A modern, simple, and economical accessory for continuous L-format measurement of steady-state fluorescence anisotropy.

In this study, the pros and cons of the most relevant L-format devices reported in the literature for measuring steady-state fluorescence polarization/anisotropy are identified. Combining all this information, and with the use of modern elements for the acquisition, treatment, and recording of signals, a modern, simple, and economical L-format accessory is implemented to rapidly and continuously record steady-state fluorescence anisotropy. This device can be adapted to the majority of the commercial spectrofluorometers (or fluorometers). During the measurement, the emission polarizer is in permanent rotation by means of a Gimbal brushless DC motor, and as a result the recorded fluorescence signal is sinusoidal. The maximums and minimums of this signal, which are obtained with the help of LabVIEW tools, allow recording the fluorescence anisotropy. The LabVIEW applications developed for this investigation are freely available, so it is not necessary to have LabVIEW software.

Assessment of fluorescence polarization assay: a candid diagnostic tool in Brucella abortus strain 19 vaccinated areas.

Brucellosis caused by the bacteria of the genus Brucella is an important zoonosis and constitutes a serious public health hazard. Brucellosis is diagnosed mainly by the Rose Bengal plate test and indirect ELISA, both of which have poor specificity because false positive serological reactions occur when screening animals that have been vaccinated with B. abortus S19. Fluorescence polarization assay (FPA) was evaluated for screening samples from cattle and buffalo calves with persistent antibody titers induced by vaccination. The standardized FPA exhibited relative sensitivity and specificity of 0.94 and 0.95, respectively, and the area under the curve, kappa and accuracy were 0.98, 0.87 and 0.95, respectively. Comparison of competitive ELISA and FPA revealed that, FPA is more specific than competitive ELISA. The high specificity, sensitivity and 95% accuracy of FPA indicate that, it is suitable for testing vaccinated animals because it can distinguish between infected from vaccinated animals.

A fluorescence anisotropy method for measuring protein concentration in complex cell culture media.

The rapid, quantitative analysis of the complex cell culture media used in biopharmaceutical manufacturing is of critical importance. Requirements for cell culture media composition profiling, or changes in specific analyte concentrations (e.g. amino acids in the media or product protein in the bioprocess broth) often necessitate the use of complicated analytical methods and extensive sample handling. Rapid spectroscopic methods like multi-dimensional fluorescence (MDF) spectroscopy have been successfully applied for the routine determination of compositional changes in cell culture media and bioprocess broths. Quantifying macromolecules in cell culture media is a specific challenge as there is a need to implement measurements rapidly on the prepared media. However, the use of standard fluorescence spectroscopy is complicated by the emission overlap from many media components. Here, we demonstrate how combining anisotropy measurements with standard total synchronous fluorescence spectroscopy (TSFS) provides a rapid, accurate quantitation method for cell culture media. Anisotropy provides emission resolution between large and small fluorophores while TSFS provides a robust measurement space. Model cell culture media was prepared using yeastolate (2.5 mg mL(-1)) spiked with bovine serum albumin (0 to 5 mg mL(-1)). Using this method, protein emission is clearly discriminated from background yeastolate emission, allowing for accurate bovine serum albumin (BSA) quantification over a 0.1 to 4.0 mg mL(-1) range with a limit of detection (LOD) of 13.8 µg mL(-1).

Multimodal assessment of nervous and immune system responses following sciatic nerve injury.

Subsequent to peripheral nerve compression and irritation, pathophysiological processes take place within nervous and immune systems. Here, we utilized a multimodal approach to comprehend peripheral and central soft tissue changes as well as alterations occurring in systemic analytes following unilateral chronic constriction injury (CCI) of the sciatic nerve in rodents. Using magnetic resonance imaging and [18F]-2-fluoro-2-deoxy-d-glucose (FDG) positron emission tomography, we demonstrated robust structural abnormalities and enhanced FDG uptake within the injured nerve and surrounding muscle, respectively. To assess whether central morphological changes were induced by nerve injury, diffusion tenor imaging was performed. A decrease in fractional anisotropy in primary motor cortex contralateral to the injury site was observed. Evaluation of a panel of circulating cytokines, chemokines, and growth factors showed decreased levels of interleukin-1ß and Fractalkine in CCI animals. Area under the receiver operating curve (ROC) calculations of analyte levels, imaging, and behavioral end points ranged from 0.786 to 1, where behavioral and peripheral imaging end points (eg, FDG uptake in muscle) were observed to have the highest discriminatory capabilities (maximum area under ROC = 1) between nerve injury and sham conditions. Lastly, performance of correlation analysis involving all analyte, behavioral, and imaging data provided an understanding of the overall association amongst these end points, and importantly, a distinction in correlation patterns was observed between CCI and sham conditions. These findings demonstrate the multidimensional pathophysiology of sciatic nerve injury and how a combined analyte, behavioral, and imaging assessment can be implemented to probe this complexity.

