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

Isono T, Yamashita K, Momose D, Kobayashi H, Kitamura M, Nishiyama Y, Hosoya T, Kanda H, Kudo A, Okada N, Yagi T, Nakata K, Mineki S, Tokunaga E - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan.

ABSTRACT
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 MeSH data available.


The 715-nm absorption peak localized within a single cell, measured by the 2D-1D conversion method.(a) Absorption spectrum of a cell suspension after under hydrogen generation conditions. (b) Position-dependent variation of absorbance spectra of 0.16 μm × 0.6 μm area (division unit) within a single cell obtained by the 2D-1D conversion method. (c) Right: The image of the cell for single-cell absorbance measurement in (b) picked up from the cell suspension with a 715 nm absorption peak. Left: A(x,y,λ = 715 nm) for the area enclosed by the red square within the cell. The local spectra at the positions A, B, and C are displayed in (b).
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pone.0128002.g005: The 715-nm absorption peak localized within a single cell, measured by the 2D-1D conversion method.(a) Absorption spectrum of a cell suspension after under hydrogen generation conditions. (b) Position-dependent variation of absorbance spectra of 0.16 μm × 0.6 μm area (division unit) within a single cell obtained by the 2D-1D conversion method. (c) Right: The image of the cell for single-cell absorbance measurement in (b) picked up from the cell suspension with a 715 nm absorption peak. Left: A(x,y,λ = 715 nm) for the area enclosed by the red square within the cell. The local spectra at the positions A, B, and C are displayed in (b).

Mentions: C. reinhardtii is attracting because of its hydrogen photoproduction property on the anaerobic condition. The most frequently used method to obtain the anaerobic condition is to use a sulfur-deprived C. reinhardtii culture [16]. In this culture, oxygenic photosynthesis is gradually deactivated under photoirradiation because reconstruction of photosynthetic proteins from photodamage is prevented. As a result, respiratory consumption of oxygen exceeds photosynthetic production of oxygen for the anaerobic condition to be realized to initiate hydrogen photoproduction. In this experiment, we found that after hydrogen generation the absorption spectra of the cell suspension change occasionally such that a new absorption peak appears around 715 nm on the longer wavelength side of Chl a (680 nm), as shown in Fig 5(a). This characteristic change in the absorption spectra do not always occur even on the same experimental conditions. Only known is that an anaerobic condition is necessary for occurrence of a 715-nm peak. The frequency is about one out of three cell suspensions. In addition, increase in the irradiation light intensity tends to enhance the probability to give rise to the 715-nm peak. Because of lacking for fully quantitative measurements so far, the necessary and sufficient condition has not yet been identified. Similar peaks were previously observed for Euglena after aging in the dark [21] or Ginkgo biloba [22], but the formation mechanism of the peak is not yet understood.


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

Isono T, Yamashita K, Momose D, Kobayashi H, Kitamura M, Nishiyama Y, Hosoya T, Kanda H, Kudo A, Okada N, Yagi T, Nakata K, Mineki S, Tokunaga E - PLoS ONE (2015)

The 715-nm absorption peak localized within a single cell, measured by the 2D-1D conversion method.(a) Absorption spectrum of a cell suspension after under hydrogen generation conditions. (b) Position-dependent variation of absorbance spectra of 0.16 μm × 0.6 μm area (division unit) within a single cell obtained by the 2D-1D conversion method. (c) Right: The image of the cell for single-cell absorbance measurement in (b) picked up from the cell suspension with a 715 nm absorption peak. Left: A(x,y,λ = 715 nm) for the area enclosed by the red square within the cell. The local spectra at the positions A, B, and C are displayed in (b).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4465668&req=5

pone.0128002.g005: The 715-nm absorption peak localized within a single cell, measured by the 2D-1D conversion method.(a) Absorption spectrum of a cell suspension after under hydrogen generation conditions. (b) Position-dependent variation of absorbance spectra of 0.16 μm × 0.6 μm area (division unit) within a single cell obtained by the 2D-1D conversion method. (c) Right: The image of the cell for single-cell absorbance measurement in (b) picked up from the cell suspension with a 715 nm absorption peak. Left: A(x,y,λ = 715 nm) for the area enclosed by the red square within the cell. The local spectra at the positions A, B, and C are displayed in (b).
Mentions: C. reinhardtii is attracting because of its hydrogen photoproduction property on the anaerobic condition. The most frequently used method to obtain the anaerobic condition is to use a sulfur-deprived C. reinhardtii culture [16]. In this culture, oxygenic photosynthesis is gradually deactivated under photoirradiation because reconstruction of photosynthetic proteins from photodamage is prevented. As a result, respiratory consumption of oxygen exceeds photosynthetic production of oxygen for the anaerobic condition to be realized to initiate hydrogen photoproduction. In this experiment, we found that after hydrogen generation the absorption spectra of the cell suspension change occasionally such that a new absorption peak appears around 715 nm on the longer wavelength side of Chl a (680 nm), as shown in Fig 5(a). This characteristic change in the absorption spectra do not always occur even on the same experimental conditions. Only known is that an anaerobic condition is necessary for occurrence of a 715-nm peak. The frequency is about one out of three cell suspensions. In addition, increase in the irradiation light intensity tends to enhance the probability to give rise to the 715-nm peak. Because of lacking for fully quantitative measurements so far, the necessary and sufficient condition has not yet been identified. Similar peaks were previously observed for Euglena after aging in the dark [21] or Ginkgo biloba [22], but the formation mechanism of the peak is not yet understood.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan.

ABSTRACT
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 MeSH data available.