Combining Raman microscopy with optical tweezers makes it possible to analyze single, live, moving cells in medium. This new combined technique, called confocal laser tweezers Raman spectroscopy (LTRS), has been extensively used in studies of optically trapped chromosomes (Ojeda et al., 2006), spores (Huang et al., 2007), Escherichia coli cells (Chen et al., 2009), and mitochondria (Tang et al., 2007). Raman spectroscopy is extraordinarily sensitive to click here the detection of carotenoids, especially when using an excitation wavelength resulting in the resonance

Raman effect, most frequently that at 514.5 nm (Vitek et al., 2009). On the other hand, photodamage may occur for living cells when using the 514.5 nm wavelength for excitation (Snook et al., 2009). The use of a longer wavelength, such as near-infrared wavelength, can substantially decrease the photodamage effect (Ashkin et al., 1987). Raman spectroscopy

has been reported to detect carotenoids from intact plants (Baranski et al., 2005), human retina (Bernstein et al., 1998), and fungal pellet (Papaioannou et al., 2009). However, most of the investigations have been performed at the tissue level, and thus do not permit further understanding of the carotenoid accumulation process in unicellular microorganisms, such as R. glutinis. These single cell analysis techniques can help to get more information, which might be buried during bulk measurements. In this paper, we developed a method based on LTRS to carry out rapid,

real time measurements of the total carotenoids, as well as nucleic selleck compound acids and lipids inside single R. glutinis cells. The LTRS technique permits the capture of a single cell suspended in a solution in the focus of a near-infrared laser beam and the subsequent analysis of this cell using Raman spectroscopy, from which the levels of carotenoids can be determined from the intensity of the 1509 cm−1 band in Raman spectra. The strain of R. glutinis was kindly provided by Ms. Lianzhu Teng at Guangxi University. Single Idoxuridine colonies of R. glutinis from YPD plates (containing 10 g of yeast extract, 20 g of peptone, 20 g of dextrose, and 15 g of agar L−1) were inoculated into a liquid YPD medium (containing no agar) and incubated at 28 °C for 16 h to obtain the preculture. The preculture in exponential phase was used as the inoculum for 50 mL of carotenoid production medium. The production medium was composed of dextrose (40 g L−1), KH2PO4 (8 g L−1), MgSO4·7H2O (0.5 g L−1), and yeast extract (5 g L−1), with a final pH of 6.0. The inoculum was placed in a 250 mL shaking flask, shaken at 200 r.p.m., and incubated at 28 °C for 72 h. A 500-μL aliquot of cells was withdrawn at 4-h intervals to measure growth and collect Raman spectra. Details of the LTRS method have been published elsewhere (Xie et al., 2002, 2005).