Contrast mechanisms in pump-probe microscopy of melanin.

Pump-probe microscopy of melanin in tumors has been proposed to improve diagnosis of malignant melanoma, based on the hypothesis that aggressive cancers disaggregate melanin structure. However, measured signals of melanin are complex superpositions of multiple nonlinear processes, which makes interpretation challenging. Polarization control during measurement and data fitting are used to decompose signals of melanin into their underlying molecular mechanisms. We then identify the molecular mechanisms that are most susceptible to melanin disaggregation and derive false-coloring schemes to highlight these processes in biological tissue. We demonstrate that false-colored images of a small set of melanoma tumors correlate with clinical concern. More generally, our systematic approach of decomposing pump-probe signals can be applied to a multitude of different samples.

Beyond intensity modulation: new approaches to pump-probe microscopy.

Pump-probe microscopy is an emerging nonlinear imaging technique based on high repetition rate lasers and fast intensity modulation. Here, we present new methods for pump-probe microscopy that keep the beam intensity constant and instead modulate the inter-pulse time delay or the relative polarization. These techniques can improve image quality for samples that have poor heat dissipation or long-lived radiative states and can selectively address nonlinear interactions in the sample. We experimentally demonstrate this approach and point out the advantages over conventional intensity modulation.

Structural, Optical, and Electronic Properties of Two Quaternary Chalcogenide Semiconductors: Ag2SrSiS4 and Ag2SrGeS4.

Quaternary chalcogenide materials have long been a source of semiconductors for optoelectronic applications. Recent studies on I2-II-IV-X4 (I = Ag, Cu, Li; II = Ba, Sr, Eu, Pb; IV = Si, Ge, Sn; X = S, Se) materials have shown particular versatility and promise among these compounds. These semiconductors take advantage of a diverse bonding scheme and chemical differences among cations to target a degree of antisite defect resistance. Within this set of compounds, the materials containing both Ag and Sr have not been experimentally studied and leave a gap in the full understanding of the family. Here, we have synthesized powders and single crystals of two Ag- and Sr-containing compounds, Ag2SrSiS4 and Ag2SrGeS4, each found to form in the tetragonal I4̅2m structure of Ag2BaGeS4. During the synthesis targeting the title compounds, two additional materials, Ag2Sr3Si2S8 and Ag2Sr3Ge2S8, have also been identified. These cubic compounds represent impurity phases during the synthesis of Ag2SrSiS4 and Ag2SrGeS4. We show through hybrid density functional theory calculations that Ag2SrSiS4 and Ag2SrGeS4 have highly dispersive band-edge states and indirect band gaps, experimentally measured as 2.08(1) and 1.73(2) eV, respectively. Second-harmonic generation measurements on Ag2SrSiS4 and Ag2SrGeS4 powders show frequency-doubling capabilities in the near-infrared range.

Disentangling the effects of geographic peripherality and habitat suitability on neutral and adaptive genetic variation in Swiss stone pine.

It is generally accepted that the spatial distribution of neutral genetic diversity within a species' native range mostly depends on effective population size, demographic history, and geographic position. However, it is unclear how genetic diversity at adaptive loci correlates with geographic peripherality or with habitat suitability within the ecological niche. Using exome-wide genomic data and distribution maps of the Alpine range, we first tested whether geographic peripherality correlates with four measures of population genetic diversity at > 17,000 SNP loci in 24 Alpine populations (480 individuals) of Swiss stone pine (Pinus cembra) from Switzerland. To distinguish between neutral and adaptive SNP sets, we used four approaches (two gene diversity estimates, FST outlier test, and environmental association analysis) that search for signatures of selection. Second, we established ecological niche models for P. cembra in the study range and investigated how habitat suitability correlates with genetic diversity at neutral and adaptive loci. All estimates of neutral genetic diversity decreased with geographic peripherality, but were uncorrelated with habitat suitability. However, heterozygosity (He ) at adaptive loci based on Tajima's D declined significantly with increasingly suitable conditions. No other diversity estimates at adaptive loci were correlated with habitat suitability. Our findings suggest that populations at the edge of a species' geographic distribution harbour limited neutral genetic diversity due to demographic properties. Moreover, we argue that populations from suitable habitats went through strong selection processes, are thus well adapted to local conditions, and therefore exhibit reduced genetic diversity at adaptive loci compared to populations at niche margins.

