State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing , China

Transcription

1 2017 International Conference on Medicine Sciences and Bioengineering (ICMSB 2017) ISBN: Raman Imaging of the Distribution of Carotenoids in Haematococcus Pluvialis under High Concentration of Sodium Chloride Qing-Ran YANG a and Wei-ping QIAN b,* State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing , China a qr_yang@seu.edu.cn, b wpingqian@163.com *Corresponding author Keywords: Raman imaging, Multivariate analysis, Carotenoids distribution. Abstract. Carotenoids have important implications for maintaining normal cell activity, antioxidant and photosynthesis. Under some extremely hard conditions, the algae cells will produce a large number of carotenoids to resist the stress. The mechanism of algae cell emergency response has attracted a lot of attention. Here, Raman spectroscopy was applied to study the distribution of carotenoids in living algae cells under high concentration of sodium chloride. Multivariate analysis (MCR) was also used to resolve the corresponding Raman spectra of different carotenoids. By MCR analysis, we succeeded in distinguishing the distribution of different pigment cells. This method of analysis can be widely used in the study of mechanism of cellular stress response. Introduction Raman spectroscopy is a spectrum based on inelastic light scattering during which the frequency of the incident laser changed. Compared to other spectroscopic techniques, the Raman spectroscopy has been receiving more and more attention among academia due to its unique characteristics [1]. Firstly, Raman spectroscopy can obtain the fingerprints of the analyte to be analyzed. For the non - polar groups such as C-C, C=C, S-S, which have weak absorption of infrared, in the Raman spectrum can be a strong absorption band. Secondly, the interference of water molecules to Raman spectroscopy is negligible, so Raman spectroscopy can be widely used in the study of moisture-containing biological systems. In addition to that, Raman spectroscopy can provide fast, simple, repeatable, and more important, noninvasive qualitative quantitative analysis [2,3]. So far, Raman spectroscopy has been widely used in organic, inorganic, polymer, biological, environmental protection, agriculture, medicine and other fields, as one of the important analytical methods and means [4]. Carotenoids play extremely important roles in maintaining normal physiological activities and against stress conditions in algae [5]. Carotenoids can serve as a light-absorbing molecule to absorb light wavelength at nm, where chlorophyll has a weak absorption there [6,7]. Exception for that, in algae cell, carotenoids also participate in the synthesis of some protein complex. Considering carotenoids are particularly rich of long-polyene carbon chain structures, they are able to quench singlet oxygen and scavenge free radicals. Therefore, the study of the distribution of carotenoids is of great significance for the better understanding of the mechanism of cell s response to stress condition and antioxidant process [8, 9]. 267

2 Materials and Methods Culturing of Haematococcus Pluvialis The Haematococcus pluvialis was purchased from Institute of Hydrobiology, Chinese Academy of Sciences. Cells of green flagellate were cultivated in BG11 medium under 25 with a circle of 12 h light/12 h dark illumination. Confocal Raman Microscopy Spectrally resolved images of Haematococcus pluvialis were measured on a Horiba HR Evolution Raman microscope. All measurements were carried out under room temperature. 532 nm laser excitation was coupled to the microscope via a 0.55 NA, 60X objective. The laser power incident on the sample is less than 1 mw. Raman signal were collected through a 100 um pinhole and dispersed by a 300 l/mm grating. In order to avoid the loss of possible information, all spectra was collected with wavenumber ranging from cm-1.and the integration time is 2 s. All spectra was collected from the cell suspension directly taking out from the medium without any other extra processing steps. Quartz substrate instead of glass substrate was used in order to avoid possible interference. Data Processing and Analysis All acquired spectra was pre-calibrated using data processing tools built-in in the instrument to avoid possible interference and unrelated background signals. For Raman imaging, spectrum from each pixel were all pre-calibrated and then filtered for a wavenumber region that corresponding to specific substances within the cell. Result and Discussion Cultivation of Haematococcus Pluvialis Optical micrographs of Haematococcus pluvialis during different stages of growth are shown in Figure 1 Figure 1A shows the image of cells of green flagellate Figure. 1B shows the beginning of the accumulation of astaxanthin, from which we can see that the cell begin turn red. Figure 1C shows the start of the accumulation of astaxanthin in the cell after 24 h culturing under high salt condition, where the cell begins turn round. After incubating with high salt condition for 100 h, the Haematococcus pluvialis cells have deform into aplanospores and the cell become even lager. Figure 1. Optical micrographs of different stages of cells. (A) green flagellate (B) begin to accumulate astaxanthin (C) after incubation under high concentration of salt for 24 h (D) after incubation under high concentration of salt for 100 h. 268

