RAMAN Imaging & Microplastics Analysis Facility

Technology that Measures Cell-by-Cell Variation in Growth Rates Could Impact Many Fields
STONY BROOK, NY, November 12, 2021 –The genomic revolution has enabled researchers to assess cell-by-cell genetic variations, but very few techniques exist to measure cell-by-cell metabolic variations, a more powerful way to understand cell responses to changing environmental conditions. Researchers from Stony Brook University’s School of Marine and Atmospheric Sciences (SoMAS), led by Gordon T. Taylor, PhD, demonstrated that Raman microspectroscopy can accurately measure cell-by-cell variations in growth rates of the bacterium E. coli grown in a broth medium. They validated the Raman-based technique against independent traditional population-based spectroscopic and mass spectrometric measurements.

“The technique emerging from our laboratory can be applied to the study of free-living and host-associated microbiomes, which could prove crucial in understanding more about their functional responses to stressors,” says Taylor, Professor and Director of the NAno Raman Molecular Laboratory (NARMIL) at SoMAS. “We also believe this is an enabling technology to examine individuality in cell populations and could have broad applications in microbiology, cell biology and biomedicine.”

Details of the technique and results are published in the American Society of Microbiology’s Applied and Environmental Microbiology (PDF). A visual of the technique is also highlighted on the cover of the journal edition.

Since our inception in 2014, we have
1) Developed protocols to interrogate single microbial cells and marine aerosol particles in the confocal Raman microspectrometer.

2) Produced sub-micrometer scale Raman maps of cells and engineered materials on the Atomic Force Microscope (AFM) stage by co-locating the Raman laser beam on the AFM tip.

3) Developed software scripts, pipelines and shortcuts to optimize routine Raman spectral analyses.

4) With Prof. Taylor’s group (SoMAS), refined techniques to map distributions of intracellular storage products, such as globular sulfur and polyhydroxy-butyrate (Fig. 1).

Figure 1. Raman map of intracellular distributions of the lipid-like energy storage product, polyhydroxybutyrate (red area with accompanying Raman spectrum) within a marine bacterium (below arrow in right panel). Blue area is relatively rich in amino acids (blue spectrum) and proteins. Cell is approximately 2.5 micrometers long.

5) For ongoing studies in Prof. Taylor’s lab (SoMAS), NARMIL improved capabilities for routine Raman-FISH single cell analyses. Technique enables recognition of photosynthetically-active phytoplankton and calculating single-cell growth rates by quantifying stable isotope incorporation into biomolecules by Raman signatures (Fig. 2) and identifying players by genetic probes (FISH = fluorescent in situ hybridization). This work is published in Frontiers in Microbiology and summarized in a recently presented poster

Figure 2. Examples of varying contributions of ¹²C-¹²C, ¹²C-¹³C, and ¹³C-¹³C isotopologues to shape, position, and areas of the n (C-C) and n (C=C) Raman spectral peaks for carotenoids of Synechococcus sp. cells assimilating varying amounts of DI¹³C. Raman spectra were obtained from individual cells grown in either 1.1% (A) or 50% (B) or 94% 13C-bicarbonate for 9 days. Spectra were baseline corrected, intensity normalized (0-1), and subjected to a full Voigt curve-fitting routine (convolution of Lorentzian and Gaussian profiles) with 5,000 iterations or a 0.00001 tolerance using Renishaw™ Wire 4.1® software.

6) Currently assisting Prof. Taylor’s group (SoMAS) in development of novel Raman and AFM techniques to follow movement of carbon from prey (Fig. 3) through protistan symbiotic associations, between hosts and viral pathogens, and from detritus through microbial communities. This new project is supported by the Gordon & Betty Moore Foundation and funds two new postdoctoral investigators.

Figure 3. Raman spectra from single heterotrophic bacterial cells grown in organic media with either natural ¹³C abundances (blue spectrum) or highly ¹³C–enriched substrates (red spectrum). Peaks experiencing significant “red-shifts” due to heavy isotope enrichment are labeled.

7) Facilitated Profs. Knopf and J. Aller group’s (SoMAS) exploration of chemical variability in sea spray aerosols and their role in cloud formation using Raman spectroscopy (Fig. 4).

