Real-time Measurement of Single Bacterium's Refractive Index Using Optofluidic Immersion Refractometry

Detection of extant microbial life on Earth and elsewhere in the Solar System requires the ability to identify and enumerate micrometer-scale, essentially featureless cells. On Earth, bacteria are usually enumerated by culture plating or epifluorescence microscopy. Culture plates require long incubation times and can only count culturable strains, and epifluorescence microscopy requires extensive staining and concentration of the sample and instrumentation that is not readily miniaturized for space. Digital holographic microscopy (DHM) represents an alternative technique with no moving parts and higher throughput than traditional microscopy, making it potentially useful in space for detection of extant microorganisms provided that sufficient numbers of cells can be collected. Because sample collection is expected to be the limiting factor for space missions, especially to outer planets, it is important to quantify the limits of detection of any proposed technique for extant life detection. Here we use both laboratory and field samples to measure the limits of detection of an off-axis digital holographic microscope (DHM). A statistical model is used to estimate any instrument's probability of detection at various bacterial concentrations based on the optical performance characteristics of the instrument, as well as estimate the confidence interval of detection. This statistical model agrees well with the limit of detection of 103 cells/mL that was found experimentally with laboratory samples. In environmental samples, active cells were immediately evident at concentrations of 104 cells/mL. Published estimates of cell densities for Enceladus plumes yield up to 104 cells/mL, which are well within the off-axis DHM's limits of detection to confidence intervals greater than or equal to 95%, assuming sufficient sample volumes can be collected. The quantitative phase imaging provided by DHM allowed minerals to be distinguished from cells. Off-axis DHM's ability for rapid low-level bacterial detection and counting shows its viability as a technique for detection of extant microbial life provided that the cells can be captured intact and delivered to the sample chamber in a sufficient volume of liquid for imaging. Key Words: In situ life detection—Extant microorganisms—Holographic microscopy—Ocean Worlds—Enceladus—Imaging. Astrobiology 17, 913–925.

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“…6c ). This is in excellent agreement with values obtained for bacteria when using other methods (Balaev et al, 2002 ; Liu et al, 2014 ).…”

Section: Results: Environmental Samplessupporting

confidence: 91%

Digital Holographic Microscopy, a Method for Detection of Microorganisms in Plume Samples from Enceladus and Other Icy Worlds

2017

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“…Since there is little difference of the refractive index of yeast cells (1.36–1.40 [ 16 ]) compared to Bacillus (1.45 [ 17 ]), this correlation is more likely determined by the shape and size of the cells. The assumption of a dependency of backward scattered light on cell morphology is supported by the drift in the calibration of K. pastoris .…”

Section: Resultsmentioning

confidence: 99%

Application of an Online-Biomass Sensor in an Optical Multisensory Platform Prototype for Growth Monitoring of Biotechnical Relevant Microorganism and Cell Lines in Single-Use Shake Flasks

Ude

Schmidt-Hager

Findeis

et al. 2014

Sensors

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In the context of this work we evaluated a multisensory, noninvasive prototype platform for shake flask cultivations by monitoring three basic parameters (pH, pO2 and biomass). The focus lies on the evaluation of the biomass sensor based on backward light scattering. The application spectrum was expanded to four new organisms in addition to E. coli K12 and S. cerevisiae [1]. It could be shown that the sensor is appropriate for a wide range of standard microorganisms, e.g., L. zeae, K. pastoris, A. niger and CHO-K1. The biomass sensor signal could successfully be correlated and calibrated with well-known measurement methods like OD600, cell dry weight (CDW) and cell concentration. Logarithmic and Bleasdale-Nelder derived functions were adequate for data fitting. Measurements at low cell concentrations proved to be critical in terms of a high signal to noise ratio, but the integration of a custom made light shade in the shake flask improved these measurements significantly. This sensor based measurement method has a high potential to initiate a new generation of online bioprocess monitoring. Metabolic studies will particularly benefit from the multisensory data acquisition. The sensor is already used in labscale experiments for shake flask cultivations.

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“…Simulation environment was in water (n water = 1.33). The refractive index of C. jejuni was estimated from that of Escherichia coli (n=1.388) 52 . Refractive index of Au is derived from Johnson and Christy (LUMERICAL database) 53 …”

Section: Methodsmentioning

confidence: 99%

Plasmonic nanoparticles assemblies templated by helical bacteria and resulting optical activity

et al. 2020

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Plasmonic nanoparticles (NPs) adsorbing onto helical bacteria can lead to formation of NP helicoids with micron scale pitch. Associated chiroptical effects can be utilized as bioanalytical tool for bacterial detection and better understanding of the spectral behavior of helical self‐assembled structures with different scales. Here, we report that enantiomerically pure helices with micron scale of chirality can be assembled on Campylobacter jejuni, a helical bacterium known for severe stomach infections. These organisms have right‐handed helical shapes with a pitch of 1–2 microns and can serve as versatile templates for a variety of NPs. The bacteria itself shows no observable rotatory activity in the visible, red, and near‐IR ranges of electromagnetic spectrum. The bacterial dispersion acquires chiroptical activity at 500–750 nm upon plasmonic functionalization with Au NPs. Finite‐difference time‐domain simulations confirmed the attribution of the chiroptical activity to the helical assembly of gold nanoparticles. The position of the circular dichroism peaks observed for these chiral structures overlaps with those obtained before for Au NPs and their constructs with molecular and nanoscale chirality. This work provides an experimental and computational pathway to utilize chiroplasmonic particles assembled on bacteria for bioanalytical purposes.

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Real-time Measurement of Single Bacterium's Refractive Index Using Optofluidic Immersion Refractometry

Cited by 45 publications

References 7 publications

Digital Holographic Microscopy, a Method for Detection of Microorganisms in Plume Samples from Enceladus and Other Icy Worlds

Digital Holographic Microscopy, a Method for Detection of Microorganisms in Plume Samples from Enceladus and Other Icy Worlds

Application of an Online-Biomass Sensor in an Optical Multisensory Platform Prototype for Growth Monitoring of Biotechnical Relevant Microorganism and Cell Lines in Single-Use Shake Flasks

Plasmonic nanoparticles assemblies templated by helical bacteria and resulting optical activity

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