Visual systems adapt to different light environments through several avenues including optical changes to the eye and neurological changes in how light signals are processed and interpreted. Spectral sensitivity can evolve via changes to visual pigments housed in the retinal photoreceptors through gene duplication and loss, differential and coexpression, and sequence evolution. Frogs provide an excellent, yet understudied, system for visual evolution research due to their diversity of ecologies (including biphasic aquatic-terrestrial life cycles) that we hypothesize imposed different selective pressures leading to adaptive evolution of the visual system, notably the opsins that encode the protein component of the visual pigments responsible for the first step in visual perception. Here, we analyze the diversity and evolution of visual opsin genes from 93 new eye transcriptomes plus published data for a combined dataset spanning 122 frog species and 34 families. We find that most species express the four visual opsins previously identified in frogs but show evidence for gene loss in two lineages. Further, we present evidence of positive selection in three opsins and shifts in selective pressures associated with differences in habitat and life history, but not activity pattern. We identify substantial novel variation in the visual opsins and, using microspectrophotometry, find highly variable spectral sensitivities, expanding known ranges for all frog visual pigments. Mutations at spectral-tuning sites only partially account for this variation, suggesting that frogs have used tuning pathways that are unique among vertebrates. These results support the hypothesis of adaptive evolution in photoreceptor physiology across the frog tree of life in response to varying environmental and ecological factors and further our growing understanding of vertebrate visual evolution.

Quantifying the growth of entomopathogenic fungi is crucial for understanding their virulence and pathogenic potential. Traditional methods for determining growth, such as biomass determination or colony growth area, are time-consuming and quantitatively and spatially limited in scope. In this study, we introduce a high-throughput method for rapidly measuring fungal growth using spectrophotometry in small-volume, liquid media cultures in 96-well microplates. Optical density (OD) changes were directly correlated with dry weight of samples for six isolates from three species of the genus Metarhizium to validate spectrophotometric growth measurements, and investigate species- and isolate-specific effects. We quantified fungal biomass from the microcultures by extracting, drying, and weighing mycelial mats. From the relationship established between OD and biomass, we generated standard curves for predicting biomass based on the OD values. The OD measurements clearly distinguished growth patterns among six isolates from three Metarhizium species. The logistic growth phase, as captured by the OD measurements, could be accurately assessed within a span of 80 h. Using isolates of M. acridum, M. brunneum, and M. guizhouense, this technique was demonstrated to be an effective, reproducible, and simple method for rapidly measuring filamentous fungal growth with high precision. This technique offers a valuable tool for studying the growth dynamics of entomopathogenic fungi and investigating the factors that influence their growth.

Dystrophic muscle is characterized by necrosis/regeneration cycles, inflammation, and fibro-adipogenic development. Conventional histological stainings provide essential topographical data of this remodeling but may be limited to discriminate closely related pathophysiological contexts. They fail to mention microarchitecture changes linked to the nature and spatial distribution of tissue compartment components. We investigated whether label-free tissue autofluorescence revealed by Synchrotron deep ultraviolet (DUV) radiation could serve as an additional tool for monitoring dystrophic muscle remodeling. Using widefield microscopy with specific emission fluorescence filters and microspectroscopy defined by high spectral resolution, we analyzed samples from healthy dogs and two groups of dystrophic dogs: naïve (severely affected) and MuStem cell-transplanted (clinically stabilized) animals. Multivariate statistical analysis and machine learning approaches demonstrated that autofluorescence emitted at 420-480 nm by the Biceps femoris muscle effectively discriminates between healthy, dystrophic, and transplanted dog samples. Microspectroscopy showed that dystrophic dog muscle displays higher and lower autofluorescence due to collagen cross-linking and NADH respectively than that of healthy and transplanted dogs, defining biomarkers to evaluate the impact of cell transplantation. Our findings demonstrate that DUV radiation is a sensitive, label-free method to assess the histopathological status of dystrophic muscle using small amounts of tissue, with potential applications in regenerative medicine.

