Diversity and Evolution of Frog Visual Opsins: Spectral Tuning and Adaptation to Distinct Light Environments.

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.

Specific and label-free endogenous signature of dystrophic muscle by Synchrotron deep ultraviolet radiation.

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.

Visible microspectrophotometry coupled with machine learning to discriminate the erythrocytic life cycle stages of P. falciparum malaria parasites in functional single cells.

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.

Enhancing the evidential value of textile fibres Part 2: Application of a database-driven fibre comparison strategy to a cold-case investigation.

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.

Reproductive aspects of freshwater unionid mussel sperm: Seasonal dynamics, male-to male variability, and cell quantification.

Freshwater unionid mussel diversity is decreasing because of species extirpation or extinction. While little can be done to recover lost species, there is an opportunity to develop techniques to save other species. This can be facilitated through gene banking and assisted reproduction. Unfortunately, limited information is available on mussel reproduction, especially relating to sperm quality. Objectives, therefore, were to quantify seasonal changes in sperm concentration and morphology for two unionid mussels, Ligumia subrostrata and Lampsilisstraminea, measure intraspecific heterogeneity for sperm morphometry, and develop an efficient method to quantify sperm concentration using a microspectrophotometer. There were no differences in sperm concentration when cells were extracted from the center or at a half centimeter on either side of the visceral mass, during the spawning season. There was a seasonal change in sperm concentration, such that concentration for L. subrostrata ranged from 1.1 × 109 to 19.60 × 109 cells/mL with there being the largest counts between 26 September to 7 November. L. straminea sperm concentration was greatest (20.0 × 109 cells/mL) on 13 September and subsequently decreased. Sperm were uniflagellated and SEM results for L. subrostrata and L. straminea showed mean head length and width (mid-spawning) were 3.38 ± 0.04 µm and 1.61 ± 0.01 µm and 3.37 ± 0.04 µm and 1.61 ± 0.01 µm, respectively. There were close (R2 ≥ 0.85) quadratic associations between hemocytometer counts and absorbance (300, 600, 700 nm). These results provide baseline information to further investigate sperm quality, fertilizing capacity, and cryopreservation for freshwater mussels.

Probing the Anti-Cancer Potency of Sulfated Galactans on Cholangiocarcinoma Cells Using Synchrotron FTIR Microspectroscopy, Molecular Docking, and In Vitro Studies.

Sulfated galactans (SG) isolated from red alga Gracilaria fisheri have been reported to inhibit the growth of cholangiocarcinoma (CCA) cells, which was similar to the epidermal growth factor receptor (EGFR)-targeted drug, cetuximab. Herein, we studied the anti-cancer potency of SG compared to cetuximab. Biological studies demonstrated SG and cetuximab had similar inhibition mechanisms in CCA cells by down-regulating EGFR/ERK pathway, and the combined treatment induced a greater inhibition effect. The molecular docking study revealed that SG binds to the dimerization domain of EGFR, and this was confirmed by dimerization assay, which showed that SG inhibited ligand-induced EGFR dimer formation. Synchrotron FTIR microspectroscopy was employed to examine alterations in cellular macromolecules after drug treatment. The SR-FTIR-MS elicited similar spectral signatures of SG and cetuximab, pointing towards the bands of RNA/DNA, lipids, and amide I vibrations, which were inconsistent with the changes of signaling proteins in CCA cells after drug treatment. Thus, this study demonstrates the underlined anti-cancer mechanism of SG by interfering with EGFR dimerization. In addition, we reveal that FTIR signature spectra offer a useful tool for screening anti-cancer drugs' effect.

Single-Cell Infrared Microspectroscopy Quantifies Dynamic Heterogeneity of Mesenchymal Stem Cells during Adipogenic Differentiation.

