Fig 1.
Experimental setup for absorbance spectral imaging of single cells.
Fig 2.
A(x,y,λ) obtained by the 2D-1D conversion method.
(a) Absorbance image of a single living cell at λ = 680 nm: A(x, y, λ = 680 nm). (b) Absorbance spectra of the cell at y = 5.7 μm from x = 0 to x = 4.0 μm: A(x, y = 5.7 μm, λ), integrated over the area of Δx × Δy = 0.10 μm × 0.60 μm for each spectrum (0.10 μm × 0.60 μm does not indicate spatial resolution but a division unit). (c) Absorbance spectra of the cell at y = 5.7 μm from x = 4.0 to x = 8.0 μm: A(x, y = 5.7 μm, λ).
Fig 3.
Absorbance spectra of 0.16 μm × 0.6 μm area (division unit) on the eyespot within two single live cells obtained by the 2D-1D conversion method.
The estimated absorbance spectra (red lines) of the eyespot are calculated by subtracting the average spectra (thick black lines) from the raw spectra (thin black lines). The average spectra within the cells are calculated by the method in Appendix 2[B]. Insets: The color photographs of the live cells, where the eyespot is located on the edge of the cell within the circle.
Fig 4.
Absorbance spectrum of the eyespot within a live cell (II).
Absorbance spectra of 0.10 μm × 0.6 μm area (division unit) within a single live cell obtained by the 2D-1D conversion method. (a) Absorbance spectra with and without an eyespot taken at the position over the red-yellow color region and over the adjacent green region, respectively, in the absorbance image of a single live cell at λ = 480 nm displayed in the inset. (b) Absorbance spectrum of the eyespot obtained as the difference spectrum between with and without the eyespot.
Fig 5.
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).
Fig 6.
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.
Fig 7.
Model for relating the single cell absorbance to the cell suspension absorbance.
Tsphere:transmittance of the chloroplast sphere as shown in the left. α:absorption coefficient within the chloroplast. d:diameter of the chloroplast sphere. nc: cell number density in the cell suspension. x:chloroplast coverage ratio in the layer of the thickness d Td:transmittance of the layer of thickness d in the cell suspension As:absorbance of the cell suspension L:thickness of the cell suspension Am:maximum local absorbance of a single cell for the light transmitted through the center of the cell
Fig 8.
Experimental data to estimate the spatial resolution of the system.
Absorbance at 480 nm around the eyespot in the inset in Fig 4 as a function of position. The FWHM of the absorbance image of the eyespot is about 1.5 μm.