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.

No abstract

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).

Euglena gracilis is an edible photosynthetic single-cell alga that can synthesize carotenoids. It is highly demanded to establish the technology to select and grow individual cells capable of synthesizing more carotenoids because it contributes to safe and inexpensive production of carotenoids. In the cells of E. gracilis, carotenoids are mainly contained in chloroplasts and eyespots, and typical carotenoids have a characteristic absorption maximum in common. E. gracilis also has an organelle resembling hematochrome, which has an appearance similar to the eyespot and the absorption band spectrally overlapping that of the carotenoid although reportedly it does not contain carotenoids. To discriminate the eyespot and hematochrome-like granules and to investigate the intracellular distribution of carotenoids, scan-free, non-invasive, absorbance spectral imaging A(x, y, λ) microscopy of single live cells was applied. It was demonstrated that this technique is a powerful tool not only for basic research on intracellular structural analysis but also for identifying difference in carotenoid content in individual cells applicable to screening of carotenoid-rich cells. By this technique, it was confirmed that carotenoids exist in chloroplasts and eyespots, and a number of characteristic absorption spectra of pigments observed specific to the eyespot or hematochrome-like granules were identified. In addition, it was found that hematochromelike granules have a characteristic absorption peak at 620 nm as well as at 676 nm, suggesting that its origin is a component of chloroplast including Chlorophyll a.

Euglena gracilis is an edible photosynthetic single-cell alga that can synthesize carotenoids. It is highly demanded to establish the technology to select and grow individual cells capable of synthesizing more carotenoids because it contributes to safe and inexpensive production of carotenoids. In the cells of E. gracilis, carotenoids are mainly contained in chloroplasts and eyespots, and typical carotenoids have a characteristic absorption maximum in common. E. gracilis also has an organelle resembling hematochrome, which has an appearance similar to the eyespot and the absorption band spectrally overlapping that of the carotenoid although reportedly it does not contain carotenoids. To discriminate the eyespot and hematochrome-like granules and to investigate the intracellular distribution of carotenoids, scan-free, non-invasive, absorbance spectral imaging A(x, y, λ) microscopy of single live cells was applied. It was demonstrated that this technique is a powerful tool not only for basic research on intracellular structural analysis but also for identifying difference in carotenoid content in individual cells applicable to screening of carotenoid-rich cells. By this technique, it was confirmed that carotenoids exist in chloroplasts and eyespots, and a number of characteristic absorption spectra of pigments observed specific to the eyespot or hematochrome-like granules were identified. In addition, it was found that hematochromelike granules have a characteristic absorption peak at 620 nm as well as at 676 nm, suggesting that its origin is a component of chloroplast including Chlorophyll a.