Nonlinear optical microscopy with vibrational contrast.

Nonlinear optical techniques which provide vibrational contrast have gained increasing attention in microscopy during the last two decades. After outlining the potential of these techniques, we give a brief introduction to coherent anti-Stokes Raman scattering, stimulated Raman scattering and sum frequency generation and discuss their suitability for contrast generation in optical microscopy. The rapid developments in these fields during the last decade have resulted in many different applications. Three exemplary application areas will therefore be presented in the last part of this manuscript.

Monitoring fluorescence of individual chromophores in peridinin-chlorophyll-protein complex using single molecule spectroscopy.

Single molecule spectroscopy experiments are reported for native peridinin-chlorophyll a-protein (PCP) complexes, and three reconstituted light-harvesting systems, where an N-terminal construct of native PCP from Amphidinium carterae has been reconstituted with chlorophyll (Chl) mixtures: with Chl a, with Chl b and with both Chl a and Chl b. Using laser excitation into peridinin (Per) absorption band we take advantage of sub-picosecond energy transfer from Per to Chl that is order of magnitude faster than the Förster energy transfer between the Chl molecules to independently populate each Chl in the complex. The results indicate that reconstituted PCP complexes contain only two Chl molecules, so that they are spectroscopically equivalent to monomers of native-trimeric-PCP and do not aggregate further. Through removal of ensemble averaging we are able to observe for single reconstituted PCP complexes two clear steps in fluorescence intensity timetraces attributed to subsequent bleaching of the two Chl molecules. Importantly, the bleaching of the first Chl affects neither the energy nor the intensity of the emission of the second one. Since in strongly interacting systems Chl is a very efficient quencher of the fluorescence, this behavior implies that the two fluorescing Chls within a PCP monomer interact very weakly with each other which makes it possible to independently monitor the fluorescence of each individual chromophore in the complex. We apply this property, which distinguishes PCP from other light-harvesting systems, to measure the distribution of the energy splitting between two chemically identical Chl a molecules contained in the PCP monomer that reaches 280 cm(-1). In agreement with this interpretation, stepwise bleaching of fluorescence is also observed for native PCP complexes, which contain six Chls. Most PCP complexes reconstituted with both Chl a and Chl b show two emission lines, whose wavelengths correspond to the fluorescence of Chl a and Chl b. This is a clear proof that these two different chromophores are present in a single PCP monomer. Single molecule fluorescence studies of PCP complexes, both native and artificially reconstituted with chlorophyll mixtures, provide new and detailed information necessary to fully understand the energy transfer in this unique light-harvesting system.

Detection and tracking of anisotropic core-shell colloids.

Optical microscopy techniques with three dimensional (3D) resolution are powerful tools for the real-space imaging of the structure and dynamics of colloidal systems. While real-space imaging of spherical particles is well established, the observation of shape anisotropic particles has only recently met a lot of interest. Apart from translation, shape anisotropic particles also possess additional rotational degrees of freedom. In this manuscript, we introduce a novel technique to find the position and the orientation of anisotropic particles in 3D. It is based on an algorithm which is applicable to core-shell particles consisting of a spherical core and a shell with arbitrary shape. We demonstrate the performance of this algorithm using PMMA/PMMA (polymethyl methacrylate) core-shell ellipsoids. The algorithm is tested on artificial images and on experimental data. The correct identification of particle positions with subpixel accuracy and of their orientations with high angular precision in dilute and dense systems is shown. In addition, we developed an advanced particle tracking algorithm that takes both translational and rotational movements of the anisotropic particles into account. We show that our 3D detection and tracking technique is suitable for the accurate and reliable detection of large and dense colloidal systems containing several thousands of particles.

Peridinin-chlorophyll-protein reconstituted with chlorophyll mixtures: preparation, bulk and single molecule spectroscopy.

Reconstitution of the 16 kDa N-terminal domain of the peridinin-chlorophyll-protein, N-PCP, with mixtures of chlorophyll a (Chl a) and Chl b, resulted in 32 kDa complexes containing two pigment clusters, each bound to one N-PCP. Besides homo-chlorophyllous complexes, hetero-chlorophyllous ones were obtained that contain Chl a in one pigment cluster, and Chl b in the other. Binding of Chl b is stronger than that of the native pigment, Chl a. Energy transfer from Chl b to Chl a is efficient, but there are only weak interactions between the two pigments. Individual homo- and hetero-chlorophyllous complexes were investigated by single molecule spectroscopy using excitation into the peridinin absorption band and scanning of the Chl fluorescence, the latter show frequently well resolved emissions of the two pigments.

