Probing the metabolic heterogeneity of live Euglena gracilis with stimulated Raman scattering microscopy

Abstract

Understanding metabolism in live microalgae is crucial for efficient biomaterial engineering, but conventional methods fail to evaluate heterogeneous populations of motile microalgae due to the labelling requirements and limited imaging speeds. Here, we demonstrate label-free video-rate metabolite imaging of live Euglena gracilis and statistical analysis of intracellular metabolite distributions under different culture conditions. Our approach provides further insights into understanding microalgal heterogeneity, optimizing culture methods and screening mutant microalgae.

Rapid in vivo lipid/carbohydrate quantification of single microalgal cell by Raman spectral imaging to reveal salinity-induced starch-to-lipid shift

“…Since our setup stability test also showed a less than 5% standard deviation for the continuous Raman measurement of standard samples (Additional file 1: Figure S1), such high standard deviation is unlikely due to the instability of our Raman setup. Furthermore, such significant deviation between the biomass accumulation levels of independent microalgal cells was reported from multiple research groups working on the analysis of cellular components at single-cell level [12, 24]. The standard deviation difference between the Raman and the conventional measurements most likely reflects the actual deviation at the single-cell and batch statistics levels.…”

Label-free biochemical quantitative phase imaging with mid-infrared photothermal effect

“…The MIR pulse fluence is ~10 pJ/µm 2 (~100 nJ over ~100 µm ×100 µm) but depends on the wavenumber. The VIS pulse fluence can be as low as ~0.1 pJ/µm 2 (~1 nJ over ~100 µm × 100 µm), which is 3 -4 orders of magnitude lower than that used in, e.g., coherent Raman imaging 10 . Figure 2b shows our MV-DH system.…”

“…Preliminary results on this method, which we term MV-sensitive QPI (MV-QPI), have been recently reported 20 . The focus of this work is to further develop the MV-QPI method, proving its practical bioimaging capability in the broadband MIR fingerprint region while also pioneering the depth-and superresolved imaging performance beyond the diffraction limit posed in other MVI techniques [6][7][8][9][10][11][12][13][14][15][16][17][18] . To highlight MV-QPI's versatility, we present two implementations of QPI scheme for the single-cell imaging application.…”

Applications of vibrational tags in biological imaging by Raman microscopy

“…More importantly, the rich spectral features that vary between different molecules makes it possible for hyperspectral imaging of multiple chemical species that are always of great interest to biologists. In recent advances with label-free techniques, for example, metabolic heterogeneity of live Euglena gracilis has been studied 10 , providing insight to microalgal research; in other studies, neurotransmitter 11 and neuronal membrane potential 12 have been visualized, adding new tools to investigate neurobiology; and brain tumor infiltration diagnostics 13, 14 has been successfully demonstrated in mouse brain and human patient samples, and an intraoperative set-up using fiber laser was then engineered 15 to assist surgery. However, although label-free vibrational imaging has been demonstrated a useful tool in biological studies, the detection specificity is usually limited – any molecules that harbor the same chemical bond of target will have severely overlapping spectrum and make it extremely hard to image a specific molecule of interest.…”