Photosynthesis in a different light: spectro-microscopy for in vivo characterization of chloroplasts.

During photosynthesis, energy conversion at the two photosystems is controlled by highly complex and dynamic adaptation processes triggered by external factors such as light quality, intensity, and duration, or internal cues such as carbon availability. These dynamics have remained largely concealed so far, because current analytical techniques are based on the investigation of isolated chloroplasts lacking full adaptation ability and are performed at non-physiologically low temperatures. Here, we use non-invasive in planta spectro-microscopic approaches to investigate living chloroplasts in their native environment at ambient temperatures.

Room temperature excitation spectroscopy of single quantum dots.

We report a single molecule detection scheme to investigate excitation spectra of single emitters at room temperature. We demonstrate the potential of single emitter photoluminescence excitation spectroscopy by recording excitation spectra of single CdSe nanocrystals over a wide spectral range of 100 nm. The spectra exhibit emission intermittency, characteristic of single emitters.

Optical imaging of excited-state tautomerization in single molecules.

Tautomerism process of single fluorescent molecules was studied by means of confocal microscopy in combination with azimuthally or radially polarized laser beams. During a tautomerism process the transition dipole moment (TDM) of a molecule changes its orientation which can be visualized by the fluorescence excitation image of the molecule. We present experimental and theoretical studies of two porphyrazine-type molecules and one type of porphyrin molecule: a symmetrically substituted metal-free phthalocyanine and porphyrin, and nonsymmetrically substituted porphyrazine.

Fluorescence intensity decay shape analysis microscopy (FIDSAM) for quantitative and sensitive live-cell imaging: a novel technique for fluorescence microscopy of endogenously expressed fusion-proteins.

Fluorescent studies of living plant cells such as confocal microscopy and fluorescence lifetime imaging often suffer from a strong autofluorescent background contribution that significantly reduces the dynamic image contrast and the quantitative access to sub-cellular processes at high spatial resolution. Here, we present a novel technique--fluorescence intensity decay shape analysis microscopy (FIDSAM)--to enhance the dynamic contrast of a fluorescence image of at least one order of magnitude. The method is based on the analysis of the shape of the fluorescence intensity decay (fluorescence lifetime curve) and benefits from the fact that the decay patterns of typical fluorescence label dyes strongly differ from emission decay curves of autofluorescent sample areas.

Imaging nanometre-sized hot spots on smooth au films with high-resolution tip-enhanced luminescence and Raman near-field optical microscopy.

A novel near-field optical microscope based on a parabolic mirror is used for recording high-resolution tip-enhanced photoluminescence (PL) and Raman images with unprecedented sensitivity and contrast. The measurements reveal small islands on the Au surface with dimensions of only a few nanometres with locally enhanced Au PL. These islands appear as nanometre-sized hot spots in tip-enhanced Raman microscopy when benzotriazole molecules adsorbed on the Au surface serve as local sensors for the optical field.