Figure 4. Raman spectrum of a single aqueous sea spray droplet (marked by red arrow in panel on right) that contains biogenic polysaccharidic and proteinaceous material obtained from a laboratory mesocosm experiment.

8) Assisted Ms. Yoonja Kang, Prof. Gobler’s student (SoMAS), to spectroscopically characterize how cellular storage products and pigment concentrations change when a harmful bloom-forming microalga (Aureoumbra lagunensis) enters a resting stage (Fig. 5).

Figure 5. Examples of single-cell Raman spectra (left) acquired from dried resting A. lagunensis cells (red) and vegetative (blue) cells under ambient laboratory conditions. Light microscope and TEM images (right). (A) light microscope image of vegetative cells at 21ºC, (B) light microscope image of resting cells formed at 35°C and enlarged image of Aureoumbra resting cell (inset), (C) TEM image of vegetative cell, (D) TEM image of resting cell. Abbreviations are C = cytoplasm, Sd = sterol-enriched droplet, Ga = Golgi apparatus, M = mitochondria, P = plastid, Pg = plastoglobuli, and Tk = thylakoids (Kang et al. 2016 J. Phycol.).

Citations published with NARMIL data:
1) Kang Soo Lee, Zachary Landry, Fátima C. Pereira, Michael Wagner, David Berry, Wei E. Huang, Gordon T. Taylor, Janina Kneipp, Juergen Popp, Meng Zhang, Ji- Xin Cheng and Roman Stocker. Raman microspectroscopy for microbiology PDF.
2) Elena Yakubovskaya, Tatiana Zaliznyak, Joaquín Martínez Martínez and Gordon T. Taylor. Raman Microspectroscopy Goes Viral: Infection Dynamics in the Cosmopolitan Microalga, Emiliania huxleyi PDF.
3) Felix Weber, Tatiana Zaliznyak, Virginia P. Edgcomb, Gordon T. Taylor. Using Stable Isotope Probing and Raman Microspectroscopy To Measure Growth Rates of Heterotrophic Bacteria PDF.
4) Luis E. Medina Faull, Tatiana Zaliznyak, Gordon T. Taylor. Assessing diversity, abundance, and mass of microplastics (~ 1–300 μm) in aquatic systems PDF.
5) Elena Yakubovskaya, Tatiana Zaliznyak, Joaquin Martínez Martínez & Gordon T. Taylor. Tear Down the fluorescent curtain: A new fluorescence Suppression Method for Raman Microspectroscopic Analyses. Scientific Reports, 9, 15785 PDF.
6) Gordon T. Taylor, Elizabeth A. Suter, Zhuo Q. Li, Stephanie Chow, Dallyce Stinton, Tatiana Zaliznyak, and Steven R. Beaupré (2017). Single-Cell Growth Rates in Photoautotrophic Populations Measured by Stable Isotope Probing and Resonance Raman Microspectrometry. Frontiers in Microbiology 8(1449) PDF.
7) Kang Y, Tang Y-Z, Taylor GT, Gobler CJ. (2017). Discovery of a resting stage in the harmful, brown tide-causing pelagophyte, Aureoumbra lagunensis: a mechanism facilitating recurring blooms and recent expansion? Journal of Phycology 53(1): 118-130 PDF.
8) Li L, Liu H, Wang L, Yue S, Tong X, Zaliznyak T, Taylor, G, Wong, SS. (2016). Chemical strategies for enhancing activity and charge transfer in ultrathin Pt nanowires immobilized onto nanotube supports for the oxygen reduction reaction. ACS Applied Materials & Interfaces 8: 34280−34294 PDF.
9) Han, J., McBean, C., Wang, L., Hoy, J., Jaye, C., Liu, H., … & Wong, S. S. (2015). Probing structure-induced optical behavior in a new class of self-activated luminescent 0D/1D CaWO4 metal oxide–CdSe nanocrystal composite heterostructures. Chemistry of Materials, 27(3), 778-792 PDF.
10) Wang, L., Han, J., Zhu, Y., Zhou, R., Jaye, C., Liu, H.,…& Wong. S. S. (2015). Probing the Dependence of Electron Transfer on Size and Coverage in Carbon Nanotube−Quantum Dot Heterostructures J. Phys. Chem. C, 119, 26327−26338 PDF.