Genomic signatures of convergent adaptation to Alpine environments in three Brassicaceae species.

It has long been discussed to what extent related species develop similar genetic mechanisms to adapt to similar environments. Most studies documenting such convergence have either used different lineages within species or surveyed only a limited portion of the genome. Here, we investigated whether similar or different sets of orthologous genes were involved in genetic adaptation of natural populations of three related plant species to similar environmental gradients in the Alps. We used whole-genome pooled population sequencing to study genome-wide SNP variation in 18 natural populations of three Brassicaceae (Arabis alpina, Arabidopsis halleri, and Cardamine resedifolia) from the Swiss Alps. We first de novo assembled draft reference genomes for all three species. We then ran population and landscape genomic analyses with ~3 million SNPs per species to look for shared genomic signatures of selection and adaptation in response to similar environmental gradients acting on these species. Genes with a signature of convergent adaptation were found at significantly higher numbers than expected by chance. The most closely related species pair showed the highest relative over-representation of shared adaptation signatures. Moreover, the identified genes of convergent adaptation were enriched for nonsynonymous mutations, suggesting functional relevance of these genes, even though many of the identified candidate genes have hitherto unknown or poorly described functions based on comparison with Arabidopsis thaliana. We conclude that adaptation to heterogeneous Alpine environments in related species is partly driven by convergent evolution, but that most of the genomic signatures of adaptation remain species-specific.

Crossed-beam pump-probe microscopy.

We present a new imaging method for pump-probe microscopy that explores non-collinear excitation. This method (crossed-beam pump-probe microscopy, or CBPM) can significantly improve the axial resolution when imaging through low-NA lenses, providing an alternative way for depth-resolved, large field-of-view imaging. We performed a proof-of-concept demonstration, characterized CBPM's resolution using different imaging lenses, and measured an enhanced axial resolution for certain types of low-NA lenses.

Genomics and the origin of species.

Speciation is a fundamental evolutionary process, the knowledge of which is crucial for understanding the origins of biodiversity. Genomic approaches are an increasingly important aspect of this research field. We review current understanding of genome-wide effects of accumulating reproductive isolation and of genomic properties that influence the process of speciation. Building on this work, we identify emergent trends and gaps in our understanding, propose new approaches to more fully integrate genomics into speciation research, translate speciation theory into hypotheses that are testable using genomic tools and provide an integrative definition of the field of speciation genomics.

Visualizing the impact of chloride addition on the microscopic carrier dynamics of MAPbI3 thin films using femtosecond transient absorption microscopy.

The spatial heterogeneity of carrier dynamics in mixed halide perovskite CH3NH3PbI3-xClx thin films with a range of different chloride additions is mapped using femtosecond transient absorption microscopy (TAM). The comparison of TAM images of fibrous and granular polycrystalline CH3NH3PbI3-xClx films indicates that the impact of chloride addition on the local heterogeneity of carrier dynamics is highly dependent on the film preparation method and the resulting morphology. In addition to signals of pristine CH3NH3PbI3, CH3NH3PbI3-xClx films with a fibrous structure show long-lived excited state absorption (ESA) signals in localized, microscopic regions. The ESA signal exhibits transient absorption with a rise time of about 5 ps after the excitation pulse, indicating that these distinct micrograins have preferential carrier trapping properties. The chemical composition of these micrograins does not differ detectably from their surroundings. In contrast, in CH3NH3PbI3-xClx films with a granular structure, Cl addition does not seem to affect the charge carrier dynamics. These results provide insight into the localized effects of halide mixing and on the resulting photophysical properties of mixed halide perovskite materials on the micrometer length scale.

Divergence in Glucosinolate Profiles between High- and Low-Elevation Populations of Arabidopsis halleri Correspond to Variation in Field Herbivory and Herbivore Behavioral Preferences.

Variation in local herbivore pressure along elevation gradients is predicted to drive variation in plant defense traits. Yet, the extent of intraspecific variation in defense investment along elevation gradients, and its effects on both herbivore preference and performance, remain relatively unexplored. Using populations of Arabidopsis halleri (Brassicaceae) occurring at different elevations in the Alps, we tested for associations between elevation, herbivore damage in the field, and constitutive chemical defense traits (glucosinolates) assayed under common-garden conditions. Additionally, we examined the feeding preferences and performance of a specialist herbivore, the butterfly Pieris brassicae, on plants from different elevations in the Alps. Although we found no effect of elevation on the overall levels of constitutive glucosinolates in leaves, relative amounts of indole glucosinolates increased significantly with elevation and were negatively correlated with herbivore damage in the field. In oviposition preference assays, P. brassicae females laid fewer eggs on plants from high-elevation populations, although larval performance was similar on populations from different elevations. Taken together, these results support the prediction that species distributed along elevation gradients exhibit genetic variation in chemical defenses, which can have consequences for interactions with herbivores in the field.

