In situ detection of live-to-dead bacteria ratio after inactivation by means of synchronous fluorescence and PCA.

The determination of live and dead bacteria is of considerable significance for preventing health care-associated infection in hospitals, field clinics, and other areas. In this study, the viable (live) and nonviable (dead) bacteria in a sample were determined by means of their fluorescence spectra and principal component analysis (PCA). Data obtained in this study show that it is possible to identify bacteria strains and determine the live/dead ratio after UV light inactivation and antibiotic treatment, in situ, within minutes. In addition, synchronous fluorescence scans enable the identification of bacterial components such as tryptophan, tyrosine, and DNA. Compared with the time-consuming plating and culturing methods, this study renders a means for rapid detection and determination of live and dead bacteria.

Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex.

Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging. Here, using fluorescence lifetime measurements empowered by a maximum entropy algorithm, we mapped the conformational heterogeneities of artificially destabilized mismatched DNA substrates of varying Rad4-binding specificities. The conformational distributions, as probed by FRET between a cytosine-analog pair exquisitely sensitive to DNA twisting/bending, reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity CCC/CCC mismatch, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound; the extent of these distortions increased with bound Rad4 and with temperature. Conversely, the non-specific TAT/TAT mismatch had a homogeneous, B-DNA-like conformation. Molecular dynamics simulations also revealed a wide distribution of conformations for CCC/CCC, complementing experimental findings. We propose that intrinsic deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating 'conformational capture' of pre-distorted damaged sites. Surprisingly, even mismatched DNA specifically bound to Rad4 remains highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions.

Fluorescence spectroscopy of rhodopsins: insights and approaches.

Fluorescence spectroscopy has become an established tool at the interface of biology, chemistry and physics because of its exquisite sensitivity and recent technical advancements. However, rhodopsin proteins present the fluorescence spectroscopist with a unique set of challenges and opportunities due to the presence of the light-sensitive retinal chromophore. This review briefly summarizes some approaches that have successfully met these challenges and the novel insights they have yielded about rhodopsin structure and function. We start with a brief overview of fluorescence fundamentals and experimental methodologies, followed by more specific discussions of technical challenges rhodopsin proteins present to fluorescence studies. Finally, we end by discussing some of the unique insights that have been gained specifically about visual rhodopsin and its interactions with affiliate proteins through the use of fluorescence spectroscopy. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.

Method of determining the optimal dilution ratio for fluorescence fingerprint of food constituents.

Quantitative determination by fluorescence spectroscopy is possible because of the linear relationship between the intensity of emitted fluorescence and the fluorophore concentration. However, concentration quenching may cause the relationship to become nonlinear, and thus, the optimal dilution ratio has to be determined. In the case of fluorescence fingerprint (FF) measurement, fluorescence is measured under multiple wavelength conditions and a method of determining the optimal dilution ratio for multivariate data such as FFs has not been reported. In this study, the FFs of mixed solutions of tryptophan and epicatechin of different concentrations and composition ratios were measured. Principal component analysis was applied, and the resulting loading plots were found to contain useful information about each constituent. The optimal concentration ranges could be determined by identifying the linear region of the PC score plotted against total concentration.

[Analysis of Chlorophyll Fluorescence Transient by Spectral Multi-exponential Approximation].

The method for analysis of chlorophyll fluorescence transient using approximation of measured signal by multi-exponential series is described. Visualization of partial sums of this series allows us to find amplitudes and characteristic times of individual phases of fluorescence induction curve. This method gives more rigid criteria of phase identification instead of semi-empirical approach currently used. Applied to Chlamidomonas reinhardtii sulfur deprivation case, it shows efficiency in finding visually undistinguishable phases of fluorescence transient for early detection of stress.

A PARAFAC-based long-term assessment of DOM in a multi-coagulant drinking water treatment scheme.