Single yeast cell vacuolar milieu viscosity assessment by fluorescence polarization microscopy with computer image analysis.

This study was undertaken to evaluate the apparent viscosity within the vacuoles of single Saccharomyces cerevisiae cells by steady-state fluorescence anisotropy measurements of quinacrine, using wide-field fluorescence polarization microscopy combined with computer image analysis. Quinacrine was shown to be rather specifically accumulated within the vacuoles of the cells. This accumulation was effectively reversed by ATP depletion of the cells, with no detectable binding of the dye within the vacuoles. Quinacrine fluorescence anisotropy in the sucrose solutions of various viscosities obeyed the Perrin equation. The fluorescence anisotropy of quinacrine was measured in the vacuoles of 39 cells. From cell to cell, this parameter changed in the range 0.032-0.086. Using the Perrin plot as a calibration curve, apparent viscosity values of the vacuolar milieu were calculated for each cell. The population of the cells studied was heterogeneous with regard to vacuolar viscosity, which was in the range 3.5 ± 0.4-14.06 ± 0.64 cP. There was a characteristic distribution of the frequencies of cells with apparent viscosities within certain limits, and cells with viscosity values in the range 5-6 cP were the most frequent. No relationship was found between the sizes of the vacuoles and their apparent viscosities.

An epifluorescence microscopy method for generalized polarization imaging.

Generalized polarization (GP) microscopy represents an excellent tool to study lipid-lipid and lipid-protein interactions in situ and in vitro. Here, we present an efficient and cost effective method to perform GP microscopy using a standard light-emitting diode (LED) epifluorescence microscope equipped with a digital color camera.

Frontal UV-visible fluorescence polarization measurement for bovine meat ageing assessment.

Among the techniques based on light interactions with biological tissues, fluorescence polarization offers a selective means of characterizing the organization of biological tissues. This paper presents a methodology for investigating the fluorescence polarization of muscle tissues in to obtain structural information, and specifically the structural modifications caused by meat ageing. A theoretical model of fluorescence anisotropy based on geometrical distribution and properties of tryptophan, the major fluorophore in muscle tissues, is proposed. Experimental data are fitted with the model and fitting parameters (C(1), C(2) and τ) are tracked during meat ageing. Results presented demonstrate how the method is able to show muscle structure modification during ageing. They highlight changes in structural proteins along the main axis of myofibrils and changes in the tryptophan environment resulting from the physicochemical and enzymatic processes at work during ageing.

Evaluation of the fluorescence polarization assay for the detection of Brucella abortus antibodies in bison in a natural setting.

Bison and elk in the greater Yellowstone area are the last-known reservoir of Brucella abortus in the United States. Diagnosis of brucellosis is challenging as there is no perfect reference test. The objectives of this study were to estimate the accuracy of the fluorescence polarization assay (FPA) for the screening of B. abortus antibodies in bison in a natural setting. Serum and tissue samples were collected and analyzed from the known brucellosis-infected bison herd in Yellowstone National Park (YNP). Additionally, serum samples from privately owned bison were serologically tested for brucellosis. While the FPA and five other tests had perfect sensitivity, all tests had substantially lower specificity in the YNP herd. However, a Bayesian analysis showed that as many as 59-74% of the culture-negative animals were most-likely truly infected. A decision-tree analysis showed that the expected cost of FPA testing was comparable to the cost of other serologic tests. The FPA was shown to be highly sensitive but may not be able to differentiate culture-positive and culture-negative animals. There is a need for long-term longitudinal studies to estimate diagnostic accuracy of tests for B. abortus in bison.

Chapter 12: Nanoscale biological fluorescence imaging: breaking the diffraction barrier.

Biological imaging has been limited by the finite resolution of light microscopy. Recent developments in ultra-high-resolution microscopy methods, many of which are based on fluorescence, are breaking the diffraction barrier; it is becoming possible to image intracellular protein distributions with resolution of tens of nanometers or better. Fluorescence photoactivation localization microscopy (FPALM) is an example of such an ultra-high-resolution method which can image living or fixed cells with demonstrated lateral resolution of better than 20 nm. A detailed description of the methods involved in FPALM imaging of biological samples is presented here, accompanied by comparison with existing methods from the literature.