Mapping Molecular Orientation with Phase Sensitive Vibrationally Resonant Sum-Frequency Generation Microscopy

We demonstrate a phase sensitive, vibrationally resonant sum-frequency generation (PSVR-SFG) microscope that combines high resolution, fast image acquisition speed, chemical selectivity, and phase sensitivity. Using the PSVR-SFG microscope, we generate amplitude and phase images of the second-order susceptibility of collagen I fibers in rat tail tendon tissue on resonance with the methylene vibrations of the protein. We find that the phase of the second-order susceptibility shows dependence on the effective polarity of the fibril bundles, revealing fibrous collagen domains of opposite orientations within the tissue. The presence of collagen microdomains in tendon tissue may have implications for the interpretation of the mechanical properties of the tissue. [Image: see text

Multiscale spectroscopic analysis of lipids in dimorphic and oleaginous Mucor circinelloides accommodate sustainable targeted lipid production

Abstract Background Oleaginous fungi have versatile metabolism and able to transform a wide range of substrates into lipids, accounting up to 20–70% of their total cell mass. Therefore, oleaginous fungi are considered as an alternative source of lipids. Oleaginous fungi can accumulate mainly acyl glycerides and free fatty acids which are localized in lipid droplets. Some of the oleaginous fungi possessing promising lipid productivity are dimorphic and can exhibit three cell forms, flat hyphae, swollen hyphae and yeast-like cells. To develop sustainable targeted fungal lipid production, deep understanding of lipogenesis and lipid droplet chemistry in these cell forms is needed at multiscale level. In this study, we explored the potential of infrared spectroscopy techniques for examining lipid droplet formation and accumulation in different cell forms of the dimorphic and oleaginous fungus Mucor circinelloides. Results Both transmission- and reflectance-based spectroscopy techniques are shown to be well suited for studying bulk fungal biomass. Exploring single cells with infrared microspectroscopy reveals differences in chemical profiles and, consequently, lipogenesis process, for different cell forms. Yeast-like cells of M. circinelloides exhibited the highest absorbance intensities for lipid-associated peaks in comparison to hyphae-like cell forms. Lipid-to-protein ratio, which is commonly used in IR spectroscopy to estimate lipid yield was the lowest in flat hyphae. Swollen hyphae are mainly composed of lipids and characterized by more uniform distribution of lipid-to-protein concentration. Yeast-like cells seem to be comprised mostly of lipids having the largest lipid-to-protein ratio among all studied cell forms. With infrared nanospectroscopy, variations in the ratios between lipid fractions triglycerides and free fatty acids and clear evidence of heterogeneity within and between lipid droplets are illustrated for the first time. Conclusions Vibrational spectroscopy techniques can provide comprehensive information on lipogenesis in dimorphic and oleaginous fungi at the levels of the bulk of cells, single cells and single lipid droplets. Unicellular spectra showed that various cell forms of M. circinelloides differs in the total lipid content and profile of the accumulated lipids, where yeast-like cells are the fatty ones and, therefore, could be considered as preferable cell form for producing lipid-rich biomass. Spectra of single lipid droplets showed an indication of possible droplet-to-droplet and within-droplet heterogeneity

Nanometre-scale infrared chemical imaging (AFM-IR) of organic matter in ultra-carbonaceous Antarctic micrometeorites (UCAMMS) and future analyses of Hayabusa 2 samples.