3 Raman Spectra of in vivo Haematococcus Pluvialis Single-cell Raman spectra of Haematococcus pluvialis under different culture condition was obtained by confocal Raman microscopy as shown in Figure 2. From Raman spectra, many important substances such as carbohydrates, lipids, proteins, nucleic acids can be identified in corresponding to the characteristic peaks in the Raman spectrum. Typical peaks of carotenoid were found around 1520 cm -1,1157 cm -1,1106 cm -1, which is assigned to C=C stretching vibrations, C-C vibrations coupled to C-CH 3 stretches or C-H in-plane bending, and CH 3 stretching modes. [10,11,12] These unique specific Raman peaks can be used for the identification of carotenoid. Comparing the intensity of Raman peak at 1520 cm -1 in Figure 2A and Figure 2B, we can find that under some extremely hard conditions, Haematococcus pluvialis cells can quickly accumulate a large number of carotenoids, while under normal condition, the accumulation of carotene is slower. This corresponds well with the research down before, that under stress conditions, algae cells produce large amounts of carotenoids to resist damage caused by reactive oxygen species (ROS). Figure 2. (A) Raman spectra of Haematococcus pluvialis under high salt condition from 0 h to 100 h. (B) Raman spectra of cells under normal condition from 0 h to 100h. MCR Analysis and Raman Image of Haematococcus Pluvialis Cell Considering the similarity of β-carotene and astaxanthin in Raman spectra, it is hard to tell one from another using normal analysis method. As shown in Figure. 3A, the Raman spectra of carotene and astaxanthin have a large overlap, hence, the separation ofβ-carotene and astaxanthin from origin spectra is crucial for the study of the distribution of all kinds of carotenoids. Here, multivariate statistical analysis (MCR) was used. By the use of MCR, massive information of various components can be extracted from a large number of overlapping spectra. The distribution of different kinds of carotenoids is shown in Figure. 3B. With the prolongation of incubation time in high salt conditions, the total amount of astaxanthin has obvious increased while the amount of chlorophyll does not. This can be attributed to the slow photosynthesis of Haematococcus pluvialis cell owing to the high salt condition. Under this condition, the Haematococcus pluvialis cells have synthesis massive of astaxanthin. From another point of view, the distribution region of astaxanthin has become bigger and bigger with the increase of incubating time. This correspond well with the fact that astaxanthin plays and important role in antioxidant. 269

4 Figure 3. (A) Raman imaging of the distribution of astaxanthin, β-carotene and chlorophyll after 0 h, 24 h, 48 h, 96 h incubation with high concentration of Nacl. (B) The Raman spectrum of astaxanthin andβ-carotene. Summary In this manuscript, with the help of MCR algorithm, we are able to distinguish β- carotene and astaxanthin. And the distribution of different pigments in living cells under high salt condition was obtained via Raman imaging, which will help the better understanding of the mechanism of the stress response of algae cell. Raman spectroscopy provides a label-free method for detecting pigment distribution in living algae cells under high salt conditions. This method can be used widely in analysis of multicomponent, complex biological systems. Acknowledgement The authors gratefully acknowledge financial support from National key research and development program of China (2017YFA ), the National Nature Science Foundation of China ( ). References [1] Moskovits M. Surface-enhanced Raman spectroscopy: a brief retrospective [J]. Journal of Raman Spectroscopy, 2005, 36(6-7): [2] Schlücker S. Surface-Enhanced raman spectroscopy: Concepts and chemical applications [J]. Angewandte Chemie International Edition, 2014, 53(19): [3] Talari A C S, Movasaghi Z, Rehman S, et al. Raman spectroscopy of biological tissues[j]. Applied Spectroscopy Reviews, 2015, 50(1):

5 [4] Kong K, Kendall C, Stone N, et al. Raman spectroscopy for medical diagnostics From in-vitro biofluid assays to in-vivo cancer detection [J]. Advanced drug delivery reviews, 2015, 89: [5] Timlin J A, Collins A M, Beechem T A, et al. Localizing and Quantifying Carotenoids in Intact Cells and Tissues[M]//Carotenoids. InTech, [6] Reyes L H, Gomez J M, Kao K C. Improving carotenoids production in yeast via adaptive laboratory evolution[j]. Metabolic engineering, 2014, 21: [7] Vishwanathan R, Kuchan M J, Sen S, et al. Lutein and preterm infants with decreased concentrations of brain carotenoids [J]. Journal of pediatric gastroenterology and nutrition, 2014, 59(5): [8] Ibarrondo I, Prieto-Taboada N, Martínez-Arkarazo I, et al. Resonance Raman imaging as a tool to assess the atmospheric pollution level: carotenoids in Lecanoraceae lichens as bioindicators[j]. Environmental Science and Pollution Research, 2016, 23(7): [9] Dementjev A, Kostkevičiene J. Applying the method of Coherent Anti-stokes Raman microscopy for imaging of carotenoids in microalgae and cyanobacteria [J]. Journal of Raman Spectroscopy, 2013, 44(7): [10] Liu J, Huang Q. Screening of Astaxanthin-Hyperproducing Haematococcus pluvialis Using Fourier Transform Infrared (FT-IR) and Raman Microspectroscopy [J]. Applied spectroscopy, 2016, 70(10): [11] Liu J, Huang Q. Screening of Astaxanthin-Hyperproducing Haematococcus pluvialis Using Fourier Transform Infrared (FT-IR) and Raman Microspectroscopy [J]. Applied spectroscopy, 2016, 70(10): [12] Keenan MR, Kotula PG (2004) Accounting for Poisson noise in the multivariate analysis of ToF-SIMS spectrum images. Surface and Interface Analysis 36: