&lt;title&gt;Refractive index of &lt;emph type="1"&gt;escherichia coli&lt;/emph&gt; cells&lt;/title&gt;

Balaev, Aleksei E.; Dvoretski, K. N.; Doubrovski, V. A.

doi:10.1117/12.475627

Cited by 25 publications

(6 citation statements)

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“…Furthermore, mCherryTYG is quite pH selective, and it is not affected by differences in monovalent salts, divalent salts, or redox (Figure S2). Alternatively, the overall decrease in lifetime is consistent with the higher index of refraction of cellular environments, which have been measured to cause lifetime shifts of up to ∼0.5 ns compared to dilute protein solutions. , Notably, in live cells, the mCherryTYG fluorescence is only partially quenched at pH 5.5, and fluorescence is still easily detectable for lifetime measurements. Thus, mCherryTYG is a well-behaved pH sensor that retains its p K a in live cells with an exceedingly large fluorescence lifetime dynamic range.…”

Section: Resultssupporting

confidence: 63%

Quantifying Acute Fuel and Respiration Dependent pH Homeostasis in Live Cells Using the mCherryTYG Mutant as a Fluorescence Lifetime Sensor

et al. 2019

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Intracellular pH plays a key role in physiology, and its measurement in living specimens remains a crucial task in biology. Fluorescent protein-based pH sensors have gained widespread use, but there is limited spectral diversity for multicolor detection, and it remains a challenge to measure absolute pH values. Here we demonstrate that mCherryTYG is an excellent fluorescence lifetime pH sensor that significantly expands the modalities available for pH quantification in live cells. We first report the 1.09 Å X-ray crystal structure of mCherryTYG, exhibiting a fully matured chromophore. We next determine that it has an extraordinarily large dynamic range with a 2 nanosecond lifetime change from pH 5.5 to pH 9.0. Critically, we find that the sensor maintains a pKa of 6.8 independent of environment, whether as the purified protein in solution or expressed in live cells. Furthermore, the lifetime measurements are ro-bustly independent of total fluorescence intensity and scatter. We demonstrate that mCherryTYG is a highly effective sensor using time resolved fluorescence spectroscopy on live-cell suspensions, which has been previously overlooked as an easily accessible approach for quantifying intracellular pH. As a red fluorescent *

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Section: Resultssupporting

confidence: 63%

Quantifying Acute Fuel and Respiration Dependent pH Homeostasis in Live Cells Using the mCherryTYG Mutant as a Fluorescence Lifetime Sensor

et al. 2019

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“…Light intensity fluctuations can be attributed to two main sources: first, the absorption of the laser light by a bacterium, and second, refraction of light at the boundary of the bacterium, caused by the difference in the refractive indices of the cell and the surrounding medium. Typical values of the refractive index for E.coli are 1.39 ± 0.05, while values for LB medium have been reported as 1.335 ± 0.03 ( 20–22 ). Light travelling through a bacterium is absorbed more than in the surrounding liquid, a property which is typically used in cell counting experiments by optical density (OD) measurements ( 23–25 ).…”

Section: Resultsmentioning

confidence: 99%

Microwell-enhanced optical rapid antibiotic susceptibility testing of single bacteria

Rosłoń

Japaridze

Rodenhuis

et al. 2023

Preprint

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Bacteria that are resistant to antibiotics present an increasing burden on healthcare. To address this emerging crisis, novel rapid Antibiotic Susceptibility Testing (AST) methods are eagerly needed. Here, we present an optical AST technique that can determine the bacterial viability within one hour down to a resolution of single bacteria. The method is based on measuring intensity fluctuations of a reflected laser focused on a bacterium in reflective microwells. Using numerical simulations, we show that both refraction and absorption of light by the bacterium contribute to the observed signal. By administering antibiotics that kill the bacteria, we show that the variance of the detected fluctuations vanishes within one hour, indicating the potential of this technique for rapid sensing of bacterial antibiotic susceptibility. We envisage the use of this method for massively parallelizable AST tests and fast detection of drug resistant pathogens.

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“…Finally, the bacterium is attached to the surface of the graphene and is likely to be in the laser beam path. The refractive index of an E. coli bacterium 39,40 (n = 1.33) is very close to that of the LB medium (n = 1.34), causing the bacteria to be nearly transparent and therefore we estimate this to have negligible impact on the nanomotion amplitude determination.…”

Section: Amplitude Calibrationmentioning

confidence: 92%

Probing nanomotion of single bacteria with graphene drums

et al. 2022

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Motion is a key characteristic of every form of life 1 . Even at the microscale, it has been reported that colonies of bacteria can generate nanomotion on mechanical cantilevers 2 , but the origin of these nanoscale vibrations has remained unresolved 3,4. Here, we present a novel technique using drums made of ultrathin bilayer graphene, where the nanomotion of single bacteria can be measured in its aqueous growth environment. A single E. coli cell is found to generate random oscillations with amplitudes of up to 60 nm, exerting forces of up to 6 nN to its environment. Using mutant strains, we are able to pinpoint the bacterial flagella as the main source of nanomotion. By real-time tracing of changes in nanomotion upon administering antibiotics, we demonstrate that graphene drums can perform antibiotic susceptibility testing with single-cell sensitivity. These findings deepen our understanding of processes underlying cellular dynamics, and pave the way towards high throughput and parallelized rapid screening of the effectiveness of antibiotics in bacterial infections with graphene devices.

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<title>Refractive index of <emph type="1">escherichia coli</emph> cells</title>

Cited by 25 publications

References 0 publications

Quantifying Acute Fuel and Respiration Dependent pH Homeostasis in Live Cells Using the mCherryTYG Mutant as a Fluorescence Lifetime Sensor

Quantifying Acute Fuel and Respiration Dependent pH Homeostasis in Live Cells Using the mCherryTYG Mutant as a Fluorescence Lifetime Sensor

Microwell-enhanced optical rapid antibiotic susceptibility testing of single bacteria

Probing nanomotion of single bacteria with graphene drums

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