International audienceIntroduction: The chemical composition of organic matter (OM) in interplanetary samples (meteorites and micrometeorites) is suitably characterized by the distribution of the different chemical bonds using infrared (IR) vibrational spectroscopy (see e.g. [1]). Classical IR microscopy provides a global view of the dust grain chemical structure content but remains limited by the diffraction, with typical spot sizes sampling a few micrometers in the mid-IR range. This spatial resolution limitation is well above that of complementary techniques such as isotopic imaging with NanoSIMS or transmission electron or X-ray microscopy techniques. These techniques reveal mineralogical, chemical and isotopic heterogeneities at the sub-micron scale but do not give full access to the distribution of the various chemical bonds. The IR diffraction limitation can be circumvented by using AFM-IR microscopy. This technique opens a new window for studies of OM at ten to tens of nanometer scales and will be of importance for studies of the samples from carbonaceous asteroid Ryugu, returned by the Hayabusa 2 space probe in December 2020. AFM-IR is now a well-established microscopy technique in the vibrational field. It combines an atomic force microscope (AFM) and a tunable IR source to detect photo-thermal effect and access chemical information down to a nanoscale resolution [2]. This technique is now applied in a wide diversity of scientific fields [3], and was recently used to analyze extraterrestrial OM [4, 5]. We report here on recent results obtained on imaging two UltraCarbonaceous Antarctic MicroMeteorites (UCAMMs) using AFM-IR [5]. A small fraction of the Antarctic micrometeorites from the Concordia collection consists in UCAMMs, particles with extreme concentrations in OM, most of them exhibiting large deuterium excesses [6]. UCAMMs are also found in Japanese interplanetary dust collections [7-9]. These UCAMMs most likely originate from the surface of small icy bodies in the outer regions of the solar system [1,6,7,10]. The large OM fraction of UCAMMs (considerably higher than in the most carbon-rich meteorites) enables direct analyses without the pre-treatment generally applied to extract the OM from other meteoritic samples, and give access to unaltered chemical maps of the intimate association of minerals and organics

Imaging and 3D morphological analysis of collagen fibrils

The recent booming of multiphoton imaging of collagen fibrils by means of second harmonic generation microscopy generates the need for the development and automation of quantitative methods for image analysis. Standard approaches sequentially analyse two-dimensional (2D) slices to gain knowledge on the spatial arrangement and dimension of the fibrils, whereas the reconstructed three-dimensional (3D) image yields better information about these characteristics. In this work, a 3D analysis method is proposed for second harmonic generation images of collagen fibrils, based on a recently developed 3D fibre quantification method. This analysis uses operators from mathematical morphology. The fibril structure is scanned with a directional distance transform. Inertia moments of the directional distances yield the main fibre orientation, corresponding to the main inertia axis. The collaboration of directional distances and fibre orientation delivers a geometrical estimate of the fibre radius. The results include local maps as well as global distribution of orientation and radius of the fibrils over the 3D image. They also bring a segmentation of the image into foreground and background, as well as a classification of the foreground pixels into the preferred orientations. This accurate determination of the spatial arrangement of the fibrils within a 3D data set will be most relevant in biomedical applications. It brings the possibility to monitor remodelling of collagen tissues upon a variety of injuries and to guide tissues engineering because biomimetic 3D organizations and density are requested for better integration of implants

A fluorescence-based assay for monitoring clinical drug resistance

BACKGROUND AND AIMS: Multidrug resistance (MDR) limits effectiveness in treating malignancy by modifying internalisation and/or externalisation of drugs through cancer cell membranes. In this study we describe an assay to monitor patients' responses to chemotherapy.METHODS: The assay is based on the fluorescent properties of doxorubicin alone as well as in combination with methotrexate, vinblastine, doxorubicin and cisplatin (MVAC). The slide-based cell imaging technique was first optimised using a panel of breast and urothelial cancer cell lines and then extended to fine needle breast aspiration biopsy and urine cytology.RESULTS: The drug fluorescence behaviour observed on smears of clinical specimens is identical to that obtained using fixed cultured cells. The fluorescence of sensitive cells to chemotherapy is mainly localised in the nucleus, whereas resistant cells show a weak fluorescence signal localised in the cytoplasm. The difference in terms of fluorescence intensity is also highlighted through fluorescence spectra.CONCLUSIONS: The results suggest that the assay provides clinically valuable information in predicting responses to doxorubicin and/or MVAC therapy. Originally set up on a confocal microscope, the assay was also effective using a standard epifluorescence microscope; as such it is technically simple, reliable and inexpensive

THE ISOTOPIC DIVERSITY OF ULTRACARBONACEOUS ANTARCTIC MICROMETEORITES, A COUPLED NANOSIMS AND AFMIR STUDY

International audienc