Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis

Confocal fluorescence correlation spectroscopy as a time-averaging fluctuation analysis combining maximum sensitivity with high statistical confidence has proved to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solutions, and in living cells. To probe the interaction of different molecular species for a detailed understanding of biologically relevant mechanisms, crosscorrelation studies on dual or multiple fluorophore assays with spectrally distinct excitation and emission are particularly promising. Despite the considerable improvement of detection specificity provided by fluorescence crosscorrelation analysis, few applications have so far been reported, presumably because of the practical challenges of properly aligning and controlling the stability of the experimental setup. In this work, we demonstrate that two-photon excitation combined with dual-color fluorescence correlation spectroscopy can be the key to simplifying simultaneous investigations of multiple fluorescent species significantly on a single-molecule scale. Two-photon excitation allows accession of common fluorophores of largely distinct emission by the same excitation wavelength, because differences in selection rules and vibronic coupling can induce considerable shifts between the one-photon and two-photon excitation spectra. The concept of dual-color two-photon fluorescence crosscorrelation analysis is introduced and experimentally demonstrated with an established assay probing the selective cleavage of dual-labeled DNA substrates by restriction endonuclease EcoRI.

Detection and selective dissociation of intact ribosomes in a mass spectrometer

Intact Escherichia coli ribosomes have been projected into the gas phase of a mass spectrometer by means of nanoflow electrospray techniques. Species with mass/charge ratios in excess of 20,000 were detected at the level of individual ions by using time-of-flight analysis. Once in the gas phase the stability of intact ribosomes was investigated and found to increase as a result of cross-linking ribosomal proteins to the rRNA. By lowering the Mg2+ concentration in solutions containing ribosomes the particles were found to dissociate into 30S and 50S subunits. The resolution of the charge states in the spectrum of the 30S subunit enabled its mass to be determined as 852,187 ± 3,918 Da, a value within 0.6% of that calculated from the individual proteins and the 16S RNA. Further dissociation into smaller macromolecular complexes and then individual proteins could be induced by subjecting the particles to increasingly energetic gas phase collisions. The ease with which proteins dissociated from the intact species was found to be related to their known interactions in the ribosome particle. The results show that emerging mass spectrometric techniques can be used to characterize a fully functional biological assembly as well as its isolated components.

Invariant size–frequency distributions along a latitudinal gradient in marine bivalves

In the most extensive analysis of body size in marine invertebrates to date, we show that the size–frequency distributions of northeastern Pacific bivalves at the provincial level are surprisingly invariant in modal and median size as well as size range, despite a 4-fold change in species richness from the tropics to the Arctic. The modal sizes and shapes of these size–frequency distributions are consistent with the predictions of an energetic model previously applied to terrestrial mammals and birds. However, analyses of the Miocene–Recent history of body sizes within 82 molluscan genera show little support for the expectation that the modal size is an evolutionary attractor over geological time.

“Coarse” stability and bifurcation analysis using time-steppers: A reaction-diffusion example

Evolutionary, pattern forming partial differential equations (PDEs) are often derived as limiting descriptions of microscopic, kinetic theory-based models of molecular processes (e.g., reaction and diffusion). The PDE dynamic behavior can be probed through direct simulation (time integration) or, more systematically, through stability/bifurcation calculations; time-stepper-based approaches, like the Recursive Projection Method [Shroff, G. M. & Keller, H. B. (1993) SIAM J. Numer. Anal. 30, 1099–1120] provide an attractive framework for the latter. We demonstrate an adaptation of this approach that allows for a direct, effective (“coarse”) bifurcation analysis of microscopic, kinetic-based models; this is illustrated through a comparative study of the FitzHugh-Nagumo PDE and of a corresponding Lattice–Boltzmann model.

Evolution of chlorophyll and bacteriochlorophyll: the problem of invariant sites in sequence analysis.

Competing hypotheses seek to explain the evolution of oxygenic and anoxygenic processes of photosynthesis. Since chlorophyll is less reduced and precedes bacteriochlorophyll on the modern biosynthetic pathway, it has been proposed that chlorophyll preceded bacteriochlorophyll in its evolution. However, recent analyses of nucleotide sequences that encode chlorophyll and bacteriochlorophyll biosynthetic enzymes appear to provide support for an alternative hypothesis. This is that the evolution of bacteriochlorophyll occurred earlier than the evolution of chlorophyll. Here we demonstrate that the presence of invariant sites in sequence datasets leads to inconsistency in tree building (including maximum-likelihood methods). Homologous sequences with different biological functions often share invariant sites at the same nucleotide positions. However, different constraints can also result in additional invariant sites unique to the genes, which have specific and different biological functions. Consequently, the distribution of these sites can be uneven between the different types of homologous genes. The presence of invariant sites, shared by related biosynthetic genes as well as those unique to only some of these genes, has misled the recent evolutionary analysis of oxygenic and anoxygenic photosynthetic pigments. We evaluate an alternative scheme for the evolution of chlorophyll and bacteriochlorophyll.