Analysis Of Intensity Fluctuations Research Articles

Pathlength-selective, interferometric diffuse correlation spectroscopy (PaLS-iDCS).

Diffuse correlation spectroscopy (DCS) is an optical method that offers non-invasive assessment of blood flow in tissue through the analysis of intensity fluctuations in diffusely backscattered coherent light. The non-invasive nature of the technique has enabled several clinical applications for deep tissue blood flow measurements, including cerebral blood flow monitoring as well as tumor blood flow mapping. While a promising technique, in measurement configurations targeting deep tissue hemodynamics, the standard DCS implementations suffer from insufficient signal-to-noise ratio (SNR), depth sensitivity, and sampling rate, limiting their utility. In this work, we present an enhanced DCS method called pathlength-selective, interferometric DCS (PaLS-iDCS), which improves upon both the sensitivity of the measurement to deep tissue hemodynamics and the SNR of the measurement using pathlength-specific coherent gain. Through interferometric detection, PaLS-iDCS can provide time-of-flight (ToF) specific blood flow information without the use of expensive time-tagging electronics and low-jitter detectors. The new technique is compared to time-domain DCS (TD-DCS), another enhanced DCS method able to resolve photon ToF in tissue, through Monte Carlo simulation, phantom experiments, and human subject measurements. PaLS-iDCS consistently demonstrates improvements in SNR (>2x) for similar measurement conditions (same photon ToF), and the SNR improvements allow for measurements at extended photon ToFs, which have increased sensitivity to deep tissue hemodynamics (~50% increase). Further, like TD-DCS, PaLS-iDCS allows direct estimation of tissue optical properties from the sampled ToF distribution without the need for a separate spectroscopic measurement. This method offers a relatively straightforward way to allow DCS systems to make robust measurements of blood flow with greatly enhanced sensitivity to deep tissue hemodynamics, enabling further applications of this non-invasive technology.

Origin of rate dispersion in translational diffusion: Distinguishing heterogeneous from homogeneous using 2D correlation analysis

Rate dispersion is commonly observed when measuring relaxation in complex media such as solution of a protein. Its origin is often associated to molecular heterogeneity in the system. However, interpretation of rate dispersion is not straightforward, and it can be exhibited by both heterogeneous (all molecules are different) and homogeneous (all molecules are identical) systems. Here, we report two-dimensional (2D) correlation analysis of intensity fluctuations originating from translational diffusion-lead fluctuations in number of particles simulated in an observation volume resembling fluorescence correlation spectroscopy experiment. Translational diffusion of particles representing a protein having two different conformations are simulated. Homogeneous and heterogeneous systems are simulated to have similar one-dimensional (1D) autocorrelation curves with comparable extent of rate dispersion, α ∼ 0.8. Time slices of 2D autocorrelation curves and rate-rate spectra obtained from them clearly demonstrate that similar extent of rate dispersion observed in 1D autocorrelation can originate from both heterogeneous and homogeneous systems. We establish that one-dimensional analysis only reports on the extent of rate dispersion. Two-dimensional analysis reports on the origin of rate dispersion and distinguishes heterogeneous from homogeneous.

Estimation of bottom attenuation in shallow water with changing depth using intensity fluctuations due to moving nonlinear internal waves

The use of analysis of intensity fluctuations in the presence of non-linear internal waves (NIWs) for estimating the bottom attenuation is demonstrated in (JASA, v.140, p.3980, 2016) using data of Shallow Water 2006. Key objects of this analysis are the time dependence and spectrum of the total intensity (summed over the entire depth of the waveguide, in other words, over all hydrophones of the vertical line array—VLA). In this paper, we consider data on the measurement of field fluctuations (frequency 224 Hz) on an acoustic track ∼ 30 km long in the presence of NVWs, obtained in the ASIAEX 2001 experiment. The acoustic track consists of two parts: “deep,” (∼ 270 m) and “shallow” (∼ 120 m). This feature leads, first, to variation of NIW parameters, propagating toward the coast, and second, it gives reason to assume different bottom properties for these sub-tracks. The paper proposes a technique for estimating the attenuation coefficients independently for two parts based on the study of spectrograms at the VLA and spectrum of total intensity, and the following fitting between experimental data and theoretical modeling. The obtained attenuation coefficients are compared with the data of other authors for the same area.

