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

Takumi Isono; Kyohei Yamashita; Daisuke Momose; Hiroki Kobayashi; Masashi Kitamura; Yusuke Nishiyama; Takahiro Hosoya; Hiroaki Kanda; Ayane Kudo; Norihide Okada; Takafumi Yagi; Kazuaki Nakata; Shigeru Mineki; Eiji Tokunaga

doi:10.1371/journal.pone.0128002

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

PLoS One. 2015 Jun 10;10(6):e0128002. doi: 10.1371/journal.pone.0128002. eCollection 2015.

Affiliations

¹ Department of Physics, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan.
² Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba-ken 278-8510, Japan.
³ Department of Physics, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan; Research Center for Green and Safety Sciences, 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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Chlamydomonas reinhardtii / chemistry
Chlorophyll / chemistry
Chlorophyta / chemistry*
Microspectrophotometry / methods*
Suspensions / chemistry

Substances

Suspensions
Chlorophyll

Grants and funding

This work was funded by Tokyo University of Science and Research Center for Green and Safety Sciences, Tokyo University of Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.