Andreas Zumbusch

Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses

In this work, we show how broad-bandwidth femtosecond pulses can be used to achieve high spectral resolution in nonlinear spectroscopy and microscopy. Our approach is based on chirping the excitation pulses in order to focus their entire bandwidth into a narrow spectral region. We show that spectral features which are 100 times narrower than the excitation light can be resolved with this simple spectral focusing. The gain in spectral selectivity and sensitivity makes its application to nonlinear microscopy very convenient. This is demonstrated with diffraction-limited coherent anti-Stokes Raman scattering microscopy.

Noninvasive Photoacoustic and Fluorescence Sentinel Lymph Node Identification using Dye-Loaded Perfluorocarbon Nanoparticles

The contrast mechanisms used for photoacoustic tomography (PAT) and fluorescence imaging differ in subtle, but significant, ways. The design of contrast agents for each or both modalities requires an understanding of the spectral characteristics as well as intra- and intermolecular interactions that occur during formulation. We found that fluorescence quenching that occurs in the formulation of near-infrared (NIR) fluorescent dyes in nanoparticles results in enhanced contrast for PAT. The ability of the new PAT method to utilize strongly absorbing chromophores for signal generation allowed us to convert a highly fluorescent dye into an exceptionally high PA contrast material. Spectroscopic characterization of the developed NIR dye-loaded perfluorocarbon-based nanoparticles for combined fluorescence and PA imaging revealed distinct dye-dependent photophysical behavior. We demonstrate that the enhanced contrast allows detection of regional lymph nodes of rats in vivo with time-domain optical and photoacoustic imaging methods. The results further show that the use of fluorescence lifetime imaging, which is less dependent on fluorescence intensity, provides a strategic approach to bridge the disparate contrast reporting mechanisms of fluorescence and PA imaging methods.

Spectral diffusion and individual two-level systems probed by fluorescence of single terrylene molecules in a polyethylene matrix

Abstract We study the influence of low-energy matrix excitations on the line widths and fluorescence correlation of single terrylene molecules in polyethylene at helium temperatures. The histogram of line widths has a cutoff at the natural line width of terrylene, showing that for some molecules dephasing and spectral diffusion are negligible on the measurement time scale. The shape of the histogram can be qualitatively interpreted by means of a simple model for spectral diffusion. The line widths of different molecules show different temperature dependences. The correlation method is then applied to a time-resolved study of the intensity fluctuations of single molecule fluorescence. Many possible shapes of correlation functions appear, spanning many decades of relaxation times. We believe single two-level systems (TLSs) are the cause of the well defined exponential steps we observe. In some cases, the two positions of a single molecules line can be identified in the spectrum. They present the same time constant in their correlation functions and jump together to a new frequency. The dependence of the correlation on exciting flux shows that jumps can be spontaneous or photo-induced. The study of the rate as a function of temperature shows clear power laws which we attribute to tunnelling of the TLS assisted by one (T1) or two (T3) acoustic phonons. In one case, we find an Arrhenius activation of the rate, which could be explained by dressing the standard TLS model with slow matrix modes.

Stark effect on single molecules in a polymer matrix

Abstract We observed spectral lines from single molecules in a new disordered system: terrylene in polyethylene. Different linewidths for different molecules and photoinduced frequency jumps are observed. Measurements of the Stark effect on single molecules are presented. The lines shift linearly under applied electric field, showing that the molecular symmetry is broken by interactions with the matrix. The shifts differ strongly according to the molecule. Some molecules show surprisingly strong induced dipole moments of up to 5 D, an illustration of how strong the solvent—solute interaction can be, even in non-polar systems.