Statistical analysis of intensity fluctuations in the second harmonic of a multimode Nd:YAG laser through a modified Pearson correlation coefficient

We apply a recently proposed modified Pearson correlation coefficient to investigate the statistical properties of intensity fluctuations in the second harmonic as well as fundamental mode of a multimode $Q$-switched Nd:yttrium aluminum garnet laser. Above threshold the system displays a photonic ferromagneticlike spontaneous mode-locking phase with replica symmetry breaking (RSB). Differently from the photonic glassy phase in random lasers in which modes do not oscillate synchronously, in the present case a relevant fraction of modes oscillate coherently, but the synchronous oscillation of some modes is frustrated with the RSB arising from the fact that a given subset of modes is randomly activated at each excitation pulse with dominating coherent oscillation, frustrating the emergence of other modes. From a single set of measurements, the modified Pearson coefficient allows to identify that the onset of the RSB phenomenon between distinct spectra as the lasing threshold is crossed coincides with the emergence of correlations between different wavelengths in the same spectrum. As different subsets of longitudinal modes are nondeterministically activated after each shot, the RSB property indicates that distinct spectra can be either correlated or anticorrelated around both the fundamental mode and the second harmonic.

Modelling Intensity Fluctuations of Rayleigh Backscattered Coherent Light in Single-Mode Fibers

This work reportsa thorough analysis of intensity fluctuations of Rayleigh backscattered coherent light in single mode optical fibers. Based on the intermediate range order of silica glasses and on residual stress of optical fibers, a theoretical model was built to calculate intensity fluctuations of Rayleigh backscattered coherent light in the optical frequency domain. Numerical simulation results were compared to experimental results in time domain, strongly supporting the conclusion that Rayleigh backscattering in single mode fibers is an ergodic process exhibiting ergodicity in the time-frequency sense so that the model can be used to determine the statistical behavior of backscattered intensity fading.

Fluorescence Intensity Fluctuation Analysis of Protein Oligomerization in Cell Membranes.

Fluorescence fluctuation spectroscopy (FFS) encompasses a bevy of techniques that involve analyzing fluorescence intensity fluctuations occurring due to fluorescently labeled molecules diffusing in and out of a microscope's focal region. Statistical analysis of these fluctuations may reveal the oligomerization (i.e., association) state of said molecules. We have recently developed a new FFS-based method, termed Two-Dimensional Fluorescence Intensity Fluctuation (2D FIF) spectrometry, which provides quantitative information on the size and stability of protein oligomers as a function of receptor concentration. This article describes protocols for employing FIF spectrometry to quantify the oligomerization of a membrane protein of interest, with specific instructions regarding cell preparation, image acquisition, and analysis of images given in detail. Application of the FIF Spectrometry Suite, a software package designed for applying FIF analysis on fluorescence images, is emphasized in the protocol. Also discussed in detail is the identification, removal, and/or analysis of inhomogeneous regions of the membrane that appear as bright spots. The 2D FIF approach is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of live cells expressing protein constructs Basic Protocol 2: Image acquisition and noise correction Basic Protocol 3: Drawing and segmenting regions of interest Basic Protocol 4: Calculating the molecular brightness and concentration of individual image segments Basic Protocol 5: Combining data subsets using a manual procedure (Optional) Alternate Protocol 1: Combining data subsets using the advanced FIF spectrometry suite (Optional; alternative to Basic Protocol 5) Basic Protocol 6: Performing meta-analysis of brightness spectrograms Alternate Protocol 2: Performing meta-analysis of brightness spectrograms (alternative to Basic Protocol 6) Basic Protocol 7: Spot extraction and analysis using a manual procedure or by writing a program (Optional) Alternate Protocol 3: Automated spot extraction and analysis (Optional; alternative to Protocol 7) Support Protocol: Monomeric brightness determination.