Though microscopy is most often intended as a technique for providing qualitative assessment of cellular and subcellular properties, when coupled with other instruments such as wavelength selectors, lasers, photoelectric devices and computers, it can perform a wide variety of quantitative measurements, which are demanding in establishing relationships between the properties and structures of biological material in all their spatial and temporal complexities. These combinations of instruments are a powerful approach to improve non-destructive investigations of cellular and subcellular properties (both physical and chemical) at a macromolecular scale resolution. Since many subcellular compartments in living cells are characterized by structurally organized molecules, this review deals with three advanced microscopy techniques well-suited for these kind of investigations, i.e., microspectrophotometry (MSP), super-resolution localization microscopy (SRLM) and holotomographic microscopy (HTM). These techniques can achieve an insight view into the role intracellular molecular organizations such as photoreceptive and photosynthetic structures and lipid bodies play in many cellular processes as well as their biophysical properties. Microspectrophotometry uses a set-up based on the combination of a wide-field microscope and a polychromator, which allows the measurement of spectroscopic features such as absorption spectra. Super resolution localization microscopy combines dedicated optics and sophisticated software algorithms to overcome the diffraction limit of light and allow the visualization of subcellular structures and dynamics in greater detail with respect to conventional optical microscopy. Holotomographic microscopy combines holography and tomography techniques into a single microscopy set-up, and allows 3D reconstruction by means of the phase separation of biomolecule condensates. This review is organized in sections, which for each technique describe some general aspects, a peculiar theoretical aspect, a specific experimental configuration and examples of applications (fish and algae photoreceptors, single labeled proteins and endocellular aggregates of lipids).

Differences in morphology, ecology, and behavior through ontogeny can result in opposing selective pressures at different life stages. Most animals, however, transition through two or more distinct phenotypic phases, which is hypothesized to allow each life stage to adapt more freely to its ecological niche. How this applies to sensory systems, and in particular how sensory systems adapt across life stages at the molecular level, is not well understood. Here, we used whole-eye transcriptomes to investigate differences in gene expression between tadpole and juvenile southern leopard frogs (Lithobates sphenocephalus), which rely on vision in aquatic and terrestrial light environments, respectively. Because visual physiology changes with light levels, we also tested the effect of light and dark exposure.
We found 42% of genes were differentially expressed in the eyes of tadpoles versus juveniles and 5% for light/dark exposure. Analyses targeting a curated subset of visual genes revealed significant differential expression of genes that control aspects of visual function and development, including spectral sensitivity and lens composition. Finally, microspectrophotometry of photoreceptors confirmed shifts in spectral sensitivity predicted by the expression results, consistent with adaptation to distinct light environments.
Overall, we identified extensive expression-level differences in the eyes of tadpoles and juveniles related to observed morphological and physiological changes through metamorphosis and corresponding adaptive shifts to improve vision in the distinct aquatic and terrestrial light environments these frogs inhabit during their life cycle. More broadly, these results suggest that decoupling of gene expression can mediate the opposing selection pressures experienced by organisms with complex life cycles that inhabit different environmental conditions throughout ontogeny.

Malaria was regarded as the most devastating infectious disease of the 21st century until the COVID-19 pandemic. Asexual blood staged parasites (ABS) play a unique role in ensuring the parasite\'s survival and pathogenesis. Hitherto, there have been no spectroscopic reports discriminating the life cycle stages of the ABS parasite under physiological conditions. The identification and quantification of the stages in the erythrocytic life cycle is important in monitoring the progression and recovery from the disease. In this study, we explored visible microspectrophotometry coupled to machine learning to discriminate functional ABS parasites at the single cell level. Principal Component Analysis (PCA) showed an excellent discrimination between the different stages of the ABS parasites. Support Vector Machine Analysis provided a 100% prediction for both schizonts and trophozoites, while a 92% and 98% accuracy was achieved for predicting control and ring staged infected RBCs, respectively. This work shows proof of principle for discriminating the life cycle stages of parasites in functional erythrocytes using visible microscopy and thus eliminating the drying and fixative steps that are associated with other optical-based spectroscopic techniques.

Identifying the formation time of a document is a problem forensic document examiners often face. It is understood that intersections with different intersecting sequences made by the same laser printer and the same gel pen are distinct from each other under coaxial light, and the appearance of an oil film or bright metallic marks in the regions of interest can be used as the basis to identify that the ink is above the toner. However, the characteristics of intersections may be influenced by a number of factors. Studying the characteristics of the intersections formed by laser-printed graphics and handwriting strokes made with a gel pen in different intersecting sequences can help us determine the formation sequence of each part on a questioned document. In this manuscript, samples were made using three pen types and two laser printers. A Leica stereo microscope M205A was used to capture the apparent morphology of the intersections. Microspectrophotometry spectra of all intersections were collected in reflectance mode using a video spectral comparator 6000 (VSC6000), and the resultant data were measured and analyzed in Statistical Product and Service Solutions 26.0 (SPSS). The results showed that 92.5% absolute accuracy was achieved from blind tests, and the intersecting sequence can be determined using the optical data of the intersections with an accuracy of up to 97.5% through discriminant analysis under ideal conditions.

