Yoshifumi Wakisaka

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

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.

Effects of Aromatic Components in Fuel on Flame Temperature and Soot Formation in Intermittent Spray Combustion

The effects of aromatic components in fuel on ignition and combustion of intermittent spray were examined experimentally. Four types of fuel with different aromatic components, and with similar cetane number and calorific value were used in this study. Fuels were injected into the high-temperature and high-pressure vessel with the injection pressures of 100 MPa and 60 MPa using an electronically controlled fuel injection system developed by the authors. Injection rate shaping applied to the experiments was rectangular, which is a typical injection rate shaping of a common rail type injection system. Images of spray flames were captured using an ICCD camera under ambient conditions corresponding to a turbo-charged diesel engine, 6.1 MPa and 1030 K. A two-color pyrometry technique was applied to the images of spray flame to quantify two-dimensional distributions of flame temperature and soot in flame. Through systematic experiments, it was explored that the aromatic components have little effects on ignition delay and combustion period. However, the aromatic components have significant effects on flame temperature and soot formation. The flame temperature and the amount of soot in flame become higher as the aromatic components in fuel increase. The injection pressure has a great effect on spray combustion, i.e., shorter combustion period and rapid soot oxidization with high injection pressure. However the effects of aromatic components on combustion were not changed qualitatively by the increased injection pressure.

Multicolor stimulated Raman imaging of live microalgal cells using fast wavelength-switched laser pulses (Conference Presentation)

High-speed label-free imaging with chemical contrast is effective for non-invasive analysis of the metabolic heterogeneity of single cells. Stimulated Raman scattering (SRS) microscopy enables high-speed label-free image acquisition with molecular vibrational specificity. While single-color SRS microscopy only acquires images at a certain vibrational frequency, multicolor SRS microscopy successively acquires SRS images at different vibrational frequencies, which then can be used to investigate the distributions of different intracellular molecules. However, its imaging speed remains an order of magnitude slower than that of single-color video-rate SRS microscopy. Previous approaches to circumvent this issue used either only two colors with limited chemical specificity or multiplex detection of SRS spectra using a photodiode array at the expense of imaging speed. Here we demonstrate high-speed four-color SRS imaging using a single photodiode by introducing fast wavelength-switched laser pulses. The fast wavelength switching is realized by the use of an optical intensity modulator as a time gate, a diffraction grating, and fiber delay lines. Using the developed system, we demonstrate motion-artifact-free multicolor SRS imaging of polymer beads and living cells. The results firmly support that our method is a powerful tool for the label-free analysis of living cells in microbiology, oncology, plant science, and medicine.

High-speed stimulated Raman scattering microscopy for studying the metabolic diversity of motile Euglena gracilis

Microalgae have been receiving great attention for their ability to produce biomaterials that are applicable for food supplements, drugs, biodegradable plastics, and biofuels. Among such microalgae, Euglena gracilis has become a popular species by virtue of its capability of accumulating useful metabolites including paramylon and lipids. In order to maximize the production of desired metabolites, it is essential to find ideal culturing conditions and to develop efficient methods for genetic transformation. To achieve this, understanding and controlling cell-to-cell variations in response to external stress is essential, with chemically specific analysis of microalgal cells including E. gracilis. However, conventional analytical tools such as fluorescence microscopy and spontaneous Raman scattering are not suitable for evaluation of diverse populations of motile microalgae, being restricted either by the requirement for fluorescent labels or a limited imaging speed, respectively. Here we demonstrate video-rate label-free metabolite imaging of live E. gracilis using stimulated Raman scattering (SRS) – an optical spectroscopic method for probing the vibrational signatures of molecules with orders of magnitude higher sensitivity than spontaneous Raman scattering. Our SRS’s highspeed image acquisition (27 metabolite images per second) allows for population analysis of live E. gracilis cells cultured under nitrogen-deficiency - a technique for promoting the accumulation of paramylon and lipids within the cell body. Thus, our SRS system’s fast imaging capability enables quantification and analysis of previously unresolvable cell-to-cell variations in the metabolite accumulation of large motile E. gracilis cell populations.

Label-free chemical imaging of live Euglena gracilis by high-speed SRS spectral microscopy(Conference Presentation)

Microbes, especially microalgae, have recently been of great interest for developing novel biofuels, drugs, and biomaterials. Imaging-based screening of live cells can provide high selectivity and is attractive for efficient bio-production from microalgae. Although conventional cellular screening techniques use cell labeling, labeling of microbes is still under development and can interfere with their cellular functions. Furthermore, since live microbes move and change their shapes rapidly, a high-speed imaging technique is required to suppress motion artifacts. Stimulated Raman scattering (SRS) microscopy allows for label-free and high-speed spectral imaging, which helps us visualize chemical components inside biological cells and tissues. Here we demonstrate high-speed SRS imaging, with temporal resolution of 0.14 seconds, of intracellular distributions of lipid, polysaccharide, and chlorophyll concentrations in rapidly moving Euglena gracilis, a unicellular phytoflagellate. Furthermore, we show that our method allows us to analyze the amount of chemical components inside each living cell. Our results indicate that SRS imaging may be applied to label-free screening of living microbes based on chemical information.