Fluorescence intensity fluctuation analysis of receptor oligomerization in membrane domains

Fluorescence micrographs of the plasma membrane of cells expressing fluorescently labeled G protein-coupled receptors (GPCRs) often exhibit small clusters of pixels (or puncta) with intensities that are higher than those of the surrounding pixels. Although studies of GPCR interactions in uniform membrane areas abound, understanding the details of the GPCR interactions within such puncta as well as the nature of the membrane formations underlying the puncta is hampered by the lack of adequate experimental techniques. Here, we introduce an enhancement of a recently developed method termed fluorescence intensity fluctuation spectrometry, which permits analysis of protein-protein interactions within the puncta in live cell membranes. We applied the novel fluorescence intensity fluctuation data analysis protocol to previously published data from cells expressing human secretin receptors and determined that the oligomer size increases with receptor concentration and duration of treatment with cognate ligand, not only within uniform regions of the membrane (in agreement with previous publications) but also within the puncta. In addition, we found that the number density and fractional area of the puncta increased after treatment with ligand. This method could be applied for probing the evolution in the time of the chain of events that begins with ligand binding and continues with coated pits formation and receptor internalization for other GPCRs and, indeed, other membrane receptors in living cells.

WITHDRAWN: Human Body Model Bone Extraction Algorithm Based On Space Segmentation and Computer Image

Abstract Segmentation of perspective bone image is a concern in many medical applications, such as detection and fracture of osteoporosis. To connect the second segment, which attachment points are extracted. The proposed technique's high processing speed and efficiency make this a promising solution in many applications to be implemented. Multi resolution wavelet binding, Fuzzy C-Means (FCM) algorithm and extraction active contour model, a model segment made from an X-ray image of the bone structure. An effective thresholding algorithm and Fuzzy C-Means segmentation and morphological operators based on proposed fracture. Compared to some of the traditions and methods of dividing the latest bone, this algorithm shows the visual assessment that it is excellent. Experimental results show this method is effective both in the manner of the divided data and speed. More accurate results for all rows divided images intensity fluctuation analysis bone region. Analyze the intensity fluctuations of the entire bone area line for more accurate segmentation of the image result.

Amyloidogenic Nanoplaques in Blood Serum of Patients with Alzheimer's Disease Revealed by Time-Resolved Thioflavin T Fluorescence Intensity Fluctuation Analysis.

Background:Biomarkers are central to current research on molecular mechanisms underlying Alzheimer’s disease (AD). Their further development is of paramount importance for understanding pathophysiological processes that eventually lead to disease onset. Biomarkers are also crucial for early disease detection, before clinical manifestation, and for development of new disease modifying therapies.Objective:The overall aim of this work is to develop a minimally invasive method for fast, ultra-sensitive and cost-effective detection of structurally modified peptide/protein self-assemblies in the peripheral blood and in other biological fluids. Specifically, we focus here on using this method to detect structured amyloidogenic oligomeric aggregates in the blood serum of apparently healthy individuals and patients in early AD stage, and measure their concentration and size.Methods:Time-resolved detection of Thioflavin T (ThT) fluorescence intensity fluctuations in a sub-femtoliter observation volume element was used to identify in blood serum ThT-active structured amyloidogenic oligomeric aggregates, hereafter called nanoplaques, and measure with single-particle sensitivity their concentration and size.Results:The concentration and size of structured amyloidogenic nanoplaques are significantly higher in the blood serum of individuals diagnosed with AD than in control subjects.Conclusion:A new method with the ultimate, single-particle sensitivity was successfully developed. The proposed approach neither relies on the use of immune-based probes, nor on the use of radiotracers, signal-amplification or protein separation techniques, and provides a minimally invasive test for fast and cost-effective early determination of structurally modified peptides/proteins in the peripheral blood, as shown here, but also in other biological fluids.

