Revealing Lipid Body Formation and its Subcellular Reorganization in Oleaginous Microalgae Using Correlative Optical Microscopy and Infrared Nanospectroscopy

Abstract

The purpose of this work is to develop an integrated imaging approach to characterize without labeling at the sub-cellular level the formation of lipid body droplets (LBs) in microalgae undergoing nitrogen starvation. First conventional optical microscopy approaches, gas chromatography, and turbidimetry measurements allowed to monitor the biomass and the total lipid content in the oleaginous microalgae Parachlorella kesslerii during the starvation process. Then a local analysis of the LBs was proposed using an innovative infrared nanospectroscopy technique called atomic force microscopy-based infrared spectroscopy (AFM-IR). This label-free technique assessed the formation of LBs and allowed to look into the LB composition thanks to the acquisition of local infrared spectra. Last correlative measurements using fluorescence microscopy and AFM-IR were performed to investigate the subcellular reorganization of LB and the chloroplasts.

Enhancing algal production strategies: strain selection, AI-informed cultivation, and mutagenesis

“…Post-isolation, strains are identified and evaluated for commercial potential based on morphological characteristics using microscopy techniques (Deniset-Besseau et al, 2021;Sonmez et al, 2023). Identification through DNA barcoding, mainly 18S rRNA gene sequencing, is a precise and widely used method for strain determination (Darienko et al, 2015;Hadi et al, 2016;Zou et al, 2016).…”

“…For instance, certain strains have demonstrated antioxidant (Yang et al, 2023), antimicrobial (Zhou et al, 2023), and anti-inflammatory properties (Leitner et al, 2022;Kurniawan et al, 2023;Manabe et al, 2023). Moreover, some strains are suited for biofuel production due to their high lipid content (Nelson et al, 2013(Nelson et al, , 2017Deniset-Besseau et al, 2021). Microalgal bioprospecting stands at the forefront of discovering novel compounds with diverse applications.…”

AFM-IR for Nanoscale Chemical Characterization in Life Sciences: Recent Developments and Future Directions

“…61 This fungus is notorious for its well-developed enzyme secretion system essential to its survival in the wild as a saprobe 62 and which has been leveraged in numerous industrial processes including recombinant protein production. 63 The strain used in this study, QM6a SecEYFP, has been modified to express yellow fluorescent protein (EYFP) under the same conditions of the main cellobiohydrolase, CBHI, and to follow the same intracellular path. Therefore, in this strain the EYFP and its respective fluorescence signal are colocalized with the cellulases and xylanases, allowing for a correlation between fluorescence intensity and the presence of proteins containing β-sheet secondary structures in the same location.…”

“…An example of whole cell imaging without resorting to chemometric models to detect the target analyte is the study by Deniset-Besseau et al of lipid body droplet formation in P. kesslerii, a microalgae used in biofuel production.…”

Emerging Technologies for the Discovery of Novel Diversity in Cyanobacteria and Algae and the Elucidation of Their Valuable Metabolites

“…This study utilized AFM-IR to monitor the production of LBs and their chemical composition in N-starved cells. Triacylglycerol (TAC), the key component of LBs, has an important application in the production of lipid-based biodiesel, a sustainable alternative to fossil fuels [27].…”