Label-free non-invasive quantitative measurement of lipid contents in individual microalgal cells using refractive index tomography

Microalgae are promising candidates for biofuel production due to their high lipid content. To facilitate utilization of the microalgae for biofuel, rapid quantification of the lipid contents in microalgae is necessary. However, conventional methods based on the chemical extraction of lipids require a time-consuming destructive extraction process. Here, we demonstrate label-free, non-invasive, rapid quantification of the lipid contents in individual micro-algal cells measuring the three-dimensional refractive index tomograms. We measure three-dimensional refractive index distributions within Nannochloropsis oculata cells and find that lipid droplets are identifiable in tomograms by their high refractive index. In addition, we alter N. oculata under nitrogen deficiency by measuring the volume, lipid weight, and dry cell weight of individual cells. Characterization of individual cells allows correlative analysis between the lipid content and size of individual cells.

[1]  J. Zeikus Chemical and fuel production by anaerobic bacteria. , 1980, Annual review of microbiology.

[2]  YongKeun Park,et al.  Simultaneous 3D visualization and position tracking of optically trapped particles using optical diffraction tomography , 2015 .

[3]  Wei Chen,et al.  A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. , 2009, Journal of microbiological methods.

[4]  T. Tornabene,et al.  TOTAL LIPID PRODUCTION OF THE GREEN ALGA NANNOCHLOROPSIS SP. QII UNDER DIFFERENT NITROGEN REGIMES 1 , 1987 .

[5]  Im,et al.  Correlative three-dimensional fluorescence and refractive index tomography : bridging the gap between molecular specificity and quantitative bioimaging , 2017 .

[6]  D. Nelson,et al.  An integrative Raman microscopy-based workflow for rapid in situ analysis of microalgal lipid bodies , 2015, Biotechnology for Biofuels.

[7]  J. Benemann,et al.  Look Back at the U.S. Department of Energy's Aquatic Species Program: Biodiesel from Algae; Close-Out Report , 1998 .

[8]  Hanwool Park,et al.  Specific light uptake rates can enhance astaxanthin productivity in Haematococcus lacustris , 2016, Bioprocess and Biosystems Engineering.

[9]  Gabriel Popescu,et al.  Optical imaging of cell mass and growth dynamics. , 2008, American journal of physiology. Cell physiology.

[10]  D. Pimentel,et al.  Ethanol Production Using Corn, Switchgrass, and Wood; Biodiesel Production Using Soybean and Sunflower , 2005 .

[11]  YongKeun Park,et al.  High-resolution three-dimensional imaging of red blood cells parasitized by Plasmodium falciparum and in situ hemozoin crystals using optical diffraction tomography , 2013, Journal of biomedical optics.

[12]  L. Rodolfi,et al.  Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low‐cost photobioreactor , 2009, Biotechnology and bioengineering.

[13]  R. Keith,et al.  A Handbook , 2006 .

[14]  E. Wolf Three-dimensional structure determination of semi-transparent objects from holographic data , 1969 .

[15]  Philip Owende,et al.  Biofuels from microalgae—A review of technologies for production, processing, and extractions of biofuels and co-products , 2010 .

[16]  Jeffrey M. Gordon,et al.  Ultrahigh bioproductivity from algae , 2007, Applied Microbiology and Biotechnology.

[17]  Kyoohyun Kim,et al.  Three-dimensional label-free imaging and quantification of lipid droplets in live hepatocytes , 2016, Scientific Reports.

[18]  YongKeun Park,et al.  Quantitative phase imaging unit. , 2014, Optics letters.

[19]  T. Huser,et al.  Label-free in vivo analysis of intracellular lipid droplets in the oleaginous microalga Monoraphidium neglectum by coherent Raman scattering microscopy , 2016, Scientific Reports.

[20]  YongKeun Park,et al.  Hyperspectral optical diffraction tomography. , 2016, Optics express.

[21]  Teresa M. Mata,et al.  Microalgae for biodiesel production and other applications: A review , 2010 .

[22]  YongKeun Park,et al.  Biomedical applications of holographic microspectroscopy [invited]. , 2014, Applied optics.

[23]  D. Firestone Physical and chemical characteristics of oils, fats, and waxes , 2013 .

[24]  YongKeun Park,et al.  Refractive index tomograms and dynamic membrane fluctuations of red blood cells from patients with diabetes mellitus , 2016, Scientific Reports.

[25]  S. D. Babacan,et al.  White-light diffraction tomography of unlabelled live cells , 2014, Nature Photonics.

[26]  Pasquale Memmolo,et al.  Tomographic flow cytometry by digital holography , 2016, Light: Science & Applications.

[27]  Yao-Xiong Huang,et al.  Dependence of Refractive Index on Concentration and Temperature in Electrolyte Solution, Polar Solution, Nonpolar Solution, and Protein Solution , 2015 .

[28]  YongKeun Park,et al.  Profiling individual human red blood cells using common-path diffraction optical tomography , 2014, Scientific Reports.