MicroRNA Detection by DNA-Mediated Liposome Fusion.

Membrane fusion is a process of fundamental importance in biological systems that involves highly selective recognition mechanisms for the trafficking of molecular and ionic cargos. Mimicking natural membrane fusion mechanisms for the purpose of biosensor development holds great potential for amplified detection because relatively few highly discriminating targets lead to fusion and an accompanied engagement of a large payload of signal‐generating molecules. In this work, sequence‐specific DNA‐mediated liposome fusion is used for the highly selective detection of microRNA. The detection of miR‐29a, a known flu biomarker, is demonstrated down to 18 nm within 30 min with high specificity by using a standard laboratory microplate reader. Furthermore, one order of magnitude improvement in the limit of detection is demonstrated by using a novel imaging technique combined with an intensity fluctuation analysis, which is coined two‐color fluorescence correlation microscopy.

Two particle tracking and detection in a single Gaussian beam optical trap.

We have studied in detail the situation wherein two microbeads are trapped axially in a single-beam Gaussian intensity profile optical trap. We find that the corner frequency extracted from a power spectral density analysis of intensity fluctuations recorded on a quadrant photodetector (QPD) is dependent on the detection scheme. Using forward- and backscattering detection schemes with single and two laser wavelengths along with computer simulations, we conclude that fluctuations detected in backscattering bear true position information of the bead encountered first in the beam propagation direction. Forward scattering, on the other hand, carries position information of both beads with substantial contribution from the bead encountered first along the beam propagation direction. Mie scattering analysis further reveals that the interference term from the scattering of the two beads contributes significantly to the signal, precluding the ability to resolve the positions of the individual beads in forward scattering. In QPD-based detection schemes, detection through backscattering, thereby, is imperative to track the true displacements of axially trapped microbeads for possible studies on light-mediated interbead interactions.

Determination of Membrane Protein Transporter Oligomerization in Native Tissue Using Spatial Fluorescence Intensity Fluctuation Analysis

Membrane transporter proteins exist in a complex dynamic equilibrium between various oligomeric states that include monomers, dimers, dimer of dimers and higher order oligomers. Given their sub-optical microscopic resolution size, the oligomerization state of membrane transporters is difficult to quantify without requiring tissue disruption and indirect biochemical methods. Here we present the application of a fluorescence measurement technique which combines fluorescence image moment analysis and spatial intensity distribution analysis (SpIDA) to determine the oligomerization state of membrane proteins in situ. As a model system we analyzed the oligomeric state(s) of the electrogenic sodium bicarbonate cotransporter NBCe1-A in cultured cells and in rat kidney. The approaches that we describe offer for the first time the ability to investigate the oligomeric state of membrane transporter proteins in their native state.

Estimate of wind velocity in the atmosphere based on an analysis of turbulent distortions of laser beam images registered by video camera

The results of an experimental method for estimating the integral along a wind velocity path, based on the determination of the variance of speed of the energy centroid displacements in the images of a laser beam passed through the path, are presented. The method is compared with optical methods of estimating the integral velocity, based on “temporal” and “spatial” variants of the correlation and spectral analysis of intensity fluctuations in the laser beam images. It is shown by the use of ten sonic anemometers placed along the sounding path that the results of the integral wind velocity estimation by all three optical methods are in agreement with each other and with the results of local measurements of wind velocity.

In Situ Measurement of Oligomerization State of NBCe1‐A in Kidney Tissues using Spatial Fluorescence Intensity Fluctuation Analysis

