microscopy using a shaper-assisted collinear SPIDER

The characterization and control of the phase of broadband femtosecond pulses in nonlinear microscopy are successfully demonstrated with a collinear configuration of spectral shear interferometry for direct electric field reconstruction (SPIDER). A femtosecond-pulse shaper is used as a dispersionless interferometer for the measurement of the spectral phase and to actively compress a broadband supercontinuum from a photonic crystal fiber. This allows in situ online phase management and enables the application of quantum control spectroscopy in microenvironments.

Time-resolving molecular vibration for microanalytics: single laser beam nonlinear Raman spectroscopy in simulation and experiment.

A single-beam implementation of coherent anti-Stokes Raman scattering (CARS) allows experimentally very much simplified and flexible approaches to time-resolved vibrational spectroscopy, with the additional benefit of microscopic spatial resolution. To achieve this, a broadband femtosecond laser is combined with a pulse shaper creating tailored pulse sequences by computer control. We discuss the theoretical foundations and technical issues of the technique in detail and show the successful implementation of different schemes for truly femtosecond time-resolved vibrational spectroscopy. Hereby, we elaborate all the details of the method shown earlier in a proof-of-principle study [Von Vacano and Motzkus, Opt. Comm., 2006, 264, 488] and greatly extend it by novel approaches relying on the use of identical double pulses or additional polarization control for background-free spectroscopy with superior robustness and signal-to-noise ratio. Perspectives and applications of the presented schemes for chemical microanalysis and high-contrast chemical imaging are examined.

Influence of agglomeration and specific lung lining lipid/protein interaction on short-term inhalation toxicity.

Abstract Lung lining fluid is the first biological barrier nanoparticles (NPs) encounter during inhalation. As previous inhalation studies revealed considerable differences between surface functionalized NPs with respect to deposition and toxicity, our aim was to investigate the influence of lipid and/or protein binding on these processes. Thus, we analyzed a set of surface functionalized NPs including different SiO2 and ZrO2 in pure phospholipids, CuroSurfTM and purified native porcine pulmonary surfactant (nS). Lipid binding was surprisingly low for pure phospholipids and only few NPs attracted a minimal lipid corona. Additional presence of hydrophobic surfactant protein (SP) B in CuroSurfTM promoted lipid binding to NPs functionalized with Amino or PEG residues. The presence of the hydrophilic SP A in nS facilitated lipid binding to all NPs. In line with this the degree of lipid and protein affinities for different surface functionalized SiO2 NPs in nS followed the same order (SiO2 Phosphate ∼ unmodified SiO2 < SiO2 PEG < SiO2 Amino NPs). Agglomeration and biomolecule interaction of NPs in nS was mainly influenced by surface charge and hydrophobicity. Toxicological differences as observed in short-term inhalation studies (STIS) were mainly influenced by the core composition and/or surface reactivity of NPs. However, agglomeration in lipid media and lipid/protein affinity appeared to play a modulatory role on short-term inhalation toxicity. For instance, lipophilic NPs like ZrO2, which are interacting with nS to a higher extent, exhibited a far higher lung burden than their hydrophilic counterparts, which deserves further attention to predict or model effects of respirable NPs.

Molecular discrimination of a mixture with single-beam Raman control.

A single beam of shaped femtosecond pulses is used to coherently control the Raman excitation and to simultaneously observe the resulting vibrations of molecules in a mixture resolved in time. This experimentally simple scheme opens up exciting new possibilities for the selective detection of dangerous chemical or bacterial species, such as spores, and will serve to enhance contrast in nonlinear Raman chemical imaging.

Artifacts by marker enzyme adsorption on nanomaterials in cytotoxicity assays with tissue cultures

We used precision cut lung slices (PCLS) to study the cytotoxicity of cobalt ferrite nanomaterials with and without bovine serum albumin (BSA) stabilization. Using mitochondrial activity as an indicator of cytotoxicity (WST-1 assay) increasing concentrations of cobalt ferrite nanomaterial caused increasing levels of cytotoxicity in PCLS irrespective of BSA stabilization. However, there was no increase in released lactate dehydrogenase (LDH) levels caused by BSA stabilized nanomaterial indicating concentration depended cytotoxictiy. Moreover, non-stabilized nanomaterial caused a decrease of background LDH levels in the PCLS culture supernatant confirmed by complementary methods. Direct characterization of the protein corona of extracted nanomaterial shows that the LDH decrease is due to adsorption of LDH onto the surface of the non-stabilized nanomaterial, correlated with strong agglomeration. Preincubation with serum protein blocks the adsorption of LDH and stabilizes the nanomaterial at low agglomeration. We have thus demonstrated the cytotoxicity of nanomaterials in PCLS does not correlate with disrupted membrane integrity followed by LDH release. Furthermore, we found that intracellular enzymes such as the marker enzyme LDH are able to bind onto surfaces of nanomaterial and thereby adulterate the detection of toxic effects. A replacement of BSA by LDH or a secondary LDH-on-BSA-corona were not observed, confirming earlier indications that the protein corona exchange rate are slow or vanishing on inorganic nanomaterial. Thus, the method(s) to assess nanomaterial-mediated effects have to be carefully chosen based on the cellular effect and possible nano-specific artifacts.

Hydrophobin-Encapsulated Quantum Dots.

The phase transfer of quantum dots to water is an important aspect of preparing nanomaterials that are suitable for biological applications, and although numerous reports describe ligand exchange, very few describe efficient ligand encapsulation techniques. In this report, we not only report a new method of phase transferring quantum dots (QDs) using an amphiphilic protein (hydrophobin) but also describe the advantages of using a biological molecule with available functional groups and their use in imaging cancer cells in vivo and other imaging applications.

Interferometrically Detected Femtosecond CARS in a Single Beam of Shaped Femtosecond Pulses

Photonic integration of functions such as excitation, probing and interferometry in shaped broadband pulses allows huge simplification of coherent anti-Stokes Raman scattering (CARS) for microspectroscopy, paving the way to cost-efficient implementations, e. g. all-fibre solutions.