Quantitative Spectroscopic Characterization of Near-UV/visible E. coli (pYAC4), B. subtilis (PY79), and Green Bread Mold Fungus Fluorescence for Diagnostic Applications

Herzog, Joshua; Sick, Volker

doi:10.1007/s10895-023-03183-6

Cited by 3 publications

(10 citation statements)

References 27 publications

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“…The optimum cutoff wavelength is chosen as the value that minimizes the relative error quantity E calculated from measured fluorescence spectra of E. coli, B. subtilis, and a wild-type green bread mold fungus using data from [27]. Specifically, for each cutoff wavelength, R and s R are calculated for each spectrum.…”

Section: Ratiometric Imaging Performancementioning

confidence: 99%

“…A quantitative, combined NADH and FAD fluorescence model is used to analyze the performance of the proposed diagnostic technique. The model is based on aqueous solutions of NADH and FAD (see fit results in [27]), but includes small changes in fit parameters to better match observed E. coli spectra which are believed to be representative of NADH and FAD in biological samples. This model provides a convenient method to calculate absorption and fluorescence spectra.…”

Section: Combined Nadh-fad Fluorescence Modelmentioning

confidence: 99%

“…The chemical environment can cause small shifts in absorption and fluorescence features and changes in collisional quenching properties, while cell morphology and inhomogeneity impact radiative transfer and Table 1. Best-fit values for NADH and FAD using 1D-CCM from [27]. NADH and FAD spectra used for fitting are from [32] and [35,36] Mathematically, each molecule is represented by a single one-dimensional configuration coordinate model implemented as a harmonic oscillator.…”

Section: Combined Nadh-fad Fluorescence Modelmentioning

confidence: 99%

“…Here, a simple, low-cost ratiometric line-of-sight fluorescence imaging technique is developed to distinguish between microbial species based on the results of a previous spectroscopic investigation [27]. In contrast to confocal and multiphoton microscopy, the line-of-sight technique is a two-dimensional path-integrated imaging measurement; the primary benefits are that it is fast, inexpensive, does not require specialized equipment, and is robust since both fluorescence bands are excited and imaged simultaneously.…”

Section: Introductionmentioning

confidence: 99%

“…In the remainder of this report, the proposed technique is analyzed using a combined NADH-FAD fluorescence model and demonstrated on 44 microbial smears across 8 species (non-pathogenic strains of Bacillus cereus, Bacillus subtilis, Escherichia coli, Micrococcus luteus, Pseudomonas fluorescens, Serratia marcescens, and a green bread-mold fungus) to illustrate the utility of the method. This work builds on the previous spectroscopic characterization [27] by developing and demonstrating a novel imaging technique that can be used to distinguish between microbial species.…”

Section: Introductionmentioning

confidence: 99%

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Design of a line-of-sight fluorescence-based imaging diagnostic for classification of microbe species

Herzog

Sick

2023

Meas. Sci. Technol.

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Fluorescence imaging of certain biochemicals, including flavins and pyridine nucleotides, has utility in characterizing the metabolic state of tissue and in discriminating between microbial species. There is significant clinical utility in this class of imaging techniques but most measurements reported to date require specialized training and equipment rendering most implementations unsuitable for routine medical imaging. Here, a low-cost and robust imaging technique is designed using ultraviolet-induced fluorescence of pyridine nucleotides (primarily NADH) and flavins (primarily FAD) in microbial samples. The diagnostic is optimized to distinguish between different microbial species based on previously reported spectral data using a ratiometric imaging approach. A detailed performance analysis is provided that relates the measured fluorescence intensity ratio (FIR) to the relative concentration ratio of NADH to FAD using a simplified spectroscopic model. Analysis suggests the technique is sensitive to changes in the NADH/FAD concentration ratio over several orders of magnitude, with better than 10% FIR precision on a per-pixel basis for microbial smears as thin as 10s of microns at a resolution of 30 mm-1 and exposures of 20 ms. Representative microbe samples from eight species were imaged to demonstrate the proposed technique. Results show that the FIR varies by an order of magnitude across different species but the intra-species variation is only ∽5% for the conditions used here. An additional imaging band may be necessary to classify species that contain red pigments or bacteriochlorophyll. Radiative trapping was discussed as a possible limitation of the technique, but no clear evidence for radiative trapping was observed here. Overall, the results suggest that the proposed approach is feasible for rapid, low-cost, and robust characterization of microbial samples.

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Section: Ratiometric Imaging Performancementioning

confidence: 99%

Section: Combined Nadh-fad Fluorescence Modelmentioning

confidence: 99%

Section: Combined Nadh-fad Fluorescence Modelmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Design of a line-of-sight fluorescence-based imaging diagnostic for classification of microbe species

Herzog

Sick

2023

Meas. Sci. Technol.

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An experimental setup for the study of Plasmopara viticola on vine leaves by fluorescence

Greco,

Leccisi,

Spagnuolo

et al. 2023

2023 IEEE International Workshop on Metrology for Agriculture and Forestry (MetroAgriFor)

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Fluorescence imaging for the anterior segment of the eye

Herzog,

Sick

2024

Front. Photon.

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Diagnostic technologies for the anterior segment of the eye, especially for hard-to-diagnose diseases such as microbial keratitis, are still lacking. Although in vivo confocal microscopy and optical coherence tomography are becoming more widely applicable to a variety of conditions, they are often prohibitively expensive, require specialized training and equipment, and are intrinsically insensitive to chemical changes. Here, ultraviolet-fluorescence imaging is proposed as a new technique to aid in investigation of the anterior segment. In this work, a novel two-color line-of-sight fluorescence imaging technique is described for imaging of the anterior segment. The technique is applied to seven ex vivo porcine eyes to illustrate the utility of the technique. The image data was used to estimate an effective fluorescence quantum yield of each eye at 370 nm. The eyes were then inoculated with bacteria to simulate microbial keratitis, a common sight-threatening infection, and the measurement was repeated. A simplified fluorescence-extinction model was developed to describe and analyze the relative intensities of the eye and biofilm fluorescence. Overall, the technique appears to have utility in clinical practice and with proper development may be suitable for detecting chemical changes in the eye, or the presence of foreign matter; however, further investigation is needed to develop the technique and analysis procedures into a quantitative diagnostic tool.

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Quantitative Spectroscopic Characterization of Near-UV/visible E. coli (pYAC4), B. subtilis (PY79), and Green Bread Mold Fungus Fluorescence for Diagnostic Applications

Cited by 3 publications

References 27 publications

Design of a line-of-sight fluorescence-based imaging diagnostic for classification of microbe species

Design of a line-of-sight fluorescence-based imaging diagnostic for classification of microbe species

An experimental setup for the study of Plasmopara viticola on vine leaves by fluorescence

Fluorescence imaging for the anterior segment of the eye

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