NBCe1‐A plays an important role in absorbing sodium bicarbonate across the basolateral membrane of the proximal tubule. We have previously showed that minimal functional unit of NBCe1‐A is a monomer, and based on in‐vitro biochemical studies in HEK293 cells, the oligomeric state of the cotransporter was shown to be predominantly dimeric with monomeric and higher oligomeric forms also present. In the present study, we developed an in situ measurement methodology to determine the oligomeric state of NBCe1‐A without requiring tissue disruption. We used fluorescent moment image analysis and spatial intensity distribution analysis (SpIDA) to study the oligomeric state of NBCe1‐A. Both methods allow for unbiased quantitative measurement of fluorescent particle densities and oligomerization states within individual images acquired with laser‐scanning microscopy. Initially we examined basal membranes of highly adherent CHO K1 cells expressing eGFP‐tagged NBCe1‐A, and showed that NBCe1‐A existed primarily in the monomeric state and negligibly in the dimeric state on the cell membrane. We immunolabeled NBCe1‐A in kidney tissues demonstrating the NBCe1‐A is present in dimeric and rarely in higher order oligomeric states. As a control, we compared the results obtained from LSM images of tissues of NBCe1‐A knockout mouse. These experiments demonstrate for the first time the in situ oligomeric state(s) of NBCe1‐A.

In Situ Measurements of Oligomerization State of NBCe1-A in Rat Kidneys Via Spatial Fluorescence Intensity Fluctuation Analysis

NBCe1-A plays an important role in absorbing sodium bicarbonate across the basolateral membrane of the proximal tubule. We have previously showed that minimal functional unit of NBCe1-A is a monomer, and based on in-vitro biochemical studies in HEK293 cells, the oligomeric state of the cotransporter was shown to be predominantly dimeric with monomeric and higher oligomeric forms also present. We developed an in situ measurement methodology to determine the oligomeric state of NBCe1-A without requiring tissue disruption using biochemical methods. We used fluorescent moment image analysis and spatial intensity distribution analysis (SpIDA) to study the oligomeric state of NBCe1-A in cultured cells expressing the cotransporter and in rat kidney tissue. Both methods allow for quantitative measurement of fluorescent particle densities and oligomerization states within individual images acquired with laser-scanning microscopy. Initially we examined basal membranes of highly adherent CHO K1 cells expressing eGFP-tagged NBCe1-A because of their large surface area. As an independent control of monomeric brightness, we used cells expressing monomeric eGFP anchored to the membrane. Taking into account the recovered values of the monomeric eGFP quantal brightness, we show that NBCe1-A exists in monomeric and dimeric states on the cell membrane. We also used an Alexa488-alpha-bungarotoxin conjugate to label cells expressing an NBCe1A-bungarotoxin binding mutant. As a monomeric control, we immobilized Alexa488 dye on cover slips. The spatial fluorescence intensity fluctuation analysis revealed a similar distribution of aggregates as shown for eGFP data. Moreover, we immunolabeled NBCe1-A in rat kidney tissues as well as in cultured HEK293 cells expressing the cotransporter demonstrating the NBCe1-A is present in monomeric, dimeric and rarely in higher order oligomeric states. These experiments demonstrate for the first time the in situ oligomeric state(s) of NBCe1-A.

Effects of Oversampling and Scanning Artifacts on the Accuracy of Spatial Fluorescence Intensity Fluctuation Analysis Methods

We present a systematic simulation and experimental study of potential measurement artifacts that arise due to raster scan acquisition for the image fluctuation analysis methods image correlation spectroscopy (ICS) and spatial intensity distribution analysis (SpIDA). With computer simulations, we show that photobleaching occurring during the sampling of sequential pixels affects the density and quantal brightness measurements obtained from single laser scanning microscopy images. This effect is even more pronounced when the images are oversampled with by using a small pixel size. The magnitude of this artifact was a function of the bleaching coefficients. The simulation results were compared to actual experimental data collected using a confocal laser-scanning microscope for a variety of dyes and sampling conditions. We analyzed images of monomeric fluorescent dyes covalently bound to coverslips, and monomeric green fluorescent protein (EGFP) transfected in mammalian cells. Moreover, we investigated the effects of non-constant emission of fluorescent probes, the presence of background noise and shot noise on the accuracy of the image fluctuation methods. We found that as the laser power increases (below saturation), some asymmetry in the spatial autocorrelation function appears which leads to systematic errors in the ICS measured number densities. SpIDA is also affected in the same manner by this artifact. Finally we looked at the accuracy of both techniques as a function of the quantal brightness of the particles. We generated computer simulated images of point emitters with different quantal brightness and then added shot noise to the images to determine accuracy and precision ranges for the measurements.