The central relevance of cellular heterogeneity to biological phenomena raises the rational needs for analytical techniques with single-cell resolution. Here, we developed a single-cell FTIR microspectroscopy-based method for the quantitative evaluation of cellular heterogeneity by calculating the cell-to-cell similarity distance of the infrared spectral data. Based on this method, we revealed the infrared phenotypes might reflect the dynamic heterogeneity changes in the cell population during the adipogenic differentiation of the human mesenchymal stem cells. These findings provide an alternative label-free optical approach for quantifying the cellular heterogeneity, and the combination with other single-cell analysis tools will be very helpful for understanding the genotype-to-phenotype relationship in cellular populations.

Raman spectroscopy-based approach to study the female gamete.

In the last years, an increasing interest has emerged on the development of new non-invasive methods for the assessment of oocyte quality in order to improve outcomes of assisted reproductive technologies (ARTs) either in medical or veterinary fields. Raman microspectroscopy (RMS) has been proposed as a promising tool for the examination of the mammalian female gamete and identification of markers of its developmental competence. This technique provides a unique spectral fingerprint indicative of molecular composition of the cell and allows probing subcellular compartments. Studies have been carried out analysing by RMS fixed or living oocytes derived from different animal models. RMS imaging has been successfully applied to discriminate the biochemical changes of the global molecular architecture of mouse oocytes at different stages of maturation and those occurring in different conditions of maturation and oocyte aging. RMS can also detect modifications of specific structural components, including the oocyte zona pellucida and F-actin subcortical cytoskeleton in fresh sheep oocytes and those underwent to vitrification procedures. Finally, the recent application of Coherent anti-Stokes Raman scattering (CARS) microscopy for examination of oocyte lipid component will be briefly discussed. CARS overcomes some limits of RMS providing vibrational and spectral information with higher sensitivity, spatial resolution which is ideal to study living oocytes. This review summarizes the research on RMS approaches for oocyte evaluation showing the high potential use, current limitations and new improvements.

Study of the intracellular nanoparticle-based radiosensitization mechanisms in F98 glioma cells treated with charged particle therapy through synchrotron-based infrared microspectroscopy.

The use of nanoparticles (NP) as dose enhancers in radiotherapy (RT) is a growing research field. Recently, the use of NP has been extended to charged particle therapy in order to improve the performance in radioresistant tumors. However, the biological mechanisms underlying the synergistic effects involved in NP-RT approaches are not clearly understood. Here, we used the capabilities of synchrotron-based Fourier Transform Infrared Microspectroscopy (SR-FTIRM) as a bio-analytical tool to elucidate the NP-induced cellular damage at the molecular level and at a single-cell scale. F98 glioma cells doped with AuNP and GdNP were irradiated using several types of medical ion beams (proton, helium, carbon and oxygen). Differences in cell composition were analyzed in the nucleic acids, protein and lipid spectral regions using multivariate methods (Principal Component Analysis, PCA). Several NP-induced cellular modifications were detected, such as conformational changes in secondary protein structures, intensity variations in the lipid CHx stretching bands, as well as complex DNA rearrangements following charged particle therapy irradiations. These spectral features seem to be correlated with the already shown enhancement both in the DNA damage response and in the reactive oxygen species (ROS) production by the NP, which causes cell damage in the form of protein, lipid, and/or DNA oxidations. Vibrational features were NP-dependent due to the NP heterogeneous radiosensitization capability. Our results provided new insights into the molecular changes in response to NP-based RT treatments using ion beams, and highlighted the relevance of SR-FTIRM as a useful and precise technique for assessing cell response to innovative radiotherapy approaches.

Connecting biology, optics and nanomechanical properties in micro-wasps.

