Analyzing the cells in various body fluids can greatly deepen the understanding of the mechanisms governing the cellular physiology. Due to the variability of physiological and metabolic states, it is important to be able to perform such studies on individual cells. Therefore, we developed an optofluidic system in which we precisely manipulated and monitored individual cells of Escherichia coli. We tested optical micromanipulation in a microfluidic chamber chip by transferring individual bacteria into the chambers. We then subjected the cells in the chambers to antibiotic cefotaxime and we observed the changes by using time-lapse microscopy. Separately, we used laser tweezers Raman spectroscopy (LTRS) in a different micro-chamber chip to manipulate and analyze individual cefotaxime-treated E. coli cells. Additionally, we performed conventional Raman micro-spectroscopic measurements of E. coli cells in a micro-chamber. We found observable changes in the cellular morphology (cell elongation) and in Raman spectra, which were consistent with other recently published observations. The principal component analysis (PCA) of Raman data distinguished between the cefotaxime treated cells and control. We tested the capabilities of the optofluidic system and found it to be a reliable and versatile solution for this class of microbiological experiments.

Center for Sepsis Control and Care Jena University Hospital Am Klinikum 1 D 07747 Jena Germany

Institute of Physical Chemistry Friedrich Schiller University Jena Helmholtzweg 4 D 07743 Jena Germany

Institute of Scientific Instruments Czech Academy of Sciences v v i Kralovopolska 147 612 64 Brno Czech Republic

Leibniz Institute of Photonic Technology Albert Einstein Str 9 D 07745 Jena Germany

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Liberale C., Cojoc G., Bragheri F., Minzioni P., Perozziello G., La Rocca R., Ferrara L., Rajamanickam V., Di Fabrizio E., Cristiani I. Integrated microfluidic device for single-cell trapping and spectroscopy. Sci. Rep. 2013;3:1258. doi: 10.1038/srep01258. PubMed DOI PMC

Redding B., Schwab M.J., Pan Y. Review: Raman spectroscopy of optically trapped single biological micro-particles. Sensors. 2015;15:19021–19046. doi: 10.3390/s150819021. PubMed DOI PMC

Gilany K., Moazeni-Pourasil R.S., Jafarzadeh N., Savadi-Shiraz A. Metabolomics fingerprinting of the human seminal plasma of asthenozoospermic patients. Mol. Reprod. Dev. 2014;81:84–86. doi: 10.1002/mrd.22284. PubMed DOI

Wang S.Y., Hasty C.E., Watson P.A., Wicksted J.P., Stith R.D., March W.F. Analysis of metabolites in aqueous solutions by using laser Raman spectroscopy. Appl. Opt. 1993;32:925–929. doi: 10.1364/AO.32.000925. PubMed DOI

Rösch P., Harz M., Schmitt M., Peschke K.-D., Ronneberger O., Burkhardt H., Motzkus H.-W., Lankers M., Hofer S., Thiele H., et al. Chemotaxonomic identification of single bacteria by micro-Raman spectroscopy: Application to clean-room-relevant biological contaminations. Appl. Environ. Microbiol. 2005;71:1626–1637. doi: 10.1128/AEM.71.3.1626-1637.2005. PubMed DOI PMC

Willemse-Erix D.F.M., Scholtes-Timmerman M.J., Jachtenberg J.-W., van Leeuwen W.B., Horst-Kreft D., Schut T.C.B., Deurenberg R.H., Puppels G.J., van Belkum A., Vos M.C., et al. Optical fingerprinting in bacterial epidemiology: Raman spectroscopy as a real-time typing method. Clin. Microbiol. 2009;47:652–659. doi: 10.1128/JCM.01900-08. PubMed DOI PMC

Stöckel S., Kirchhoff J., Neugebauer U., Rösch P., Popp J. The application of Raman spectroscopy for the detection and identification of microorganisms. J. Raman Spectrosc. 2016;47:89–109. doi: 10.1002/jrs.4844. DOI

Xie C., Goodman C., Dinno M.A., Li Y.Q. Real-time Raman spectroscopy of optically trapped living cells and organelles. Opt. Express. 2004;25:6208–6214. doi: 10.1364/OPEX.12.006208. PubMed DOI

