Martin Winterhalder

DMD-based LED-illumination Super-resolution and optical sectioning microscopy

Super-resolution three-dimensional (3D) optical microscopy has incomparable advantages over other high-resolution microscopic technologies, such as electron microscopy and atomic force microscopy, in the study of biological molecules, pathways and events in live cells and tissues. We present a novel approach of structured illumination microscopy (SIM) by using a digital micromirror device (DMD) for fringe projection and a low-coherence LED light for illumination. The lateral resolution of 90 nm and the optical sectioning depth of 120 μm were achieved. The maximum acquisition speed for 3D imaging in the optical sectioning mode was 1.6×107 pixels/second, which was mainly limited by the sensitivity and speed of the CCD camera. In contrast to other SIM techniques, the DMD-based LED-illumination SIM is cost-effective, ease of multi-wavelength switchable and speckle-noise-free. The 2D super-resolution and 3D optical sectioning modalities can be easily switched and applied to either fluorescent or non-fluorescent specimens.

Compact coherent anti-Stokes Raman scattering microscope based on a picosecond two-color Er:fiber laser system

We present a compact coherent anti-Stokes Raman scattering microscope based on a widely tunable picosecond Er:fiber laser. Intense and bandwidth-limited 1 ps pump pulses at a center wavelength of 775 nm are generated via frequency mixing within the broadband fundamental at 1.55 microm. Narrowband Stokes pulses are obtained by frequency shifting of solitons in a highly nonlinear bulk fiber and subsequent second-harmonic generation. The tuning range from 850 nm to 1100 nm gives access to vibrational resonances between 1150 cm(-1) and 3800 cm(-1). A first imaging application in the spectral region of CH stretch vibrations is demonstrated.

Tailor-Made Conjugated Polymer Nanoparticles for Multicolor and Multiphoton Cell Imaging

Sonogashira coupling of dibromo- and diethynyl-substituted benzenes and fluorenes in aqueous mini-emulsion afforded colloidally stable dispersions of highly fluorescent 60-120 nm particles of poly(arylene ethynylene)s of molecular weights M(n) 4 × 10(4)-2 × 10(5) g mol(-1) with solids contents up to 15 wt %. By covalent incorporation of diethynyl pyrrolo-pyrrole or diethynyl fluorenone in the mini-emulsion polymerization the emission color of these photostable nanoparticles can be tuned from blue (λ(em max) 470) to orange at a given excitation wavelength due to an efficient energy transfer to these acceptors. Two-photon action cross sections of the particles amount to 10(6) to 10(7) GM. The particles from emulsion polymerization are taken up by HeLa cells without an adverse effect on the cells. Differentiation of the location in cells of particle species varying in emission color was demonstrated for both linear and two-photon excitation microscopy in the NIR regime.

Ultrabroadband background-free coherent anti-Stokes Raman scattering microscopy based on a compact Er:fiber laser system

We demonstrate a scheme for efficient coherent anti-Stokes Raman scattering (CARS) microscopy free of nonresonant background. Our method is based on a compact Er:fiber laser source. Impulsive excitation of molecular resonances is achieved by an 11 fs pulse at 1210 nm. Broadband excitation gives access to molecular resonances from 0 cm(-1) up to 4000 cm(-1). Time-delayed narrowband probing at 775 nm enables sensitive and high-speed spectral detection of the CARS signal free of nonresonant background with a resolution of 10 cm(-1).