Spectroscopic Differentiation and Microscopic Imaging of Red Organic Pigments Using Optical Pump-Probe Contrast.

Analysis of red organic pigments in artworks (and in forensics applications) is challenging, because conventional nondestructive mapping techniques provide little contrast, and most chemical analyses with high specificity require sample removal. Here we demonstrate a new optical approach, pump-probe microscopy, for the analysis of red organic pigments. We investigate Carmine naccarat, Lac dye, purpurin, alizarin, madder lake, and eosin Y and show that their intrinsic photophysical properties produce distinctive pump-probe spectra. We utilize this contrast for high-resolution, three-dimensional imaging without the need for physical sample removal. Lastly, we highlight the potential of pump-probe microscopy as an analytical tool for forensics of other types of organic colorants by investigating a series of automotive paints.

The genomic basis of adaptation to calcareous and siliceous soils in Arabidopsis lyrata.

Edaphic conditions are important determinants of plant fitness. While much has been learnt in recent years about plant adaptation to heavy metal contaminated soils, the genomic basis underlying adaptation to calcareous and siliceous substrates remains largely unknown. We performed a reciprocal germination experiment and whole-genome resequencing in natural calcareous and siliceous populations of diploid Arabidopsis lyrata to test for edaphic adaptation and detect signatures of selection at loci associated with soil-mediated divergence. In parallel, genome scans on respective diploid ecotypes from the Arabidopsis arenosa species complex were undertaken, to search for shared patterns of adaptive genetic divergence. Soil ecotypes of A. lyrata display significant genotype-by-treatment responses for seed germination. Sequence (SNPs) and copy-number variants (CNVs) point towards loci involved in ion transport as the main targets of adaptive genetic divergence. Two genes exhibiting high differentiation among soil types in A. lyrata further share trans-specific single nucleotide polymorphisms with A. arenosa. This work applies experimental and genomic approaches to study edaphic adaptation in A. lyrata and suggests that physiological response to elemental toxicity and deficiency underlies the evolution of calcareous and siliceous ecotypes. The discovery of shared adaptive variation between sister species indicates that ancient polymorphisms contribute to adaptive evolution.

Estimating genomic diversity and population differentiation - an empirical comparison of microsatellite and SNP variation in Arabidopsis halleri.

BACKGROUND: Microsatellite markers are widely used for estimating genetic diversity within and differentiation among populations. However, it has rarely been tested whether such estimates are useful proxies for genome-wide patterns of variation and differentiation. Here, we compared microsatellite variation with genome-wide single nucleotide polymorphisms (SNPs) to assess and quantify potential marker-specific biases and derive recommendations for future studies. Overall, we genotyped 180 Arabidopsis halleri individuals from nine populations using 20 microsatellite markers. Twelve of these markers were originally developed for Arabidopsis thaliana (cross-species markers) and eight for A. halleri (species-specific markers). We further characterized 2 million SNPs across the genome with a pooled whole-genome re-sequencing approach (Pool-Seq). RESULTS: Our analyses revealed that estimates of genetic diversity and differentiation derived from cross-species and species-specific microsatellites differed substantially and that expected microsatellite heterozygosity (SSR-H e) was not significantly correlated with genome-wide SNP diversity estimates (SNP-H e and θ Watterson) in A. halleri. Instead, microsatellite allelic richness (A r) was a better proxy for genome-wide SNP diversity. Estimates of genetic differentiation among populations (F ST) based on both marker types were correlated, but microsatellite-based estimates were significantly larger than those from SNPs. Possible causes include the limited number of microsatellite markers used, marker ascertainment bias, as well as the high variance in microsatellite-derived estimates. In contrast, genome-wide SNP data provided unbiased estimates of genetic diversity independent of whether genome- or only exome-wide SNPs were used. Further, we inferred that a few thousand random SNPs are sufficient to reliably estimate genome-wide diversity and to distinguish among populations differing in genetic variation. CONCLUSIONS: We recommend that future analyses of genetic diversity within and differentiation among populations use randomly selected high-throughput sequencing-based SNP data to draw conclusions on genome-wide diversity patterns. In species comparable to A. halleri, a few thousand SNPs are sufficient to achieve this goal.

