ROS signaling: the new wave?

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues

The interaction of cells and bacteria with surfaces structured at the nanometre scale.

The ability to dynamically image features deep within living organisms, permitting real-time analysis of cellular structure and function, is important for biological science. This Review article discusses multiphoton microscopy capable of such analysis, along with technologies that are pushing the limits of phenomena that can be quantitatively imaged.

/pdf/advances-in-multiphoton-microscopy-technology-42gqk18ef1.pdf

Advances in multiphoton microscopy technology

Spine Dynamics: Are They All the Same?

http://www1.udel.edu/chem/bahnson/chem645/Martini.pdf

Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time

Detailed and quantitative information about structure-function relation, concentrations and interaction kinetics of biological molecules and subcellular components is a key prerequisite to understand and model cellular organisation and temporal dynamics. In systems nanobi-ology, cellular processes are quantitatively investigated at the sensitivity level of single molecules and cells. This approach provides direct access to biomolecular information without being statistically ensemble-averaged, their associated distribution functions, and possible subpopulations. Moreover at the single cell level, the interplay of regulated genomic information and proteomic variabilities can be investigated and attributed to functional peculiarities. These requirements necessitate the development of novel and ultrasensitive methods and instruments for single molecule detection, microscopy and spectroscopy for analysis without the need of amplification and preconcentration. In this chapter, we present three methodological applications that demonstrate how quantitative informations can be accessed that are representative for cellular processes or single cell analysis like gene expression regulation, intracellular protein translocation dynamics, and single cell protein fingerprinting. First, the interaction kinetics of transcriptionally regulated DNA-protein interaction can be quantitatively investigated with single molecule force spectroscopy allowing a molecular affinity ranking. Second, intracellular protein dynamics for a transcription regulator migrating form the nucleus to the cytoplasm can be quantitatively monitored by photoactivable GFP and two-photon laser scanning microscopy. And third, a microfluidic-based method for label-free single cell proteomics and fingerprinting and first label-free single cell electropherograms are presented which include the manipulation and steering of single cells in a microfluidic device.

Systems nanobiology: from quantitative single molecule biophysics to microfluidic-based single cell analysis.

Analytics of single biological cells allows quantitative investigation from a structural, functional and dynamical point of view and opens novel possibilities to an unamplified subcellular analysis. In this article, we report on three different experimental methods and their applications to single cellular systems with a subcellular sensitivity down to the single molecule level. First, the subcellular surface structure of living bacteria (Corynebacterium glutamicum) was investigated with atomic force microscopy (AFM) at the resolution of individual surface layer (S-layer) proteins; discrimination of bacterial strains that lack the expression of hexagonally packed surface layer proteins was possible. Second, quantitative measurement of individual recognition events of membrane-bound receptors on living B-cells was achieved in single cell manipulation and probing experiments with optical tweezers (OT) force spectroscopy. And third, intracellular dynamics of translocating photoactivatable GFP in plant protoplasts (Nicotiana tabacum BY-2) was quantitatively monitored by two-photon laser scanning microscopy (2PLSM).

/pdf/analysis-of-subcellular-surface-structure-function-and-4jfpqlg78i.pdf

Analysis of subcellular surface structure, function and dynamics.

We have developed a new descanned parallel 32-fold pinhole and photomultiplier detection array for multifocal multipho- ton microscopy that effectively reduces the blurring effect originating from scattered fluorescence photons in strongly scattering biological media. With this method, we achieve a fourfold improvement in pho- ton statistics for detecting ballistic photons and an increase in spatial resolution by 21% in the lateral and 35% in the axial direction com- pared to single-beam non-descanned multiphoton microscopy. The new detection concept has been applied to plant leaves and pollen grains to verify the improvements in imaging quality. © 2007 Society of

/pdf/scattering-suppression-and-confocal-detection-in-multifocal-46sqpq4seu.pdf

Scattering suppression and confocal detection in multifocal multiphoton microscopy.

Hyalin cartilage was investigated with 2-photon laser scanning microscope . NIR-2-photon-excitation and a specialized long distance objective lens allowed for autofluorescence and SHG measurements of the extracellular matrix up to 400 µm inside the sample.

/pdf/2-photon-laser-scanning-microscopy-on-native-human-cartilage-3vb43pnu41.pdf

Joerg Martini

Papers

Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time

Systems nanobiology: from quantitative single molecule biophysics to microfluidic-based single cell analysis.

Analysis of subcellular surface structure, function and dynamics.

Scattering suppression and confocal detection in multifocal multiphoton microscopy.

2-photon laser scanning microscopy on native human cartilage