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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.


Comparison between single cell absorbance calculated from cell suspension absorbance and that averaged over 100 single-cell measurements.(a) Black: absorbance of a cell suspension of 5-mm path length measured with an integrating sphere. Red: maximum local absorbance calculated from the absorbance of the 5-mm cell suspension. (b) The maximum local absorbance of a single living cell, (black line) averaged over 100 single live cells measurement assuming a chloroplast of average diameter 7.42 μm, and (red line) calculated from the absorbance of the cell suspension.
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pone.0128002.g006: Comparison between single cell absorbance calculated from cell suspension absorbance and that averaged over 100 single-cell measurements.(a) Black: absorbance of a cell suspension of 5-mm path length measured with an integrating sphere. Red: maximum local absorbance calculated from the absorbance of the 5-mm cell suspension. (b) The maximum local absorbance of a single living cell, (black line) averaged over 100 single live cells measurement assuming a chloroplast of average diameter 7.42 μm, and (red line) calculated from the absorbance of the cell suspension.

Mentions: It is remarkable that the peak local absorbance in a single living cell is sometimes close to unity as shown in Fig 5(b), well comparable with that of cell suspensions as shown in Fig 6(a) (black line). We measured the spatially resolved absorbance A(x, y, λ) of 100 single live cells from the suspension to obtain the maximum local absorbance of the single cell, averaged over 100 single live cells in Fig 6(b) (black line) [Appendix 2 and Supporting Information (S1 Text, S1–S3 Figs)].


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)

Comparison between single cell absorbance calculated from cell suspension absorbance and that averaged over 100 single-cell measurements.(a) Black: absorbance of a cell suspension of 5-mm path length measured with an integrating sphere. Red: maximum local absorbance calculated from the absorbance of the 5-mm cell suspension. (b) The maximum local absorbance of a single living cell, (black line) averaged over 100 single live cells measurement assuming a chloroplast of average diameter 7.42 μm, and (red line) calculated from the absorbance of the cell suspension.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128002.g006: Comparison between single cell absorbance calculated from cell suspension absorbance and that averaged over 100 single-cell measurements.(a) Black: absorbance of a cell suspension of 5-mm path length measured with an integrating sphere. Red: maximum local absorbance calculated from the absorbance of the 5-mm cell suspension. (b) The maximum local absorbance of a single living cell, (black line) averaged over 100 single live cells measurement assuming a chloroplast of average diameter 7.42 μm, and (red line) calculated from the absorbance of the cell suspension.
Mentions: It is remarkable that the peak local absorbance in a single living cell is sometimes close to unity as shown in Fig 5(b), well comparable with that of cell suspensions as shown in Fig 6(a) (black line). We measured the spatially resolved absorbance A(x, y, λ) of 100 single live cells from the suspension to obtain the maximum local absorbance of the single cell, averaged over 100 single live cells in Fig 6(b) (black line) [Appendix 2 and Supporting Information (S1 Text, S1–S3 Figs)].

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.