Xie C., Chen D., Li Y.-Q. Raman sorting and identification of single living micro-organisms with optical tweezers. Opt. Lett. 2005;30:1800–1802. doi: 10.1364/OL.30.001800. PubMed DOI

Neuman K.C., Chadd E.H., Liou G.F., Bergman K., Block S.M. Characterization of photodamage to Escherichia coli in optical traps. Biophys. J. 1999;77:2856–2863. doi: 10.1016/S0006-3495(99)77117-1. PubMed DOI PMC

Chan J.W., Winhold H., Corzett M.H., Ulloa J., Cosman M., Balhorn R., Huser T. Monitoring dynamic protein expression in living E. coli bacterial cells by laser tweezers Raman spectroscopy. Cytom. Part A. 2007;71A:468–474. doi: 10.1002/cyto.a.20407. PubMed DOI

Dai J., Hamon M., Jambovane S. Microfluidics for antibiotic susceptibility and toxicity testing. Bioengineering. 2016;3:25. doi: 10.3390/bioengineering3040025. PubMed DOI PMC

Assmann C., Kirchhoff J., Beleites C., Hey J., Kostudis S., Pfister W., Schlattmann P., Popp J., Neugebauer U. Identification of vancomycin interaction with Enterococcus faecalis within 30 min of interaction time using Raman spectroscopy. Anal. Bioanal. Chem. 2015;407:8343–8352. doi: 10.1007/s00216-015-8912-y. PubMed DOI

Schröder U., Kirchhoff J., Hübner U., Mayer G., Glaser U., Henkel T., Pfister W., Fritzsche W., Popp J., Neugebauer U. On-Chip spectroscopic assessment of microbial susceptibility to antibiotics within 3½ hours. J. Biophotonic. 2017;10:1547–1557. doi: 10.1002/jbio.201600316. PubMed DOI

Schröder U.-C., Beleites C., Assmann C., Glaser U., Hübner U., Pfister W., Fritzsche W., Popp J., Neugebauer U. Detection of vancomycin resistances in enterococci within 3½ hours. Sci. Rep. 2015;5:8271. doi: 10.1038/srep08217. PubMed DOI PMC

Kirchhoff J., Glaser U., Bohnert J.A., Pletz M., Popp J., Neugebauer U. Simple ciprofloxacin resistance test and determination of minimal inhibitory concentration (MIC) within two hours using Raman spectroscopy. Anal. Chem. 2018;90:1811–1818. doi: 10.1021/acs.analchem.7b03800. PubMed DOI

Schröder U.-C., Ramoji A., Glaser U., Sachse S., Leiterer C., Cszaki A., Huebner U., Fritzsche W., Pfister W., Bauer M., et al. Combined dielectrophoresis-Raman setup for the classification of pathogens recovered from the urinary tract. Anal. Chem. 2013;85:10717–10724. doi: 10.1021/ac4021616. PubMed DOI

Schröder U.-C., Bokeloh F., O’Sullivan M., Glaser U., Wolf K., Pfister W., Popp J., Ducreé J., Neugebauer U. Rapid, culture-independent, optical diagnostics of centrifugally captured bacteria from urine samples. Biomicrofluidics. 2015;9:044118. doi: 10.1063/1.4928070. PubMed DOI PMC

Pilát Z., Jonáš A., Ježek J., Zemánek P. Effects of infrared optical trapping on Saccharomyces cerevisiae in a microfluidic system. Sensors. 2017;17:2640. doi: 10.3390/s17112640. PubMed DOI PMC

Jonáš A., Pilát Z., Ježek J., Bernatová S., Fořt T., Zemánek P., Aas M., Kiraz A. Thermal tuning of spectral emission from optically trapped liquid-crystal droplet resonators. JOSA B. 2017;34:1855–1864. doi: 10.1364/JOSAB.34.001855. DOI

Samek O., Jonáš A., Pilát Z., Zemánek P., Nedbal L., Tříska J., Kotas P., Trtílek M. Raman Microspectroscopy of individual algal Cells: Sensing unsaturation of storage lipids in vivo. Sensors. 2010;10:8635–8651. doi: 10.3390/s100908635. PubMed DOI PMC

Pilát Z., Bernatová S., Ježek J., Šerý M., Samek O., Zemánek P., Nedbal L., Trtílek M. Raman microspectroscopy of algal lipid Bodies: Beta-carotene as a sensor. SPIE Proc. 2011;8306:83060L. doi: 10.1117/12.912264. DOI

