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

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.

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.

Three-dimensional time-resolved fluorescence imaging by multifocal multiphoton microscopy for a photosensitizer study in living cells.

Two-photon fluorescence microscopy is widely applied to biology and medicine to study both the structure and dynamic processes in living cells. The main issue is the slow acquisition rate due to the point scanning approach limiting the multimodal detection (x, y, z, t). To extend the performances of this powerful technique, we present a time-resolved multifocal multiphoton microscope (MMM) based on laser amplitude splitting. An array of 8 x 8 foci is created on the sample that gives a direct insight of the fluorescence localization. Four-dimensional (4D) imaging is obtained by combining simultaneous foci scanning, time-gated detection, and z displacement. We illustrate time-resolved MMM capabilities for 4D imaging of a photosensitizer inside living colon cancer cells. The aim of this study is to have a better understanding of the photophysical processes implied in the photosensitizer reactivity.