Coloration in insects provides a fruitful opportunity for interdisciplinary research involving both physics and biology, and for a better understanding of the design principles of biological structures. In this research we used nanometric and micrometric analyses to investigate the morphological and mechanical properties of the black-orange-black (BOB) color pattern in scelionid wasps, which has never been studied. The primary objective of the present investigation was to explore the structural and mechanical differences in the mesoscutum of four species: Baryconus with an orange mesosoma (i.e. BOB pattern), all black Baryconus, Scelio with an orange mesosoma (i.e. BOB pattern), and all black Scelio. The most outstanding findings include the absence of multilayer structures that generate structural color, a pigment concentrated in the upper surface of the epicuticle, and surprising differences between the four species. Three of the four species showed an accordion-like structure in the furrow (notaulus), whereas the adjacent mesoscutum was different in each species. Moreover, the normalized color component spectra for blue, green and red colors of the black mesoscutum of each genus showed the same spectral dependence while the orange color manifested small changes in the dominant wavelength, resulting in slightly different orange tones.

Visual Opsin Diversity in Sharks and Rays.

The diversity of color vision systems found in extant vertebrates suggests that different evolutionary selection pressures have driven specializations in photoreceptor complement and visual pigment spectral tuning appropriate for an animal's behavior, habitat, and life history. Aquatic vertebrates in particular show high variability in chromatic vision and have become important models for understanding the role of color vision in prey detection, predator avoidance, and social interactions. In this study, we examined the capacity for chromatic vision in elasmobranch fishes, a group that have received relatively little attention to date. We used microspectrophotometry to measure the spectral absorbance of the visual pigments in the outer segments of individual photoreceptors from several ray and shark species, and we sequenced the opsin mRNAs obtained from the retinas of the same species, as well as from additional elasmobranch species. We reveal the phylogenetically widespread occurrence of dichromatic color vision in rays based on two cone opsins, RH2 and LWS. We also confirm that all shark species studied to date appear to be cone monochromats but report that in different species the single cone opsin may be of either the LWS or the RH2 class. From this, we infer that cone monochromacy in sharks has evolved independently on multiple occasions. Together with earlier discoveries in secondarily aquatic marine mammals, this suggests that cone-based color vision may be of little use for large marine predators, such as sharks, pinnipeds, and cetaceans.

Diffusive structural colour in Hoplia argentea.

Nature's nanostructures can bring about vivid and iridescent colours seen in many insects, notably in beetles and butterflies. While the intense structural colours can be advantageous for display purposes, they may also be appealing to predators and therefore constitute an evolutionary disadvantage. Animals often employ absorption and scattering in order to reduce the directionality of the reflected light and thereby enhance their camouflage. Here, we investigated the monkey beetle Hoplia argentea using microspectrophotometry, electron microscopy, fluorimetry and optical modelling. We show that the dull green dorsal colour comes from the nanostructured scales on the elytra. The nanostructure consists of a multi-layered photonic structure covered by a filamentous layer. The filamentous layer acts as a spatial diffuser of the specular reflection from the multilayer and suppresses the iridescence. This combination leads to a colour-stable and angle-independent green reflection that probably enhances the camouflage of the beetles in their natural habitat.

Vision in the snapping shrimp Alpheus heterochaelis.

Snapping shrimp engage in heterospecific behavioral associations in which their partners, such as goby fish, help them avoid predators. It has been argued that snapping shrimp engage in these partnerships because their vision is impaired by their orbital hood, an extension of their carapace that covers their eyes. To examine this idea, we assessed the visual abilities of snapping shrimp. We found the big claw snapping shrimp, Alpheus heterochaelis, has spatial vision provided by compound eyes with reflecting superposition optics. These eyes view the world through an orbital hood that is 80-90% as transparent as seawater across visible wavelengths (400-700 nm). Through electroretinography and microspectrophotometry, we found the eyes of A. heterochaelis have a temporal sampling rate of >40 Hz and have at least two spectral classes of photoreceptors (λmax=500 and 519 nm). From the results of optomotor behavioral experiments, we estimate the eyes of A. heterochaelis provide spatial vision with an angular resolution of ∼8 deg. We conclude that snapping shrimp have competent visual systems, suggesting the function and evolution of their behavioral associations should be re-assessed and that these animals may communicate visually with conspecifics and heterospecific partners.

Unusual A1/A2-visual pigment conversion during light/dark adaptation in marine fish.