Pilát Z., Bernatová S., Ježek J., Šerý M., Samek O., Zemánek P., Nedbal L., Trtílek M. Raman microspectroscopy of algal lipid Bodies: Beta-carotene quantification. J. Appl. Phycol. 2012;24:541–546. doi: 10.1007/s10811-011-9754-4. DOI

Bernatová S., Samek O., Pilát Z., Šerý M., Ježek J., Jákl P., Šiler M., Krzyžánek V., Zemánek P., Holá V., et al. Following the mechanisms of bacteriostatic versus bactericidal action using Raman spectroscopy. Molecules. 2013;18:13188–13199. doi: 10.3390/molecules181113188. PubMed DOI PMC

Samek O., Mlynáriková K., Bernatová S., Ježek J., Krzyžánek V., Šiler M., Zemánek P., Růžička F., Holá V., Mahelová M. Candida parapsilosis biofilm identification by Raman spectroscopy. Int. J. Mol. Sci. 2014;15:23924–23935. doi: 10.3390/ijms151223924. PubMed DOI PMC

Samek O., Bernatová S., Ježek J., Šiler M., Šerý M., Krzyžánek V., Hrubanová K., Zemánek P., Holá V., Růžička F. Identification of individual biofilm-forming bacterial cells using Raman tweezers. J. Biomed. Opt. 2015;20:051038. doi: 10.1117/1.JBO.20.5.051038. PubMed DOI

Mlynáriková K., Samek O., Bernatová S., Růžička F., Ježek J., Hároniková A., Šiler M., Zemánek P., Holá V. Influence of culture media on microbial fingerprints using Raman spectroscopy. Sensors. 2015;15:29635–29647. doi: 10.3390/s151129635. PubMed DOI PMC

Petrov D.V. Raman spectroscopy of optically trapped particles. J. Opt. A Pure Appl. Opt. 2007;9:139–156. doi: 10.1088/1464-4258/9/8/S06. DOI

Jonáš A., Zemánek P. Light at work: The use of optical forces for particle manipulation, sorting, and analysis. Electrophoresis. 2008;29:4813–4851. doi: 10.1002/elps.200800484. PubMed DOI

Xia Y., Whitesides G.M. Soft lithography. Ann. Rev. Mater. Sci. 1998;28:153–184. doi: 10.1146/annurev.matsci.28.1.153. DOI

Pilát Z., Ježek J., Šerý M., Trtílek M., Nedbal L., Zemánek P. Optical trapping of microalgae at 735–1064 nm: Photodamage assessment. J. Photochem. Photobiol. B Biol. 2013;121:27–31. doi: 10.1016/j.jphotobiol.2013.02.006. PubMed DOI

Brandt N.N., Brovko O.O., Chikishev A.Y., Paraschuk O.D. Optimization of the Rolling-Circle Filter for Raman Background Subtraction. Appl. Spectrosc. 2006;60:288–293. doi: 10.1366/000370206776342553. PubMed DOI

Choi J., Yoo J., Lee M., Kim E.-G., Lee J.S., Lee S., Joo S., Song S.H., Kim E.-C., Lee J.C., et al. A rapid antimicrobial susceptibility test based on single-cell morphological analysis. Sci. Transl. Med. 2014;6:267ra174. doi: 10.1126/scitranslmed.3009650. PubMed DOI

Long D.A. The Raman Effect: A Unified Treatment of the Theory of Raman Scattering by Molecules. John Wiley & Sons Ltd.; Chichester, UK: 2002. pp. 21–22. ISBNs 0-471-49028-8 (Hardback), 0-470-84576-7 (Electronic)

Harz M., Rosch P., Peschke K.-D., Ronneberger O., Burkhardt H., Popp J. Micro-Raman spectroscopic identification of bacterial cells of the genus Staphylococcus and dependence on their cultivation conditions. Analyst. 2005;130:1543–1550. doi: 10.1039/b507715j. PubMed DOI

Dutta R.K., Sharma P.K., Pandey A.C. Surface enhanced Raman spectra of Escherichia coli cells using ZnO nanoparticles. Dig. J. Nanomater. Biostruct. 2009;4:83–87.

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