Equivalent path lengths in an integrating cavity: comment

Fry, Edward S.; Kattawar, George W.; Strycker, Benjamin D.; Zhai, Peng-Wang

doi:10.1364/ao.49.000575

Cited by 22 publications

(17 citation statements)

References 16 publications

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“…Apparent absorbance spectra (typically 6.2 s −1 ) were then collected until any visible absorbance changes had ceased. Raw apparent absorbance values were converted to absorbance values per cm using Fry’s method (Fry et al, 2010) as described in the text.…”

Section: Methodsmentioning

confidence: 99%

In situ spectroscopy on intact Leptospirillum ferrooxidans reveals that reduced cytochrome 579 is an obligatory intermediate in the aerobic iron respiratory chain

Blake

Griff

2012

Front. Microbio.

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Electron transfer reactions among colored cytochromes in intact bacterial cells were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scatter light. The aerobic iron respiratory chain of Leptospirillum ferrooxidans was dominated by the redox status of an abundant cellular cytochrome that had an absorbance peak at 579 nm in the reduced state. Intracellular cytochrome 579 was reduced within the time that it took to mix a suspension of the bacteria with soluble ferrous iron at pH 1.7. Steady state turnover experiments were conducted where the initial concentrations of ferrous iron were less than or equal to that of the oxygen concentration. Under these conditions, the initial absorbance spectrum of the bacterium observed under air-oxidized conditions was always regenerated from that of the bacterium observed in the presence of Fe(II). The kinetics of aerobic respiration on soluble iron by intact L. ferrooxidans conformed to the Michaelis–Menten formalism, where the reduced intracellular cytochrome 579 represented the Michaelis complex whose subsequent oxidation appeared to be the rate-limiting step in the overall aerobic respiratory process. The velocity of formation of ferric iron at any time point was directly proportional to the concentration of the reduced cytochrome 579. Further, the integral over time of the concentration of the reduced cytochrome was directly proportional to the total concentration of ferrous iron in each reaction mixture. These kinetic data obtained using whole cells were consistent with the hypothesis that reduced cytochrome 579 is an obligatory steady state intermediate in the iron respiratory chain of this bacterium. The capability of conducting visible spectroscopy in suspensions of intact cells comprises a powerful post-reductionist means to study cellular respiration in situ under physiological conditions for the organism.

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Section: Methodsmentioning

confidence: 99%

In situ spectroscopy on intact Leptospirillum ferrooxidans reveals that reduced cytochrome 579 is an obligatory intermediate in the aerobic iron respiratory chain

Blake

Griff

2012

Front. Microbio.

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“…The resulting absorbance changes were stable for the entire time that raw data were collected. These raw absorbance values were subsequently converted to equivalent absorbance values per cm using Fry’s method (Fry et al, 2010) with analysis software provided by OLIS, Inc.…”

Section: Methodsmentioning

confidence: 99%

In situ Spectroscopy Reveals that Microorganisms in Different Phyla Use Different Electron Transfer Biomolecules to Respire Aerobically on Soluble Iron

et al. 2016

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Absorbance spectra were collected on 12 different live microorganisms, representing six phyla, as they respired aerobically on soluble iron at pH 1.5. A novel integrating cavity absorption meter was employed that permitted accurate absorbance measurements in turbid suspensions that scattered light. Illumination of each microorganism yielded a characteristic spectrum of electrochemically reduced colored prosthetic groups. A total of six different patterns of reduced-minus-oxidized difference spectra were observed. Three different spectra were obtained with members of the Gram-negative eubacteria. Acidithiobacillus, representing Proteobacteria, yielded a spectrum in which cytochromes a and c and a blue copper protein were all prominent. Acidihalobacter, also representing the Proteobacteria, yielded a spectrum in which both cytochrome b and a long-wavelength cytochrome a were clearly visible. Two species of Leptospirillum, representing the Nitrospirae, both yielded spectra that were dominated by a cytochrome with a reduced peak at 579 nm. Sulfobacillus and Alicyclobacillus, representing the Gram-positive Firmicutes, both yielded spectra dominated by a-type cytochromes. Acidimicrobium and Ferrimicrobium, representing the Gram-positive Actinobacteria, also yielded spectra dominated by a-type cytochromes. Acidiplasma and Ferroplasma, representing the Euryarchaeota, both yielded spectra dominated by a ba3-type of cytochrome. Metallosphaera and Sulfolobus, representing the Crenarchaeota, both yielded spectra dominated by the same novel cytochrome as that observed in the Nitrospirae and a new, heretofore unrecognized redox-active prosthetic group with a reduced peak at around 485 nm. These observations are consistent with the hypothesis that individual acidophilic microorganisms that respire aerobically on iron utilize one of at least six different types of electron transfer pathways that are characterized by different redox-active prosthetic groups. In situ absorbance spectroscopy is shown to be a useful complement to existing means of investigating the details of energy conservation in intact microorganisms under physiological conditions.

