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.
Highlights
Microalgae, photosynthetic unicellular organisms, are collecting global attention from their high potentials for resources of biofuel and food [1,2,3,4]
A spatially resolved absorbance image of a single living cell is shown in Fig 2(a) at a selected wavelength, λ = 680 nm, of the absorbance peak of chlorophyll a (Chl a) Q-band
One can see that Chl a is nonuniformly distributed within the cell [15]
Summary
Microalgae, photosynthetic unicellular organisms, are collecting global attention from their high potentials for resources of biofuel and food [1,2,3,4]. It is well known that a suspension of absorbing cells which contain densely packed pigments exhibit a flattened absorbance spectrum compared with that of a solution containing the same average number density of pigments as homogeneous dispersion;the higher the absorption of the individual cells, the stronger the flattening. Absorbance Spectral Imaging of Single Live Algal Cells nonlinearity results in the ‘package’ effect [7, 8], which can be seen as a reduction in the absorption of pigmented cells relative to the absorption of the same pigments in solution [9]. Detailed theoretical modeling of light attenuation properties including scattering effects by phytoplanktonic cells was previously presented [10], but single-cell absorbance is usually left for an unknown fitting parameter because of lack of a knowledge on detailed absorptive properties of single live cells
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