Spatial activity mapping of ß-mannanase on soybean seeds.

For farm animals the supplementation of exogenous enzymes, like ß-mannanase, to soybean-based diets is beneficial to improve feed digestibility. In order to unravel the effect of ß-mannanase on soybean meal's cell structure, a novel imaging concept was developed which allows visualizing the spatial activity pattern of ß-mannanase with high sensitivity by fluorescence microscopy before any visible degradation of the cellular structure occurs. It is based on fluorescence labeling of newly formed reducing ends of ß-mannanase-hydrolyzed polysaccharides after the native reducing ends of all polysaccharides present were chemically reduced. It was revealed that ß-mannanase is not only active at the cell wall but also at previously unknown sites, like the middle lamella and, most prominently, at an intracellular matrix enclosing the protein storage vacuoles. Based on these findings it can be hypothesized that the evaluated ß-mannanase can degrade the enclosing matrix of encapsulated proteins and the cell wall structure and thereby improves efficiency of feed utilization.

Near-degenerate four-wave-mixing microscopy.

Fluorescence microscopy has been widely used to explore the nanoscale world because of its superb sensitivity, but it is limited to fluorescent samples. Hence, various spectroscopic contrasts have been explored for imaging nonfluorescent species. Here we report a multiphoton microscopy based on single-beam near-degenerate four wave mixing (ND-FWM), by detecting a coherent signal generated by the sample at frequencies close to the "edge" of the spectrally "truncated" incident femtosecond pulses. ND-FWM microscopy allows label-free biomedical imaging with high sensitivity and spatial resolution. In particular, by achieving a nearly perfect phase matching condition, ND-FWM generates almost the highest nonlinear coherent signal in a bulk medium and provides a contrast mechanism different from other nonlinear imaging techniques. More importantly, we developed an electronic resonant version of ND-FWM for absorbing but nonfluorescent molecules. Ultrasensitive chromophore detection (approximately 50 molecules) and hemoglobin imaging are demonstrated, by harnessing a fully (triply) resonant enhancement of the nonlinear polarization and using optical heterodyne detection.

Properties of coherence-gated wavefront sensing.

Coherence-gated wavefront sensing (CGWS) allows the determination of wavefront aberrations in strongly scattering tissue and their correction by adaptive optics. This allows, e.g., the restoration of the diffraction limit in light microscopy. Here, we develop a model, based on ray tracing of ballistic light scattered from a set of discrete scatterers, to characterize CGWS performance as it depends on coherence length, scatterer density, coherence-gate position, and polarization. The model is evaluated by using Monte Carlo simulation and verified against experimental measurements. We show, in particular, that all aberrations needed for adaptive wavefront restoration are correctly sensed if circularly polarized light is used.

Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing.

The image quality of a two-photon microscope is often degraded by wavefront aberrations induced by the specimen. We demonstrate here that resolution and signal size in two-photon microcopy can be substantially improved, even in living biological specimens, by adaptive wavefront correction based on sensing the wavefront of coherence-gated backscattered light (coherence-gated wavefront sensing, CGWS) and wavefront control by a deformable mirror. A nearly diffraction-limited focus can be restored even for strong aberrations. CGWS-based wavefront correction should be applicable to samples with a wide range of scattering properties and it should be possible to perform real-time pixel-by-pixel correction even at fast scan speeds.