A parallel factor (PARAFAC) analysis approach was used to study the character and composition of dissolved organic matter (DOM) in a multicoagulant (two aluminum-based coagulants) full scale drinking water treatment plant. A three year, long-term assessment was conducted based on deconstruction of the excitation-emission matrices (EEM) of over 1000 water samples collected before and after parallel coagulation treatment basins. Two humic moieties and a protein-like group were identified in the raw and treated waters. Apportionment of fluorophores was established using a novel approach based on the overall fluorescence intensity (OFI) of PARAFAC components. Uncorrected matrix correlation (UMC) revealed minimal changes of the fluorescence moieties after treatment (UMC > 0.98), and a comparable effect of both coagulants on the structure (UMC > 0.99) and distribution of these groups. Coagulation increased the proportion of the protein-like fluorophore and preferentially removed a humic-like group irrespective of the coagulant. Preference for this moiety was supported by a coagulant-affinity factor derived from the association between PARAFAC components after treatment. The suitability of a PARAFAC-based approach for coagulant evaluation/selection was demonstrated when compared to a dissolved organic carbon (DOC)-based criterion. This paper contributes to the understanding of the behavior of PARAFAC components in water treatment processes and presents several approaches for the future monitoring and control of coagulation at full scale treatment facilities.

Simulating the peptide folding kinetic related spectra based on the Markov State Model.

Optical spectroscopic tools are used to monitor protein folding/unfolding dynamics after a fast triggering such as the laser induced temperature jump. These techniques provide new opportunities for comparison between theory and simulations and atom-level understanding protein folding mechanism. However, the direct comparison still face two main challenges: a gap between folding relevant timescales (microseconds or above) and length of molecular dynamics simulations (typically tens to hundreds of nanoseconds), and difficulty in directly calculating spectroscopic observables from simulation configurations. Markov State Model (MSM) approach is one of the most powerful means which can increase simulations timescale up to microsecond or even millisecond. We address progress on modeling infrared and fluorescence spectroscopic signals of temperature jump induced unfolding dynamics for a few small proteins. The harmoniousness between experiment and theoretical can both improve our understanding of protein folding mechanisms and provide direct validation of those theoretical models.

Accuracy and precision in camera-based fluorescence correlation spectroscopy measurements.

Imaging fluorescence correlation spectroscopy (FCS) performed using array detectors has been successfully used to quantify the number, mobility, and organization of biomolecules in cells and organisms. However, there have not been any systematic studies on the errors in these estimates that are introduced due to instrumental and experimental factors. State-of-the-art array detectors are still restricted in the number of frames that can be recorded per unit time, sensitivity and noise characteristics, and the total number of frames that can be realistically recorded. These limitations place constraints on the time resolution, the signal-to-noise ratio, and the total measurement time, respectively. This work addresses these problems by using a combination of simulations and experiments on lipid bilayers to provide characteristic performance parameters and guidelines that govern accuracy and precision of diffusion coefficient and concentration measurements in camera-based FCS. We then proceed to demonstrate the effects of these parameters on the capability of camera-based FCS to determine membrane heterogeneity via the FCS diffusion laws, showing that there is a lower length scale limit beyond which membrane organization cannot be detected and which can be overcome by choosing suitable experimental parameters. On the basis of these results, we provide guidelines for an efficient experimental design for camera-based FCS to extract information on mobility, concentration, and heterogeneity.

Demonstration of a single-wavelength spectral-imaging-based Thai jasmine rice identification.

A single-wavelength spectral-imaging-based Thai jasmine rice breed identification is demonstrated. Our nondestructive identification approach relies on a combination of fluorescent imaging and simple image processing techniques. Especially, we apply simple image thresholding, blob filtering, and image subtracting processes to either a 545 or a 575 nm image in order to identify our desired Thai jasmine rice breed from others. Other key advantages include no waste product and fast identification time. In our demonstration, UVC light is used as our exciting light, a liquid crystal tunable optical filter is used as our wavelength seclector, and a digital camera with 640 active pixels × 480 active pixels is used to capture the desired spectral image. Eight Thai rice breeds having similar size and shape are tested. Our experimental proof of concept shows that by suitably applying image thresholding, blob filtering, and image subtracting processes to the selected fluorescent image, the Thai jasmine rice breed can be identified with measured false acceptance rates of <22.9% and <25.7% for spectral images at 545 and 575 nm wavelengths, respectively. A measured fast identification time is 25 ms, showing high potential for real-time applications.

