Figs 2(b) and 2(c) show the position dependent spectra of 0.10 = 680 nm: = 680 nm). suspension absorbance and that averaged over 100 single-cell measurements, the same as in Fig 6(b).(DOC) pone.0128002.s004.doc (445K) GUID:?1BE8D748-968F-46F3-9C0E-C9E56A60E537 S4 Fig: a: 0th order CCD image of the 1D fiber array around the slit. b: AZD-5069 1st order diffraction (spectrally dispersed) image of light from the 1D fiber array. c: Absorbance spectral image of the cell having 715 nm peak in Fig 3(c). d: The area enclosed by the red circle is usually magnified to show a rectangle unit constituting the image.(DOC) pone.0128002.s005.doc (439K) GUID:?DBAD8F52-8CC6-4C47-9B00-50CDD8C2EE89 Data Availability StatementAll relevant data AZD-5069 are within the paper and its Supporting Information files. Abstract Label-free, non-invasive, rapid absorbance spectral imaging was investigated to find a single 715-nm absorption peak was locally distributed within single cells. The formula to calculate the absorbance of cell AZD-5069 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 usually significantly underestimated when it is evaluated from the absorbance of the cell suspensions due to the package effect. Introduction Microalgae, photosynthetic unicellular organisms, are collecting global attention from their high potentials for resources of biofuel and food [1C4]. Precise knowledge of absorptive properties of them to sunlight is usually vitally important for seeking for efficient photoproduction of renewable energy from microalgae [5, 6]. 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 made up of the same average number density of pigments as homogeneous dispersion;the higher the absorption of the individual cells, the stronger the flattening. This nonlinearity results in the package effect [7, 8], which also can be seen as a reduction in the absorption of pigmented cells relative to the absorption of the same pigments in answer [9]. However, there has been no fully quantitative evaluation of absorbance of cell suspensions on the basis of absorbance of single cells. Detailed theoretical modeling Klf6 of light attenuation properties including scattering effects by phytoplanktonic cells was also 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. For early 1960s, there was a pioneering work on absorption spectroscopy of a single live cell [11], but afterward advances in dynamic live-cell imaging based on fluorescence confocal microscopy are so impressive and successful in medical and biological science while absorption imaging is not fully explored except for a few examples such as one of variations of hyperspectral approach [12, 13]. In this paper, we introduce a live-cell imaging method using absorption microspectroscopy. In addition to characterization of absorptive properties of cells, there are several reasons which necessitate absorbance spectral imaging of live algal cells: Firstly, plant cells have cell walls which make it difficult to introduce fluorescent labels into the cells. Secondly, presence of photosynthetic pigments, chlorophyll, which fluoresce red, prevent the use of red fluorescent labels. Third, what is most important, absorption spectra contain much more information than fluorescence spectra about the excited says of cellular organisms and pigments, because the latter usually give only the information of the lowest (calm) excited state. Fourth, there are no fluorescent labels (fluorophores) needed, which may affect biochemical properties of the cells, to realize a noninvasive measurement in the true sence of the word. Fluorescence imaging has, of course, fundamental advantages over absorbance imaging in that fluorescence from single molecules is usually detectable while absorbance of single molecules is usually hard to be detected because the former is usually background-free measurement while the latter suffers from noise of background light. Therefore variations based on single-molecule fluorescence such as PALM(photoactivated localization microscopy) [14] are advantages of fluorescence imaging. Materials and Methods The sample used was the green alga Dangeard NIES-2238 (IAM C-541), which is one of the model photosynthetic micro-organisms [15] and also attractive in view of its ability of hydrogen photoproduction [16, 17]. The absorbance spectra of cell suspensions were measured with an absorption spectrophotometer using an integrating sphere (SolidSpec-3700DUV, Shimadzu). All the measurements were performed at room temperature..