Femtosecond pump-probe microscopy generates virtual cross-sections in historic artwork.

The layering structure of a painting contains a wealth of information about the artist's choice of materials and working methods, but currently, no 3D noninvasive method exists to replace the taking of small paint samples in the study of the stratigraphy. Here, we adapt femtosecond pump-probe imaging, previously shown in tissue, to the case of the color palette in paintings, where chromophores have much greater variety. We show that combining the contrasts of multispectral and multidelay pump-probe spectroscopy permits nondestructive 3D imaging of paintings with molecular and structural contrast, even for pigments with linear absorption spectra that are broad and relatively featureless. We show virtual cross-sectioning capabilities in mockup paintings, with pigment separation and nondestructive imaging on an intact 14th century painting (The crucifixion by Puccio Capanna). Our approach makes it possible to extract microscopic information for a broad range of applications to cultural heritage.

Label-Free Imaging of Female Genital Tract Melanocytic Lesions With Pump-Probe Microscopy: A Promising Diagnostic Tool.

OBJECTIVES: Melanomas of the female genital tract present a unique clinical challenge. Not only are these lesions in an anatomically sensitive area, but also they tend to be multifocal and have high recurrence rates. Furthermore, several benign melanocytic proliferations resemble early-stage melanoma clinically and/or histopathologically. Thus, there is a significant need for additional tools that can help correctly diagnose and stage these lesions. Here, we quantitatively and nondestructively analyze the chemical composition of melanin in excised pigmented lesions of the female genital tract using pump-probe microscopy, a high-resolution optical imaging technique that is sensitive to many biochemical properties of melanin. MATERIALS AND METHODS: Thirty-one thin (~5 µm) tissue sections previously excised from female genital tract melanocytic lesions were imaged with pump-probe microscopy and analyzed. RESULTS: We find significant quantitative differences in melanin type and structure between melanoma and nonmalignant melanocytic proliferations. Our analysis also suggests a link between the molecular signatures of melanins and lesion-specific genetic mutations. Finally, significant differences are found between metastatic and nonmetastatic melanomas. The limitations of this work include the fact that molecular information is restricted to melanin pigment and the sample size is relatively small. CONCLUSIONS: Pump-probe microscopy provides unique information regarding the biochemical composition of genital tract melanocytic lesions, which can be used to improve the diagnosis and staging of vulvar melanomas.

Dispersion-based stimulated Raman scattering spectroscopy, holography, and optical coherence tomography.

Stimulated Raman scattering (SRS) enables fast, high resolution imaging of chemical constituents important to biological structures and functional processes, both in a label-free manner and using exogenous biomarkers. While this technology has shown remarkable potential, it is currently limited to point scanning and can only probe a few Raman bands at a time (most often, only one). In this work we take a fundamentally different approach to detecting the small nonlinear signals based on dispersion effects that accompany the loss/gain processes in SRS. In this proof of concept, we demonstrate that the dispersive measurements are more robust to noise compared to amplitude-based measurements, which then permit spectral or spatial multiplexing (potentially both, simultaneously). Finally, we illustrate how this method may enable different strategies for biochemical imaging using phase microscopy and optical coherence tomography.

Femtosecond pulse shaping enables detection of optical Kerr-effect (OKE) dynamics for molecular imaging.

We apply femtosecond pulse shaping to generate optical pulse trains that directly access a material's nonlinear refractive index (n2) and can thus determine time-resolved optical Kerr-effect (OKE) dynamics. Two types of static pulse trains are discussed: The first uses two identical fields delayed in time, plus a pump field at a different wavelength. Time-resolved OKE dynamics are retrieved by monitoring the phase of the interference pattern produced by the two identical fields in the Fourier-domain (FD) as a function of pump-probe-time-delay (where the probe is one of the two identical fields). The second pulse train uses three fields with equal time delays, but with the center field phase shifted by π/2. In this pulse scheme, changes on a sample's nonlinear refractive index produce a new frequency in the FD signal, which in turn yields background-free intensity changes in the conjugate (time) domain and provides superior signal-to-noise ratios. The demonstrated sensitivity improvements enable, for the first time to our knowledge, molecular imaging based on OKE dynamics.