Selective NIR chromophores : Bis(Pyrrolopyrrole) Cyanines

New applications have recently led to an increased interest in near infrared (NIR) dyes. Both, strong NIR absorption and fluorescence emission are important phenomena. While fluorescence is exploited especially for labelling purposes in microscopy, NIR absorption has a broad range of applications mainly in material science. Examples of growing fields of interest are NIR light emitting diodes and the use of NIR-photosensitizers in dye-sensitized solar cells. In the latter cases, the aim is to utilize the NIR part of the solar spectrum in order to generate photocurrents. In many applications, especially selective NIR absorbers which absorb strongly in the NIR while exhibiting negligible absorption in the visible spectral range are required. Dyes with these properties find use as NIR absorbers in paints and windows to block off heat, in laser protecting glasses, as dyes for laser polymer welding of transparent components, or as anti-forgery markers. In all these examples it is required that the dye does not influence the spectral properties of the components in the visible. Thus, photostable dyes with strong, narrowbandwidth NIR absorptions and negligible absorption in the visible spectral range are needed. Common strategies to achieve bathochromic shifts of the absorption maximum of a dye are the extension of the chromophoric system or the introduction of donor and acceptor groups into the chromophore. Cyanine dyes and the rylene dyes are the most popular classes showing significant bathochromic shifts upon extension of their π-system. In cyanine dyes, however, the chemical stability decreases with increasing extension. By contrast, rylene dyes are chemically very stable, but, due to comparably large bondlength changes accompanying the S0→S1 transition, they show rather intense 01 and 02 vibronic bands (Franck-Condon principle). Thus, their S0→S1 absorption is extended into the visible range. Other important classes of NIR dyes, showing strong and narrowband NIR absorption are squarine dyes, bodipys and some aza[18]annulene derivatives such as porphyrins phthalocyanines and especially naphtolocyanines. We recently described pyrrolopyrrole cyanine (PPCy) dyes as a new class of NIR dyes and fluorophores. PPCys are synthesized by a condensation reaction of diketopyrrolopyrrole (DPP) 1 and heteroaromatic acetonitriles (HAA) 3. Complexation with either BF2 or BPh2 yields fluorophores which exhibit narrowband absorption between 650 and 900 nm and fluorescence emission with high quantum yields. By isolation of the monoactivated DPP 2, selectivly one of the carbonyl groups of the DPP can be reacted with a HAA 3 to the half converted products 4. Further reaction with another acetonitrile derivative 3 leads to PPCys with an asymmetric substitution pattern. This stepwise reaction scheme allows for the introduction of just one functional group. Here, we describe the synthesis of a new class of NIR chromophores with unprecedented spectral properties. They have very strong and narrowband NIR absorption while featuring negligible visible absorption. The absorption coefficients of these dyes in the NIR belong to the strongest ever reported for organic fluorophores, being twice as high as those of recently reported rylene dyes. Our previous approaches to shift the absorption of the PPCys bathochromically concerned the modification of the heteroaromatic endgroup (A) and/or the complexation agent. The selective synthesis of the halfconverted DPPs 4 opens a strategy to enlarge the chromophore of the PPCys by reacting two equivalents of 4 with a bifunctional bridging heteroaromatic acetonitrile such as 5 (Scheme 1.) The resulting condensation products contain two pyrrolopyrrole moieties and can therefore be called bis-pyrrolopyrrole cyanine dyes.

Pyrrolopyrrole cyanine dyes: a new class of near-infrared dyes and fluorophores.

Pyrrolopyrrole cyanine (PPCy) dyes are presented as a novel class of near-infrared (NIR) chromophores, which are synthesized in a condensation reaction of diketopyrrolopyrrole with heteroarylacetonitrile compounds. Their optical properties are marked by strong and narrow-band NIR absorptions. Complexation products with BF(2) and BPh(2) show strong NIR fluorescence and hardly any absorption in the visible range. We synthesized a series of new PPCys that differ only in the heterocyclic peripheral groups of the chromophore. With this strategy, the absorption spectra can be tuned between 684 and 864 nm, while high fluorescence quantum yields are maintained. The influence of the heterocycle on the optical properties of the dyes is discussed.

Remodeling of Lipid Droplets during Lipolysis and Growth in Adipocytes

Background: Micro-lipid droplets (mLDs) appear in adipocytes upon lipolytic stimulation. LDs may grow by spontaneous, homotypic fusion. Results: Scavenging of fatty acids prevents mLD formation. LDs grow by a slow transfer of lipids between LDs. Conclusion: mLDs form due to fatty acid overflow. LD growth is a controlled process. Significance: Novel mechanistic insights into LD remodeling are provided. Synthesis, storage, and turnover of triacylglycerols (TAGs) in adipocytes are critical cellular processes to maintain lipid and energy homeostasis in mammals. TAGs are stored in metabolically highly dynamic lipid droplets (LDs), which are believed to undergo fragmentation and fusion under lipolytic and lipogenic conditions, respectively. Time lapse fluorescence microscopy showed that stimulation of lipolysis in 3T3-L1 adipocytes causes progressive shrinkage and almost complete degradation of all cellular LDs but without any detectable fragmentation into micro-LDs (mLDs). However, mLDs were rapidly formed after induction of lipolysis in the absence of BSA in the culture medium that acts as a fatty acid scavenger. Moreover, mLD formation was blocked by the acyl-CoA synthetase inhibitor triacsin C, implicating that mLDs are synthesized de novo in response to cellular fatty acid overload. Using label-free coherent anti-Stokes Raman scattering microscopy, we demonstrate that LDs grow by transfer of lipids from one organelle to another. Notably, this lipid transfer between closely associated LDs is not a rapid and spontaneous process but rather occurs over several h and does not appear to require physical interaction over large LD surface areas. These data indicate that LD growth is a highly regulated process leading to the heterogeneous LD size distribution within and between individual cells. Our findings suggest that lipolysis and lipogenesis occur in parallel in a cell to prevent cellular fatty acid overflow. Furthermore, we propose that formation of large LDs requires a yet uncharacterized protein machinery mediating LD interaction and lipid transfer.

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.