Changes in visual pigments were studied in two marine fish species, the masked greenling Hexagrammos octogrammus and the prickleback Pholidapus dybowskii. A microspectrophotometric (MSP) analysis showed that the rods and cones of the fish collected from the natural marine environment in summer or kept in a tank at a high illumination level predominantly contained porphyropsins based on chromophore A2. As a result, λmax of the double cones significantly shifted to longer wavelengths, reaching 625 and 609 nm, respectively. After several weeks of dark adaptation, the spectra of all the photoreceptor types shifted to shorter wavelengths, as the A1:A2 ratio switched to A1. The MSP data from the fish kept under controlled light conditions were confirmed by chromatography (HPLC), which showed that the changes in the chromophore ratio were reversible and independent of the water temperature. After the preliminary deep dark adaptation, the first noticeable shift in the pigment ratio from A1 to A2 occurred within two weeks of exposure to bright light. A novel finding in this study was a reverse polarity of A1/A2 changes, unlike the case in most other fish species, where A2 chromophore predominated after the dark exposure. This demonstration of the unusual phenomenon of visual pigment transformation suggests a modification or a new way for the activation of specific biochemical mechanisms of A1:A2 conversion at both high and low illumination levels.

Método microespectrofotométrico para la determinación de valores de referencia de la fosfomanosa isomerasa / A Microspectrophotometric Method for the Determination of Reference Values of Phosphomannose Isomerase / Método microespectrofotométrico para determinar valores de referência da fosfomanose isomerase

Resumen: Los desórdenes congénitos de glicosilación son un conjunto de defectos genéticos de tipo multisistémico que afectan la función de la proteína. Se han descrito cerca de 75 enfermedades desde sus primeros estudios. En el presente estudio se desarrolló un método microespectrofotométrico para el diagnóstico de la enzima citosólica fosfomanosa isomerasa EC 5.3.1.8 (PMI), se analizaron 32 muestras de individuos con rango de edad de 0,6 a 27 años y se estableció el intervalo y el valor de referencia de actividad enzimática específica. Este estudio permitirá iniciar el diagnóstico de pacientes deficientes de la PMI de forma temprana y oportuna, lo cual la convierte en una posible enzima candidata para pruebas de tamizaje neonatal, ya que esta patología tiene un tratamiento fácil y de bajo costo, que consiste en la suplementación de manosa en forma oral. El diagnóstico clínico de este desorden metabólico beneficiará al paciente y a su familia al mejorar su calidad de vida, como también al sistema de salud colombiano.

Abstract: Congenital glycosylation disorders are a set of multi-systemic genetic defects affecting protein function. About 75 diseases have been described since early studies. This study developed a microspectrophotometric method for the diagnosis of the cytosolic enzyme phosphomannose isomerase (PMI) (EC 5.3.1.8), analyzed 32 samples of individuals ranging between 0.6 and 27 years old, and established the interval and reference value of specific enzyme activity. This study will allow early and timely diagnosis of PMI deficient patients, which makes this enzyme a potential candidate for neonatal screening tests since this pathology has an easy, low-cost treatment (oral administration of mannose supplements). Clinical diagnosis of this metabolic disorder will benefit the patient and his family by improving his quality of life, as well as the Colombian healthcare system.

Resumo: Os defeitos congénitos de glicosilação são um conjunto de defeitos genéticos de tipo mul-tissistêmico que afetam a função da proteína. Foram descritas 75 doenças desde seus primeiros estudos. Neste estudo, foi desenvolvido um método microespectrofotométrico para diagnosticar a enzima citosólica fosfomanose isomerase EC 5.3.1.8 (PMI); foram analisadas 32 amostras de indivíduos entre 0,6 e 27 anos e estabelecidos o intervalo e o valor de referência de atividade enzimática específica. Este estudo permitirá iniciar o diagnóstico de pacientes deficientes da PMI de forma precoce e oportuna, o que a converte em uma possível enzima candidata para testes genéticos de rastreio pré-natal, já que essa patologia tem um tratamento fácil e de baixo custo, que consiste na suplementação de manose por via oral. O diagnóstico clínico desse defeito metabólico beneficiará o paciente e sua família ao melhorar a qualidade de vida e o sistema de saúde colombiano.

