Absorbance spectra of the hematochrome-like granules and eyespot of Euglena gracilis by scan-free absorbance spectral imaging A(x, y, λ) within the live cells.

Euglena gracilis has an organelle resembling hematochrome, with an appearance similar to the eyespot and the absorption band spectrally overlapped with that of the carotenoid. To discriminate the hematochrome-like granules and eyespot, scan-free, non-invasive, absorbance spectral imaging A(x, y, λ) microscopy of single live cells, where A(x, y, λ) means absorbance at a position (x, y) on a two-dimensional image at a specific wavelength λ was applied. This technique was demonstrated to be a powerful tool for basic research on intracellular structural analysis. By this method, characteristic absorption spectra specific to the hematochrome-like granule or eyespot were identified among a variety of spectra observed depending on the location inside the organelles. The hematochrome-like granule was dark orange and deep green in its outline and had a characteristic absorption peak at 620 nm as well as at 676 to 698 nm, suggesting that its origin is a component of chloroplast including chlorophyll a. Furthermore, the representative spectra of these organelles were derived by principal component analysis of the absorbance and its position in absorbance image, indicating that they can be distinguished from each other and other regions. It was also confirmed that even in areas where these organelles and chloroplasts overlap, one can distinguish them from each other. The present research clarified the absorption spectra of the eyespot with 1 × 1 µm spatial resolution and those unpublished of hematochrome-like granules of E. gracilis, and indicated that one can statistically distinguish these organelles by this method.

Hydrogen photoproduction in green algae Chlamydomonas reinhardtii sustainable over 2 weeks with the original cell culture without supply of fresh cells nor exchange of the whole culture medium.

Unicellular green algae Chlamydomonas reinhardtii are known to make hydrogen photoproduction under the anaerobic condition with water molecules as the hydrogen source. Since the hydrogen photoproduction occurs for a cell to circumvent crisis of its survival, it is only temporary. It is a challenge to realize persistent hydrogen production because the cells must withstand stressful conditions to survive with alternation of generations in the cell culture. In this paper, we have found a simple and cost-effective method to sustain the hydrogen production over 14 days in the original culture, without supply of fresh cells nor exchange of the culture medium. This is achieved for the cells under hydrogen production in a sulfur-deprived culture solution on the {anaerobic, intense light} condition in a desiccator, by periodically providing a short period of the recovery time (2 h) with a small amount of TAP(+S) supplied outside of the desiccator. As this operation is repeated, the response time of transition into hydrogen production (preparation time) is shortened and the rate of hydrogen production (build up time) is increased. The optimum states of these properties favorable to the hydrogen production are attained in a few days and stably sustained for more than 10 days. Since generations are alternated during this consecutive hydrogen production experiment, it is suggested that the improved hydrogen production properties are inherited to next generations without genetic mutation. The properties are reset only when the cells are placed on the {sulfur-sufficient, aerobic, moderate light} conditions for a long time (more than 1 day at least).

Scan-Free Absorbance Spectral Imaging A(x, y, λ) of Single Live Algal Cells for Quantifying Absorbance of Cell Suspensions.

Label-free, non-invasive, rapid absorbance spectral imaging A(x,y,λ) microscopy of single live cells at 1.2 µm × 1.2 µm resolution with an NA = 0.85 objective was developed and applied to unicellular green algae Chlamydomonas reinhardtii. By introducing the fiber assembly to rearrange a two-dimensional image to the one-dimensional array to fit the slit of an imaging spectrograph equipped with a CCD detector, scan-free acquisition of three-dimensional information of A(x,y,λ) was realized. The space-resolved absorbance spectra of the eyespot, an orange organelle about 1 µm, were extracted from the green-color background in a chlorophyll-rich single live cell absorbance image. Characteristic absorbance change in the cell suspension after hydrogen photoproduction in C. reinhardtii was investigated to find a single 715-nm absorption peak was locally distributed within single cells. The formula to calculate the absorbance of cell suspensions from that of single cells was presented to obtain a quantitative, parameter-free agreement with the experiment. It is quantitatively shown that the average number of chlorophylls per cell is significantly underestimated when it is evaluated from the absorbance of the cell suspensions due to the package effect.