[29]  Y. Li-Beisson,et al.  Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves , 2011, BMC biotechnology.

[30]  Mor Habaza,et al.  Tomographic phase microscopy with 180° rotation of live cells in suspension by holographic optical tweezers. , 2015, Optics letters.

[31]  R. Barer,et al.  Refractometry of Living Cells , 1952, Nature.

[32]  Youngchan Kim,et al.  Common-path diffraction optical tomography for investigation of three-dimensional structures and dynamics of biological cells. , 2014, Optics express.

[33]  Kyoohyun Kim,et al.  Label-free characterization of white blood cells by measuring 3D refractive index maps. , 2015, Biomedical optics express.

[34]  Kyoohyun Kim,et al.  High-Resolution 3-D Refractive Index Tomography and 2-D Synthetic Aperture Imaging of Live Phytoplankton , 2014 .

[35]  Björn Kemper,et al.  Tomographic phase microscopy of living three-dimensional cell cultures , 2014, Journal of biomedical optics.

[36]  Jaeduck Jang,et al.  Polarization holographic microscopy for extracting spatio-temporally resolved Jones matrix. , 2012, Optics express.

[37]  W. Yunus,et al.  Refractive index of solutions at high concentrations. , 1988, Applied optics.

[38]  Francisco E. Robles,et al.  Optical Spectroscopy of Biological Cells , 2012 .

[39]  C. Lan,et al.  Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans , 2008, Applied Microbiology and Biotechnology.

[40]  M. Takeda,et al.  Fourier-transform method of fringe-pattern analysis for computer-based topography and interferometry , 1982 .

[41]  Jonghee Yoon,et al.  A Bacteria‐Based Remotely Tunable Photonic Device , 2017 .

[42]  Jochen Guck,et al.  Bacterial infection of macrophages induces decrease in refractive index , 2013, Journal of biophotonics.

[43]  R. Barer Determination of Dry Mass, Thickness, Solid and Water Concentration in Living Cells , 1953, Nature.

[44]  Mladen Bošnjaković,et al.  Biodiesel from algae , 2013 .

[45]  Barry R. Masters,et al.  Quantitative Phase Imaging of Cells and Tissues , 2012 .

[46]  Kyoohyun Kim,et al.  Optical diffraction tomography techniques for the study of cell pathophysiology , 2016, 1603.00592.

[47]  Michael Hannon,et al.  Biofuels from algae: challenges and potential , 2010, Biofuels.

[48]  Jong Chul Ye,et al.  Comparative study of iterative reconstruction algorithms for missing cone problems in optical diffraction tomography. , 2015, Optics express.

[49]  S. Wani,et al.  Biology and genetic improvement of Jatropha curcas L.: A review , 2010 .

[50]  YongKeun Park,et al.  Real-time quantitative phase imaging with a spatial phase-shifting algorithm. , 2011, Optics letters.

[51]  Rob Lee,et al.  Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli , 2013, Proceedings of the National Academy of Sciences.

[52]  Ya. E. Sergeeva,et al.  Lipids of filamentous fungi as a material for producing biodiesel fuel , 2008, Applied Biochemistry and Microbiology.

[53]  C. Fang-Yen,et al.  Optical diffraction tomography for high resolution live cell imaging. , 2009, Optics express.

[54]  P. Marquet,et al.  Marker-free phase nanoscopy , 2013, Nature Photonics.

[55]  J. Obbard,et al.  Improved Nile Red staining of Nannochloropsis sp. , 2011, Journal of Applied Phycology.

[56]  YoungJu Jo,et al.  Quantitative Phase Imaging Techniques for the Study of Cell Pathophysiology: From Principles to Applications , 2013, Sensors.

[57]  Jonghee Yoon,et al.  Optical diffraction tomography using a digital micromirror device for stable measurements of 4D refractive index tomography of cells , 2016, SPIE BiOS.

[58]  Annika Enejder,et al.  Imaging of Lipids in Microalgae with Coherent Anti-Stokes Raman Scattering Microscopy1[OPEN] , 2015, Plant Physiology.

[59]  Jong Chul Ye,et al.  Real-time Visualization of 3-d Dynamic Microscopic Objects Using Optical Diffraction Tomography References and Links , 2022 .

[60]  D. Nikogosyan,et al.  Properties of Optical and Laser-Related Materials: A Handbook , 1997 .

[61]  YongKeun Park,et al.  Holotomography: refractive index as an intrinsic imaging contrast for 3-D label-free live cell imaging , 2017, bioRxiv.

[62]  Byung-Kwan Cho,et al.  Elucidation of the growth delimitation of Dunaliella tertiolecta under nitrogen stress by integrating transcriptome and peptidome analysis. , 2015, Bioresource technology.

[63]  Robert C. Wolpert,et al.  A Review of the , 1985 .

[64]  S. Godtfredsen,et al.  Ullmann ' s Encyclopedia of Industrial Chemistry , 2017 .

[65]  Jean-Paul Cadoret,et al.  The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae , 2015, Biotechnology for Biofuels.