Absorbance spectra of the hematochrome-like granules and eyespot of Euglena gracilis by scan-free absorbance spectral imaging A(x, y, λ) within the live cells.

Euglena gracilis has an organelle resembling hematochrome, with an appearance similar to the eyespot and the absorption band spectrally overlapped with that of the carotenoid. To discriminate the hematochrome-like granules and eyespot, scan-free, non-invasive, absorbance spectral imaging A(x, y, λ) microscopy of single live cells, where A(x, y, λ) means absorbance at a position (x, y) on a two-dimensional image at a specific wavelength λ was applied. This technique was demonstrated to be a powerful tool for basic research on intracellular structural analysis. By this method, characteristic absorption spectra specific to the hematochrome-like granule or eyespot were identified among a variety of spectra observed depending on the location inside the organelles. The hematochrome-like granule was dark orange and deep green in its outline and had a characteristic absorption peak at 620 nm as well as at 676 to 698 nm, suggesting that its origin is a component of chloroplast including chlorophyll a. Furthermore, the representative spectra of these organelles were derived by principal component analysis of the absorbance and its position in absorbance image, indicating that they can be distinguished from each other and other regions. It was also confirmed that even in areas where these organelles and chloroplasts overlap, one can distinguish them from each other. The present research clarified the absorption spectra of the eyespot with 1 × 1 µm spatial resolution and those unpublished of hematochrome-like granules of E. gracilis, and indicated that one can statistically distinguish these organelles by this method.

Rejection of the biophoton hypothesis on the origin of photoreceptor dark noise.

Rod photoreceptors of the vertebrate retina produce, in darkness, spontaneous discrete current waves virtually identical to responses to single photons. The waves comprise an irreducible source of noise (discrete dark noise) that may limit the threshold sensitivity of vision. The waves obviously originate from acts of random activation of single rhodopsin molecules. Until recently, it was generally accepted that the activation occurs due to the rhodopsin thermal motion. Yet, a few years ago it was proposed that rhodopsin molecules are activated not by heat but rather by real photons generated within the retina by chemiluminescence. Using a high-sensitive photomultiplier, we measured intensities of biophoton emission from isolated retinas and eyecups of frogs (Rana ridibunda) and fish (sterlet, Acipenser ruthenus). Retinal samples were placed in a perfusion chamber and emitted photons collected by a high-aperture quartz lens. The collected light was sent to the photomultiplier cathode through a rotating chopper so that a long-lasting synchronous accumulation of the light signal was possible. The absolute intensity of bio-emission was estimated by the response of the measuring system to a calibrated light source. The intensity of the source, in turn, was quantified by measuring rhodopsin bleaching with single-rod microspectrophotometry. We also measured the frequency of discrete dark waves in rods of the two species with suction pipette recordings. Expressed as the rate constant of rhodopsin activation, it was 1.2 × 10-11/s in frogs and 7.6 × 10-11/s in sterlets. Approximately two thirds of retinal samples of each species produced reliably measurable biophoton emissions. However, its intensity was ≥100 times lower than necessary to produce the discrete dark noise. We argue that this is just a lower estimate of the discrepancy between the hypothesis and experiment. We conclude that the biophoton hypothesis on the origin of discrete dark noise in photoreceptors must be rejected.

Synchrotron macro ATR-FTIR microspectroscopy for high-resolution chemical mapping of single cells.

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy has been used widely for probing the molecular properties of materials. Coupling a synchrotron infrared (IR) beam to an ATR element using a high numerical aperture (NA) microscope objective enhances the spatial resolution, relative to transmission or transflectance microspectroscopy, by a factor proportional to the refractive index (n) of the ATR element. This work presents the development of the synchrotron macro ATR-FTIR microspectroscopy at Australian Synchrotron Infrared Microspectroscopy (IRM) Beamline, and demonstrates that high quality FTIR chemical maps of single cells and tissues can be achieved at an enhanced spatial resolution. The so-called "hybrid" macro ATR-FTIR device was developed by modifying the cantilever arm of a standard Bruker macro ATR-FTIR unit to accept germanium (Ge) ATR elements with different facet sizes (i.e. 1 mm, 250 µm and 100 µm in diameter) suitable for different types of sample surfaces. We demonstrated the capability of the technique for high-resolution single cell analysis of malaria-infected red blood cells, individual neurons in a brain tissue and cellular structures of a Eucalyptus leaf. The ability to measure a range of samples from soft membranes to hard cell wall structures demonstrates the potential of the technique for high-resolution chemical mapping across a broad range of applications in biology, medicine and environmental science.

A methodology study on chemical and molecular structure imaging in modified forage leaf tissue with cutting-edge synchrotron-powered technology (SR-IMS) as a potential research tool.

To date there is no any study on imaging molecular chemistry and chemical structure of biotech-modified plant tissue on a molecular basis. The objective of this methodology study was to apply a non-invasive and non-destructive synchrotron powered technology - SR-IMS to image molecular chemistry of the modified forage leaf tissue. The infrared molecular vibrational microspectroscopy powered with synchrotron light at Advanced Light Source (ALS, Lawrence Berkeley National Lab, Berkeley, California, Dept. of Energy, USA) were applied. The synchrotron beamline time was arranged by National Synchrotron Light Source (Scientist Dr. Lisa Miller, Brookhaven National Lab, Dept. of Energy, USA). The various molecular functional groups in the forage tissue included CH symmetric and asymmetric regions, amides I and II regions, structure and non-structure CHO regions, carbonyl ester region with peak areas at ca. 3644-3000 cm-1, ca 3005-2979 cm-1, ca. 1722-1483 cm-1, ca. 1488-1412 cm-1, ca. 1296-1189 cm-1, and ca. 1194-951 cm-1. The spectral peak area ratio imaging of chemical functional groups were also studied which included the ratio of peak area under ca. 1722-1483 cm-1 to peak area under ca. 3005-2979 cm-1 and the ratio of peak area under ca. 1722-1483 cm-1 to peak area under ca. 1194-951 cm-1. The results showed that the advanced synchrotron-based technology - SR-IMS was able to image the forage tissue at an ultra-highly resolution within intact tissue within cellular and subcellular dimension. It revealed the forage tissue in a molecular chemical sense and provided an insight on nutrient properties and their molecular structure as well as chemical features. In conclusion, the synchrotron-radiation SR-IMS is able to image molecular structure of the forage leaf tissue at an ultra-highly resolution. The advanced SR-IMS technique could provide leaf tissue four kinds of information simultaneously: tissue structure, tissue chemistry, tissue nutrients, and tissue environment of forage.

Probing primary mesenchymal stem cells differentiation status by micro-Raman spectroscopy.

We have employed micro-Raman spectroscopy to get insight into intrinsic biomolecular profile of individual mesenchymal stem cell isolated from periodontal ligament. Furthermore, these cells were stimulated towards adipogenic, chondrogenic, and osteogenic lineages and their status of differentiation was assessed using micro-Raman spectroscopy. In both cases, glass coverslips were used as substrates, due to their wide availability and cost effectiveness. In all sample groups, the same type of behavior was observed, manifested as changes in Raman spectra: the increase of relative intensity of protein/lipid bands and decrease of nucleic acid bands. Comprehensive statistical analysis in the form of principal component analysis was performed, which revealed noticeable grouping of cells with the similar features. Despite the inhomogeneity of primary stem cells and their differentiated lineages, we demonstrated that micro-Raman spectroscopy is sufficient for distinguishing cells' status, which can be valuable for medical and clinical application.