Improving autofluorescent proteins: comparative studies of the effective brightness of Green Fluorescent Protein (GFP) mutants.

We study the photophysical behavior of 8 mutants of Green Fluorescent Protein (GFP) using fluorescence correlation spectroscopy (FCS) on the single molecule level and double resonance excitation of bulk samples. Experimental data reported here and the previously published data on the RH/R(-) equilibrium and fluorescence quantum yields Phi(Fl) (Jung et al., 2005; Biophys J 88:1932-1947) are analyzed with respect to single molecule as well as conventional fluorescence microscopy. The fraction of GFP molecules in a dark state, [D], reduces the effective absorption cross section under photostationary conditions. The determination of the excitable fraction [B] and its fluorescence quantum yield Phi(Fl) gives the effective brightness Phi(eff). Our results show that in its wavelength range, eGFP is, among the GFPs, the best fluorophore for most microscopic applications. However, in the red shifted YFP-proteins, there is still potential for improvement, since a pronounced dark state population is detectable in all mutants investigated so far. We propose to use the mutant T203Y/E222Q in imaging studies, whenever the expression yield is not a limiting factor. In FCS experiments, where the useful concentration range of the expressed molecules is restricted to concentrations below micromolarity, our data suggest the use of wt-GFP or mutant T203Y, as these represent photochemical buffers. Both mutants might surpass the limitations given by out-of-focus bleaching in live cell microscopy.

Single-molecule microscopy of the green fluorescent protein using simultaneous two-color excitation.

In vivo microscopy of the Green Fluorescent Protein (GFP), the most important label in cell biology, with single-molecule sensitivity is hampered by an insufficient signal-to-noise ratio. A significant improvement is obtained with a novel two-color excitation technique. The picture clearly shows the increased brightness of GFP in in vitro single-molecule assays and in live-cell microscopy under two-color illumination (upper cell) as compared to normal illumination (lower cell).

Birth of healthy children after intracytoplasmic sperm injection in two couples with male Kartagener's syndrome.

OBJECTIVE: To describe two cases in which intracytoplasmic sperm injection (ICSI) was successful for patients with infertility due to Kartagener's syndrome. DESIGN: Case report. SETTING: Private hospital for gynecology department of reproductive medicine, and university hospital center for andrology. PATIENT(S): Two couples with primary infertility due to Kartagener's syndrome in the male. INTERVENTION(S): ICSI. MAIN OUTCOME MEASURE(S): Pregnancy and birth after ICSI. RESULT(S): In both couples, ICSI was successful in the first cycle. The uncomplicated pregnancies resulted in the birth of three healthy children. One female and male/female twins. CONCLUSION(S): In couples with infertility due to Kartagener's syndrome in the male, ICSI has proved to be a successful therapy resulting in clinically healthy offspring. This knowledge may improve our understanding of the involvement of paternally inherited centrosomes, which nucleate microtubules, in human reproduction.

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.

Indistinguishable photons from a single molecule.

We report the results of coincidence counting experiments at the output, of a Michelson interferometer using the zero-phonon-line emission of a single molecule at 1.4 K. Under continuous wave excitation, we observe the absence of coincidence counts as an indication of two-photon interference. This corresponds to the observation of Hong-Ou-Mandel correlations and proves the suitability of the zero-phonon-line emission of single molecules for applications in linear optics quantum computation.

Spectroscopy of single phycoerythrocyanin monomers: dark state identification and observation of energy transfer heterogeneities.

Phycoerythrocyanin (PEC) is part of the light harvesting system of cyanobacteria. The PEC monomer contains one phycoviolobilin chromophore, which transfers excitation energy onto two phycocyanobilin chromophores. Many spectroscopical methods have been used in the past to study the bulk properties of PEC. These methods average over many molecules. Therefore, differences in the behavior of individual molecules remain hidden. The energy transfer within photosynthetic complexes is however sensitive to changes in the spectroscopic properties of the participating subunits. Knowledge about heterogeneities is therefore important for the description of the energy transfer in photosynthetic systems. Here, the recording of the fluorescence emission of single PEC molecules is used as a tool to obtain such information. Spectrally resolved detection as well as double resonance excitation of single PEC molecules is used to investigate their bleaching behavior. The trans isomer of the phycoviolobilin chromophore is identified as a short-lived dark state of monomeric PEC. Polarization sensitive single molecule detection is used for the direct observation of the energy transfer in individual PEC molecules. The experiments reveal that more than one-half of the PEC molecules exhibit an energy transfer behavior significantly different from the bulk. These heterogeneities persist on a time scale of several seconds. Model calculations lead to the conclusion that they are caused by minor shifts in the spectra of the chromophores.