30) to form a single-beam optical trap. A R. glutinis cell in the phosphate-buffered
saline (PBS) was trapped about 10 μm above the bottom of cover slip with a gradient force generated by the focused beam. The same laser beam was used to excite Raman scattering from molecules inside the trapped cell. The spectrum was obtained by a liquid-nitrogen-cooled charge-coupled detector. The spectral resolution of our Raman system was about 6 cm−1. The Raman measurement of an individual cell was performed with a 10-s exposure time and 30 mW excitation power. The Raman spectra of 100 cells were collected for each time point. The PBS background spectrum was recorded with the same acquisition condition without the trapped cells and subtracted from the Ion Channel Ligand Library ic50 spectra of individual cells. The subtracted spectra were then smoothed using the Adjacent-Averaging filter method. Preprocessing of spectral data was performed using matlab 7.0 software. The total carotenoid level in an individual cell was estimated from the peak intensity at 1509 cm−1 in its Raman spectrum. β-Carotene standard (purchased from Sigma-Aldrich) was dissolved in chloroform and diluted into a series of concentrations: 62.5, 125, 187.5, 250, 312.5, 375, 437.5, and 500 mg L−1. For each measurement, a 150-μL aliquot of β-carotene solution was added to the sealed holder and its
Raman spectrum was acquired with the same experimental parameters used for determining the cell spectra. The Raman spectrum of the pure chloroform was taken as background and subtracted from the above-mentioned spectra. A standard curve Selleck Ku 0059436 for carotenoid
quantification was linearly fitted by correlating the β-carotene concentration tetracosactide with the peak intensity at 1518 cm−1 in its Raman spectrum. Carotenoids are a family of isoprenoids containing a characteristic polyene chain of conjugated double bonds. In R. glutinis cells, carotenoid pigments predominantly consist of β-carotene, torulene, and torularhodin (Sakaki et al., 2002). In this work, the Raman spectra of R. glutinis cells cultivated for 12 and 32 h, as well as the pure β-carotene standard were acquired in order to verify the existence of carotenoids in the investigated stain (Fig. 1). The three fundamental carotenoid bands at 1505–1520 cm−1 assigned to C=C (ν1) in-phase stretching, 1156 cm−1 assigned to C–C (ν2) stretching and 1005 cm−1 assigned to δ(C=CH) in-plane rocking modes of CH3 groups were clearly visible in all of the spectra. Thus, to a high degree of certainty, these peaks resulted from carotenoid compounds. The intensity of these peaks for R. glutinis cells cultivated for 32 h was more than 30 times higher than those for cells cultivated for just 12 h. It is noteworthy that the C=C (ν1) peak was at 1509 cm−1 for carotenoids present in cells, while it was at 1518 cm−1 for the β-carotene standard. This difference may be attributed to the fact that carotenoids usually bind to proteins or lipids in R.
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