Recent advances in nonlinear microscopy: Deep insights and polarized revelations.

Nonlinear microscopy is a technique that utilizes nonlinear interactions between light and matter to image fluorescence and scattering phenomena in biological tissues. Very high peak intensities from focused short pulsed lasers are required for nonlinear excitation due to the extremely low probability of the simultaneous arrival of multiple photons of lower energy to excite fluorophores or interact with selective structures for harmonic generation. Combined with reduced scattering from the utilization of longer wavelengths, the inherent spatial confinement associated with achieving simultaneous arrival of photons within the focal volume enables deep imaging with low out-of-focus background for nonlinear imaging. This review provides an introduction to the different contrast mechanisms available with nonlinear imaging and instrumentation commonly used in nonlinear microscopy. Furthermore, we discuss some recent advances in nonlinear microscopy to extend the imaging penetration depth, conduct histopathological investigations on fresh tissues and examine the molecular order and orientation of molecules using polarization nonlinear microscopy.

Reproductive physiology of the female Magellanic penguin (Spheniscus magellanicus): Insights from the study of a zoological colony.

Eight captive female Magellanic penguins (Spheniscus magellanicus) were monitored over a 10week period, commencing at 5weeks prior to egg lay (EL), to increase our understanding of the species' reproductive biology. Females in cordoned nest sites underwent cloacal artificial insemination (AI) every 4-7days with different semen donors for each insemination. The EL interval was 97.9±3.6h (range: 84-108h) and paternity analyses revealed that conceptive inseminations occurred from 11.5 to 4.5days before oviposition. A biphasic pattern of estradiol, testosterone, progesterone and the biochemical analytes triglyceride, iron, calcium and phosphorus occurred in relation to EL, with values increasing (P<0.05) to maximal concentrations during the three weeks preceding oviposition, then decreasing (P<0.05) rapidly after oviposition completion. In comparison with post-lay (baseline) values, concentrations of estradiol and testosterone relative to the first oviposition were elevated at Week-5, and those of triglyceride, a yolk formation index, as well as iron, calcium and phosphorus, became elevated at Week-4 (P<0.05). Collective data indicate an estimated total egg formation interval of 29days, with oviducal transit of the ovulated ovum occurring over the majority of the ∼4day EL interval. These findings indicate that egg formation is prolonged with folliculogenesis initiated at 5weeks or more prior to oviposition. Consequently, the period of folliculogenesis and egg formation is estimated to overlap with the final ∼3weeks that wild females spend at sea prior to returning to land for breeding.

In vivo imaging of specific drug-target binding at subcellular resolution.

The possibility of measuring binding of small-molecule drugs to desired targets in live cells could provide a better understanding of drug action. However, current approaches mostly yield static data, require lysis or rely on indirect assays and thus often provide an incomplete understanding of drug action. Here, we present a multiphoton fluorescence anisotropy microscopy live cell imaging technique to measure and map drug-target interaction in real time at subcellular resolution. This approach is generally applicable using any fluorescently labelled drug and enables high-resolution spatial and temporal mapping of bound and unbound drug distribution. To illustrate our approach we measure intracellular target engagement of the chemotherapeutic Olaparib, a poly(ADP-ribose) polymerase inhibitor, in live cells and within a tumour in vivo. These results are the first generalizable approach to directly measure drug-target binding in vivo and present a promising tool to enhance understanding of drug activity.

Nuclear DNA variation in spider monkeys (Ateles).

Phylogenetic relationships based on DNA sequence variation for the aldolase A intron V nuclear genomic region were evaluated and compared to phylogenies based on mitochondrial DNA sequence variation among spider monkeys (Ateles). Samples of Ateles ranging from Central America throughout the Amazon Basin were sequenced to determine phylogenetic relationships among geographically widely distributed populations. Analysis of nuclear DNA sequences using parsimony, maximum-likelihood, and genetic distance analyses produced similar phylogenies. Four previously proposed monophyletic species of spider monkeys were: (1) Ateles paniscus, composed of haplotypes from the northeastern Amazon Basin; (2) A. belzebuth, found in the western and southern Amazon Basin; (3) A. hybridus, located primarily along the Magdalena River valley of Colombia; and (4) A. geoffroyi, including all haplotypes found in the Choco region of South America and throughout Central America. The nuclear phylograms were analyzed based on associated bootstrap support and confidence probabilities. Support from the nuclear DNA genome was less robust than support from the mitochondrial DNA data, most likely due to a level of sequence variation, which was 90% less than that of the mitochondrial DNA genome. Nuclear DNA congruencies with mitochondrial DNA-based phylogenies, as supported by the incongruence length difference and winning sites tests, provide further support for the suggested revisions in Ateles taxonomy that are contradictory to long-held taxonomies based on pelage variation.

Kinetics of cytoplasmic aspartate aminotransferase from three genotypes of the deer mouse (Peromyscus maniculatus).

Three genotypic forms of the cytoplasmic enzyme, aspartate aminotransferase from the deer mouse (Peromyscus maniculatus) were each analyzed kinetically after partial purification. Kmapp and Vmax for aspartate decreased in value as temperature changed from 37.9 to 15 degrees C for all three forms. For the AA genotype, the binding enthalpies were highest at 37.9 degrees C and lowest at 25 degrees C, while for the A'A' form, they were lowest at 37.9 degrees and highest at 25 degrees C. Results for the heterozygote were generally intermediate reflecting the properties of both subunits. Inhibition by glyceraldehyde-3-phosphate was of a non-competitive type for all three genotypes.