Femtosecond pulse train shaping improves two-photon excited fluorescence measurements.

Measurements of two-photon absorption (TPA) cross sections are greatly confounded by even very weak linear absorption, for example from hot bands. In this case, the experimental power dependence of fluorescence from amplified and mode-locked laser systems can differ drastically, even if the peak intensity is adjusted to be the same in both cases. A simple pulse train shaping method suppresses linear contributions and extracts the nonlinear absorption cross section, demonstrated here for a meso-to-meso ethyne-bridged bis[(porphinato)zinc(II)] fluorophore (DD) at 800 nm. This approach permits reliable TPA cross-section measurement, even with standard modelocked lasers under conditions identical to that used for multiphoton microscopy.

Cost-effective, open-source light shutters with Arduino control.

In optical experiments, shutters are devices that open or close a path of light. They are often used to limit the duration of light exposure onto a target or onto a detector to reduce possible light-induced damage. Many commercial shutters are available for different applications - some provide very fast opening and closing times, some can handle large optical powers, and others allow for fail-safe operation. Many of these devices are costly and offer limited control options. Here we provide an open-source design for a low-cost, general purpose shutter system based on ubiquitous actuators (servo motors or solenoids) that are connected to an Arduino-based controller. Several shutters can be controlled by one controller, further reducing system cost. The state of the shutters can be controlled via a display built into the controller, by serial commands via USB, or by electrical control lines. The use of a microcontroller makes the shutter controller adaptable - only control options that are used need to be included, and the design accommodates a selection of display and actuator options. We provide designs for all required components, including 3D print files for the actuator holders and cases, the Arduino code, libraries for serial communication (C and python), and example graphical user interfaces for testing.

Minimally destructive hDNA extraction method for retrospective genetics of pinned historical Lepidoptera specimens.

The millions of specimens stored in entomological collections provide a unique opportunity to study historical insect diversity. Current technologies allow to sequence entire genomes of historical specimens and estimate past genetic diversity of present-day endangered species, advancing our understanding of anthropogenic impact on genetic diversity and enabling the implementation of conservation strategies. A limiting challenge is the extraction of historical DNA (hDNA) of adequate quality for sequencing platforms. We tested four hDNA extraction protocols on five body parts of pinned false heath fritillary butterflies, Melitaea diamina, aiming to minimise specimen damage, preserve their scientific value to the collections, and maximise DNA quality and yield for whole-genome re-sequencing. We developed a very effective approach that successfully recovers hDNA appropriate for short-read sequencing from a single leg of pinned specimens using silica-based DNA extraction columns and an extraction buffer that includes SDS, Tris, Proteinase K, EDTA, NaCl, PTB, and DTT. We observed substantial variation in the ratio of nuclear to mitochondrial DNA in extractions from different tissues, indicating that optimal tissue choice depends on project aims and anticipated downstream analyses. We found that sufficient DNA for whole genome re-sequencing can reliably be extracted from a single leg, opening the possibility to monitor changes in genetic diversity maintaining the scientific value of specimens while supporting current and future conservation strategies.

Population genomic footprints of selection and associations with climate in natural populations of Arabidopsis halleri from the Alps.

Natural genetic variation is essential for the adaptation of organisms to their local environment and to changing environmental conditions. Here, we examine genomewide patterns of nucleotide variation in natural populations of the outcrossing herb Arabidopsis halleri and associations with climatic variation among populations in the Alps. Using a pooled population sequencing (Pool-Seq) approach, we discovered more than two million SNPs in five natural populations and identified highly differentiated genomic regions and SNPs using FST -based analyses. We tested only the most strongly differentiated SNPs for associations with a nonredundant set of environmental factors using partial Mantel tests to identify topo-climatic factors that may underlie the observed footprints of selection. Possible functions of genes showing signatures of selection were identified by Gene Ontology analysis. We found 175 genes to be highly associated with one or more of the five tested topo-climatic factors. Of these, 23.4% had unknown functions. Genetic variation in four candidate genes was strongly associated with site water balance and solar radiation, and functional annotations were congruent with these environmental factors. Our results provide a genomewide perspective on the distribution of adaptive genetic variation in natural plant populations from a highly diverse and heterogeneous alpine environment.