Vibrational Spectroscopic Imaging and Multiphoton Microscopy of Spinal Cord Injury

Spinal cord injury triggers a series of complex biochemical alterations of nervous tissue. Up to now, such cellular events could not be studied without conventional tissue staining. The development of optical, label-free imaging techniques could provide powerful monitoring tools with the potential to be applied in vivo. In this work, we assess the ability of vibrational spectroscopy to generate contrast at molecular level between normal and altered regions in a rat model of spinal cord injury. Using tissue sections, we demonstrate that Fourier transform infrared (FT-IR) spectroscopy and spontaneous Raman spectroscopy are able to identify the lesion, the surrounding scar, and unharmed normal tissue, delivering insight into the biochemical events induced by the injury and allowing mapping of tissue degeneration. The FT-IR and Raman spectroscopic imaging provides the basis for fast multimodal nonlinear optical microscopy (coherent anti-Stokes Raman scattering, endogenous two-photon fluorescence, and second harmonic generation). The latter proves to be a fast tool for imaging of the lesion on unstained tissue samples, based on the alteration in lipid content, extracellular matrix composition, and microglia/macrophages distribution pattern. The results establish these technologies in the field of regeneration in central nervous system, with the long-term goal to extend them to intravital use, where fast and nonharmful imaging is required.

Fast and long term lipid droplet tracking with CARS microscopy

Photobleaching of organic fluorophores commonly used in fluorescence microscopy puts a limit to the number of images which can be acquired. Label-free imaging techniques therefore offer advantages both for rapid image acquisition and for long-term observations. CARS microscopy is a label-free imaging technique offering molecule specific contrast. Here we demonstrate that CARS microscopy allows video-rate tracking of intracellular transport of lipid droplets, but also continuous long-term observation of cells over several hours.

Giant lipid vesicles under electric field pulses assessed by non invasive imaging

We present experimental results regarding the effects of electric pulses on giant unilamellar vesicles (GUVs). We have used phase contrast and coherent anti-Stokes Raman scattering (CARS) microscopy as relevant optical approaches to gain insight into membrane changes under electropermeabilization. No addition of exogenous molecules (lipid analogue, fluorescent dye) was needed. Therefore, experiments were performed on pure lipid systems avoiding possible artefacts linked to their use. Structural membrane changes were assessed by loss of contrast inside the GUVs due to sucrose and glucose mixing. Our observations, performed at the single vesicle level, indicate these changes are under the control of the number of pulses and field intensity. Larger number of pulses enhances membrane alterations. A threshold value of the field intensity must be applied to allow exchange of molecules between GUVs and the external medium. This threshold depends on the size of the vesicles, the larger GUVs being affected at lower electric field strengths than the smaller ones. Our experimental data are well described by a simple model in which molecule entry is driven by direct exchange. The CARS microscopic study of the effect of pulse duration confirms that pulses, in the ms time range, induce loss of lipids and membrane deformations facing the electrodes.

Video-rate wide-field coherent anti-Stokes Raman scattering microscopy with collinear nonphase-matching illumination.

A simple scheme for video-rate wide-field coherent anti-Stokes Raman scattering (CARS) microscopy is presented. The method is based on collinear nonphase-matching illumination. The mechanisms leading to CARS signal generation are investigated. We find that refraction-mediated phase-matching is the main effect. Video-rate wide-field CARS microscopy of polystyrene beads and CARS wide-field images of C. elegans embryos are shown, and the capabilities and the limitations of the scheme are discussed.

Beyond the borders — Biomedical applications of non-linear Raman microscopy ☆

Raman spectroscopy offers great promise for label free imaging in biomedical applications. Its use, however, is hampered by the long integration times required and the presence of autofluorescence in many samples which outshines the Raman signals. In order to overcome these limitations, a variety of different non-linear Raman imaging techniques have been developed over the last decade. This review describes biomedical applications of these novel but already mature imaging techniques.

Toward far-field vibrational spectroscopy of single molecules at room temperature

We propose a new scheme for the extraction of chemically sensitive vibrational information from a single fluorescent molecule at room temperature. Our approach is based on a three-photon fluorescence excitation scheme, with selectivity in the production of a vibrational population of the ground state. We estimate the expected signal in perturbation theory for a standard dye molecule, compare its magnitude qualitatively to noise and various background sources, and discuss the experimental realization of this scheme.