Experimental design approach for development of spectrofluorimetric method for determination of favipiravir; a potential therapeutic agent against COVID-19 virus: Application to spiked human plasma.

The present work describes development of rapid, robust, sensitive and green spectrofluorimetric method for determination of favipiravir (FAV). Different factors affecting fluorescence were carefully studied and Box Behnken Design was applied to optimize experimental parameters. The proposed method is based on measuring native fluorescence of FAV in 0.2 M borate buffer (pH 8.0) at 432 nm after excitation at 361 nm. There was a linear relationship between FAV concentration and relative fluorescence intensity over the range 40-280 ng/mL with limit of detection of 9.44 ng/mL and quantitation limit of 28.60 ng/mL. The method was successfully implemented for determination of FAV in its pharmaceutical formulation with mean % recovery of 99.26 ± 0.87. Moreover, the high sensitivity of the method allowed determination of FAV in spiked human plasma over a range of 48-192 ng/mL. The proposed spectrofluorimetric method was proved to be eco-friendly according to analytical eco-scale.

Characterization of coupled ground state and excited state equilibria by fluorescence spectral deconvolution.

Fluorescence probes with multiparametric response based on the relative variation in the intensities of several emission bands are of great general utility. An accurate interpretation of the system requires the determination of the number, positions and intensities of the spectral components. We have developed a new algorithm for spectral deconvolution that is applicable to fluorescence probes exhibiting a two-state ground-state equilibrium and a two-state excited-state reaction. Three distinct fluorescence emission bands are resolved, with a distribution of intensities that is excitation-wavelength-dependent. The deconvolution of the spectrum into individual components is based on their representation as asymmetric Siano-Metzler log-normal functions. The application of the algorithm to the solvation response of a 3-hydroxychromone (3HC) derivative that exhibits an H-bonding-dependent excited-state intramolecular proton transfer (ESIPT) reaction allowed the separation of the spectral signatures characteristic of polarity and hydrogen bonding. This example demonstrates the ability of the method to characterize two potentially uncorrelated parameters characterizing dye environment and interactions.

Identification of fluorescent beads using a coded aperture snapshot spectral imager.

We apply a coded aperture snapshot spectral imager (CASSI) to fluorescence microscopy. CASSI records a two-dimensional (2D) spectrally filtered projection of a three-dimensional (3D) spectral data cube. We minimize a convex quadratic function with total variation (TV) constraints for data cube estimation from the 2D snapshot. We adapt the TV minimization algorithm for direct fluorescent bead identification from CASSI measurements by combining a priori knowledge of the spectra associated with each bead type. Our proposed method creates a 2D bead identity image. Simulated fluorescence CASSI measurements are used to evaluate the behavior of the algorithm. We also record real CASSI measurements of a ten bead type fluorescence scene and create a 2D bead identity map. A baseline image from filtered-array imaging system verifies CASSI's 2D bead identity map.

Measurement of two-photon excitation spectra of fluorescent proteins with nonlinear Fourier-transform spectroscopy.

We present measurements of two-photon excitation (TPE) spectra of various fluorescent proteins with nonlinear Fourier-transform spectroscopy. By using an ultrabroadband laser pulse with a spectrum ranging from 700 to 1100 nm, the absolute TPE spectra of six typical fluorescent proteins (SeBFP, Sapphire, eGFP, eCFP, Venus, DsRed) were measured with high spectral resolution.

Visualization of molecular interactions using bimolecular fluorescence complementation analysis: characteristics of protein fragment complementation.