Signature examination is the most common examination performed by any document examiner. Determination of the authenticity of a handwritten signature on a questioned document is an important task for forensic document examiners in the forensic science field. As a result of continuous developments in technology, a signature stamp can now be created using a photosensitive seal to enable the reproduction of a handwritten signature. These stamps are commonly used in China and several other countries. In this study, 10 types of black photosensitive stamp-pad ink, 10 brands of fountain pen ink, 15 types of black gel ink and six types of black erasable gel ink found on the Chinese domestic market were collected and 10 photosensitive signature stamps were created using the signatures of 10 people. Microscopic analysis, infrared (IR) and fluorescence analyses and microspectrophotometry (MSP) techniques were used to examine the resulting photosensitive signature stamp impressions when applied to printing papers, writing papers and invoice papers. By comparing the printing and spectral characteristics of the photosensitive signature stamp impressions with those of the signatures executed using the fountain pens, gel pens and erasable gel pens, it was possible to determine whether each signature was written or stamped using a photosensitive signature stamp. To validate these results, a 96.7% absolute accuracy and a 99.3% detection rate were achieved over a total of 150 blind tests conducted by five forensic document examiners, thus demonstrating that a combination of the four analysis methods used in this work can provide a more scientific approach and improve the accuracy and the detection rate of the examination process.KEY POINTSA signature stamp is a photosensitive seal made in the style of a handwritten signature.Although microscopic analysis can usually provide better examination results, a comprehensive examination method that includes microscopic analysis and ink composition analysis is required to improve the accuracy and the detection rate of the examination process.This study collected and tested photosensitive stamp-pad inks, fountain pen inks, gel inks and erasable inks.Infrared and fluorescence analyses and microspectrophotometry were able to distinguish the photosensitive ink from both erasable ink and fountain pen ink. Supplemental data for this article are available online at https://doi.org/10.1080/20961790.2021.1898755.

This paper describes the involvement of our laboratory in a Western Australian \'cold-case\' investigation that spanned 24 years. The investigation was widely considered to be the largest in Australian history. During the investigative phase, our laboratory was tasked with the collection of trace evidence of all types in connection with a sexual assault and two homicides that were suspected to be related. Textile fibres represented the vast majority of trace evidence recovered. A much greater quantity of fibres (>10,800) was collected than would be typical for a routine case, as fibres of any colour or type were potentially of investigative value. The investigation was unprecedented in its scale, and presented numerous challenges in terms of evidence recovery, analysis, interpretation, reporting, and provision of testimony. A textile fibre microspectrophotometric (MSP) database was developed specifically for the interpretation of data in connection with the investigation. The database currently contains over 25,000 normalised and first derivative spectra of casework, validation and reference textile fibres. A fibre comparison strategy was devised, involving the identification of preliminary fibre groups on the basis of corresponding/similar MSP spectra, and verification of these groups via brightfield and fluorescence comparison microscopy. A potential link to an automotive source was identified for one of the homicide victims during the investigative phase. After identification of a suspect, a total of 98 fibres recovered from victims and from a seized motor vehicle were found to correspond in properties to six different fibre types from known textile sources in connection with the suspect. A highly publicised criminal trial was held, and textile fibre evidence provided a major contribution to the trial findings, in which the accused was found guilty of two homicides.

Non-iridescent, structural coloration in birds originates from the feather\'s internal nanostructure (the spongy matrix) but melanin pigments and the barb\'s cortex can affect the resulting color. Here, we explore how this nanostructure is combined with other elements in differently colored plumage patches within a bird. We investigated the association between light reflectance and the morphology of feathers from the back and belly plumage patches of male swallow tanagers (Tersina viridis), which look greenish-blue and white, respectively. Both plumage patches have a reflectance peak around 550 nm but the reflectance spectrum is much less saturated in the belly. The barbs of both types of feathers have similar spongy matrices at their tips, rendering their reflectance spectra alike. However, the color of the belly feather barbs changes from light green at their tips to white closer to the rachis. These barbs lack pigments and their morphology changes considerably throughout. Toward the rachis, the barb is almost hollow, with a reduced area occupied by spongy matrix, and has a flattened shape. By contrast, the blue back feathers\' barbs have melanin underneath the spongy matrix resulting in a much more saturated coloration. The color of these barbs is also even along the barbs\' length. Our results suggest that the color differences between the white and greenish-blue plumage are mostly due to the differential deposition of melanin and a reduction of the spongy matrix near the rachis of the belly feather barbs and not a result of changes in the characteristics of the spongy matrix.