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“…Apparent absorbance values for 1-mL samples were recorded relative to a 1 PBS baseline at the heme Soret peaks of 400 nm for the standard solutions (which contained methemoglobin) and 410 nm for cell samples. Fry correction (19) was carried out to normalize enhanced pathlength values to 1 cm using SpectralWorks software (Olis, Inc.). The Soret peak baseline was then determined for the corrected spectra by linear interpolation.…”

Section: Cell Culturementioning

confidence: 99%

“…The light is trapped within the reflective confines of the cuvette until it encounters the output port; thus, its effective pathlength is increased significantly. The enhanced pathlength is inversely related to the detected (apparent) sample absorbance, and this nonlinear effect is corrected by converting the apparent absorbance to absorbance per centimeter using first principles and the Fry Equation (19). The resulting sensitivity of the CLARiTY to light absorption in turbid samples is overwhelmingly superior to that of traditional transmission spectrophotometers (Figure 1B).…”

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confidence: 99%

“…(B) Overlay of absorbance spectra for MEL cells washed and resuspended in 1 PBS following a 72-h induction in regular media containing 1.5% DMSO. The apparent absorbance scan of a 1-mL cell sample on the RSM-CLARiTY is decreased upon Fry correction (19) for the increased pathlength within the cuvette. A traditional transmission spectrophotometer (Cary 1G by Varian) was used to scan a 1:10 dilution of the same cell resuspension analyzed on the CLARiTY.…”

mentioning

confidence: 99%

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Rapid and Sensitive Quantitation of Heme in Hemoglobinized Cells

et al. 2016

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Rapid and accurate heme quantitation in the research lab has become more desirable as the crucial role that intracellular hemoproteins play in metabolism continues to emerge. Here, the time-honored approaches of pyridine hemochromogen and fluorescence heme assays are compared with direct absorbance-based technologies using the CLARiTY spectrophotometer. All samples tested with these methods were rich in hemoglobin-associated heme, including buffered hemoglobin standards, whole blood from mice, and murine erythroleukemia (MEL) and K562 cells. While the pyridine hemochromogen assay demonstrated the greatest linear range of heme detection, all 3 methods demonstrated similar analytical sensitivities and normalized limits of quantitation of ∼1 µM. Surprisingly, the fluorescence assay was only shown to be distinct in its ability to quantitate extremely small samples. Using the CLARiTY system in combination with pyridine hemochromogen and cell count data, a common hemoglobin extinction coefficient for blood and differentiating MEL and K562 cells of 0.46 µM-1 cm-1 was derived. This value was applied to supplemental experiments designed to measure MEL cell hemoglobinization in response to the addition or removal of factors previously shown to affect heme biosynthesis (e.g., L-glutamine, iron).

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Equivalent path lengths in an integrating cavity: comment

Cited by 22 publications

References 16 publications

In situ spectroscopy on intact Leptospirillum ferrooxidans reveals that reduced cytochrome 579 is an obligatory intermediate in the aerobic iron respiratory chain

In situ spectroscopy on intact Leptospirillum ferrooxidans reveals that reduced cytochrome 579 is an obligatory intermediate in the aerobic iron respiratory chain

In situ Spectroscopy Reveals that Microorganisms in Different Phyla Use Different Electron Transfer Biomolecules to Respire Aerobically on Soluble Iron

Rapid and Sensitive Quantitation of Heme in Hemoglobinized Cells

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