Investigations of the molecular processes that sustain life must include studies of these processes in their normal cellular environment. The bimolecular fluorescence complementation (BiFC) assay provides an approach for the visualization of protein interactions and modifications in living cells. This assay is based on the facilitated association of complementary fragments of a fluorescent protein that are fused to interaction partners. Complex formation by the interaction partners tethers the fluorescent protein fragments in proximity to each other, which can facilitate their association. The BiFC assay enables sensitive visualization of protein complexes with high spatial resolution. The temporal resolution of BiFC analysis is limited by the time required for fluorophore formation, as well as the stabilization of complexes by association of the fluorescent protein fragments. Many modifications and enhancements to the BiFC assay have been developed. The multicolor BiFC assay enables simultaneous visualization of multiple protein complexes in the same cell, and can be used to investigate competition among mutually exclusive interaction partners for complex formation in cells. The ubiquitin-mediated fluorescence complementation (UbFC) assay enables visualization of covalent ubiquitin family peptide conjugation to substrate proteins in cells. The BiFC assay can also be used to visualize protein binding to specific chromatin domains, as well as other molecular scaffolds in cells. BiFC analysis therefore provides a powerful approach for the visualization of a variety of processes that affect molecular proximity in living cells. The visualization of macromolecular interactions and modifications is of great importance owing to the central roles of proteins, nucleic acids and other macromolecular complexes in the regulation of cellular functions. This tutorial review describes the BiFC assay, and discusses the advantages and disadvantages of this experimental approach. The review will be of interest to scientists interested in the investigation of macromolecular interactions or modifications who need exquisite sensitivity for the detection of their complexes or conjugates of interest.

Potentiality of using front face fluorescence spectroscopy for quantitative analysis of cow milk adulteration in buffalo milk.

In current study, synchronous front-face fluorescence spectroscopy together with partial least squares regression (PLSR) is used to predict the adulteration of cow and buffalo milk quantitatively. Fresh (unprocessed milk) samples of cow and buffalo were collected from local dairy farms. Fluorescence emission from milk samples mixed in different concentrations, show intensity variations at wavelengths 370-380 nm, 410 nm, 442 nm and 520-560 nm. Among them, the emissions at band position of 442 nm and 525 nm are highly selective between the two species and could help in finding adulteration of cow milk in buffalo milk and vice versa. The emissions at these wavelength positions correspond to fat-soluble vitamin-A as well as ß-carotene. PLS regression is used as a statistical prediction model, which is developed by training with the emission spectra of milk samples having known level of adulterations. The developed model predicts the unknown level of adulterations by means of their spectral data. The goodness of the model is determined by the correlation coefficient R-square (r2) value, which in our case is 0.99. Furthermore, the model root mean square error in cross validation (RMSECV) and in prediction (RMSECP) remains 1.16 and 6.24 respectively. This approach can effectively be applied to determine milk adulterations among other species as well as in detecting external agents (fraudulent) added into milk and other dairy products by further studies.

A simple and useful method for evaluation of oxidative stress in vivo by spectrofluorometric estimation of urinary pteridines.

Pteridine derivatives are intermediate metabolites of folic acid and its cofactors. Oxidized-form pteridines, but not reduced-form pteridines, are fluorescent substances. The purpose of this study was to clarify whether oxidized-form pteridine level in urine, estimated by spectrofluorometry, reflects oxidative stress in vivo. The subjects were healthy middle-aged men (n = 258). Urinary pteridine level was estimated by spectrofluorometry with an excitation wavelength of 360 nm and an emission wavelength of 450 nm. Relationships of urinary pteridines with oxidative stress markers (urinary DNA/RNA oxidation products and 15-isoprostane F2t) and with smoking were analyzed. Concentrations of pteridines, DNA/RNA oxidation products and 15-isoprostane F2t were used after logarithmic transformation in linear analyses. Pteridine levels were significantly correlated with levels of DNA/RNA oxidation products (Pearson's correlation coefficient: 0.626, p < 0.01) and 15-isoprostane F2t (Pearson's correlation coefficient: 0.695, p < 0.01). These correlations were not confounded by age, body mass index, history of smoking and estimated glomerular filtration rate in multivariate analysis. The mean urinary pteridine level was significantly higher in heavy smokers (16 cigarettes or more per day) than in nonsmokers and light smokers (less than 16 cigarettes per day) and was higher in light smokers than in nonsmokers. Thus, urinary fluorometric pteridine levels were shown to be associated with known biomarkers of oxidative stress as well as smoking, which causes oxidative stress in vivo. We propose spectrofluorometrical estimation of urinary pteridines as a simple and useful method for evaluation of oxidative stress in vivo.

Determination of flibanserin in the presence of confirmed degradation products by a third derivative emission spectrofluorometric method: Application to pharmaceutical formulation.

Flibanserin is a new drug used for the treatment of hypoactive sexual desire disorder. This work is considered the first study concerning the fluorimetric behaviour of flibanserin and its new florescent degradation products. A fast, cost-effective, stability-indicating spectrofluorometric method was developed and validated for the determination of flibanserin in the presence of oxidative degradation products. Stability studies are performed to predict the behaviour of substances under various harsh conditions. Thus, flibanserin was subjected to degradation using hydrogen peroxide. The stability-indicating method was developed and validated per ICH guidelines; it was linear in the range of 0.1-3 µg/mL. The method was accurate and precise as it showed good recoveries between 98.50 and 100.90% and relative standard deviation less than 2%, respectively, and no significant differences were found after statistical comparison with the in-house HPLC method. In addition, the structures of the oxidative degradation products were confirmed using infrared spectroscopy and mass spectrometry, and the proposed degradation pathway was predicted.

A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling.

Fluorescent proteins (FPs) with far-red excitation and emission are desirable for multicolor labeling and live-animal imaging. We describe E2-Crimson, a far-red derivative of the tetrameric FP DsRed-Express2. Unlike other far-red FPs, E2-Crimson is noncytotoxic in bacterial and mammalian cells. E2-Crimson is brighter than other far-red FPs and matures substantially faster than other red and far-red FPs. Approximately 40% of the E2-Crimson fluorescence signal is remarkably photostable. With an excitation maximum at 611 nm, E2-Crimson is the first FP that is efficiently excited with standard far-red lasers. We show that E2-Crimson has unique applications for flow cytometry and stimulated emission depletion (STED) microscopy.

Potentiality of front-face fluorescence and mid-infrared spectroscopies coupled with partial least square regression to predict lipid oxidation in pound cakes during storage.

The potentialities of front-face fluorescence (FFF) and mid-infrared (MIR) spectroscopies coupled with partial least square regression (PLSR) were compared to predict the lipid oxidation of pound cakes. The level of lipid oxidation in pound cakes determined using classical methods showed some changes. Similarly, the fluorescence emission (305-490 nm) and excitation (252-390 nm) spectra and MIR spectra scanned in the 4000-700 cm-1 region showed some changes in pound cakes as a function of both storage time and the type of oil used in the formulation. The application of PLSR to the MIR spectra, provided excellent predictive results for free fatty acid (R2 = 0.97) and peroxide values (R2 = 0.87). Similar results were obtained from both tryptophan and MIR spectra for the prediction of TOTOX (R2 > 0.86) demonstrating the efficiency of the MIR and FFF spectroscopies to qualify and quantify the level of lipid oxidation in pound cakes.

Chromophoric dissolved organic matter influence correction of algal concentration measurements using three-dimensional fluorescence spectra.

In view of the adverse effects of CDOM (chromophoric or colored dissolved organic matter) on in vivo algal pigment concentration measurements in natural water bodies, a CDOM influence correction method for algal concentration measurements based on three-dimensional fluorescence spectra is investigated. The three-dimensional fluorescence spectra of five common species of algae belonging to five categories, HA (humic acid), and natural water sampled from the Dongpu reservoir, Hefei were analyzed, and the spectral similarity of endogenous/exogenous CDOM in the algal fluorescence spectra region was compared. HA was selected to represent the CDOM spectrum group. The CDOM modified algal pigment concentration measurement method was developed using three-dimensional fluorescence spectra coupled with non-negative weighted least squares linear regression analysis. The results show that under the presence of CDOM interference factors, the recognition accuracy rate of Pyrrophyta, Bacillariophyta, Cyanophyta, and Chlorophyta increased 100%, 100%, 40%, and 40%, respectively. The average recovery rate of Cryptomonas, Pyrrophyta, Bacillariophyta, and Chlorophyta increased 162.7%, 50.3%, 106.4%, and 19.1%, respectively. In addition, the classification accuracy of Pyrrophyta, Bacillariophyta, Cyanophyta, Chlorophyta increased 83.9%, 100%, 38.2%, and 48%, respectively. This was concluded by comparing these results with the results of the algal pigment concentration measurement method without the CDOM modification. This study provides an experimental basis for the development of accurate phytoplankton fluorescence classification monitoring technology.