Statistical analysis of intensity fluctuations in single molecule SERS spectra

We have investigated the fluctuations of the intensity and the line intermittency in the surface enhanced Raman spectra of a single iron-protoporphyrin IX molecule. A statistical analysis has revealed a high correlation between the intensity of each frequency couple in the spectrum. Removal of the continuum background has led to a suppression of the correlation at those frequencies where no Raman lines are present. Conversely, we have observed the persistence of a strong correlation at the intensities corresponding to the vibrational modes of the molecule. Further evidence of correlation between the intensities and the background signal indicates that the background is involved in the enhancement mechanism. Moreover, analysis of the Raman line intermittency reveals a random activation of the different molecular vibrational modes. These results can be generally put into relationship to the presence of two different contributions to the intensity fluctuations: one, strictly related to the continuum background, and affecting the whole spectrum, and another one which selectively acts on the various vibrational modes of the molecule.

Visible Emission of a Photoproduct from Tryptophan Solution Induced by Multiphoton Excitation: An Investigation by Intensity Fluctuation Analysis

Strong visible emission from a tryptophan methylester solution was observed by a confocal microscope with a 795-nm femtosecond pulsed laser. The emission was presumably similar to that observed in ...

Dynamic light scattering study of the interface evolution in porous media

The interface evolution during the evaporation of a liquid from a saturated layer of porous medium (paper) was experimentally studied using spectral analysis of intensity fluctuations of a laser radiation scattered by the layer. The data obtained were compared with the results of modeling the irreversible growth in three-dimensional lattices. The dependences of the spectral halfwidth of intensity fluctuations on the drying time demonstrate the characteristic features of drying front evolution, which proved to be similar to those found in the modeling of irreversible growth front. A comparison of the maximal halfwidths for two different saturating liquids suggests that the motion of local interfaces during the liquid evaporation from a layer of porous medium is close to the “classical” diffusion.

Further analysis of intensity fluctuations from a 3252-km acoustic propagation experiment in the eastern North Pacific Ocean

In the Acoustic Thermometry of Ocean Climate (ATOC) program’s Acoustic Engineering Test (AET), broadband 75-Hz center frequency transmissions were recorded on a 700-m-long vertical array, 3252 km distant from a midwater source suspended from R/P FLIP. The transmissions occurred over a 6-day period. Previously reported results from the AET using 12.7-min averaged data by Colosi et al. [J. Acoust. Soc. Am. 105(6), 3202–3218 (1999)], hereafter referred to as Colosi99) revealed surprisingly weak acoustic scattering for early arriving identifiable wavefronts. Colosi99 found pulse time spreads on the order of 0–5 ms and the probability density function (PDF) of peak intensity was close to log normal. In this paper these results are confirmed using 1.8-min averaged data. It is also shown that scintillation index (SI) is a strong function of position along the pulse with the smallest values occurring at the peak and larger values occurring at the tails. Intensity PDFs of identifiable wavefronts are reanalyzed in terms of both peak intensity and integrated pulse energy (IE) where the integration is over ±50 ms from the wavefront peaks. While SI for the IE are somewhat smaller than for the peak intensity, the PDFs are both very closely log normal. Regarding multipathing along the wavefronts, it is found that on average there are 1.7 peaks per wavefront segment per hydrophone and the intensity PDF of all multipath peaks is log normal. The combined observation of weak scattering and multipathing is a novel result. A reanalysis of the scintillations in the AET transmission finale where no wavefronts are evident is presented. Colosi99 analyzed the finale in terms of peak scintillations and found a near log-normal intensity PDF. Reprocessing the full field without limiting data to intensity peaks and accounting for mean intensity nonstationarity yields an intensity PDF which is much closer to the exponential distribution associated with full saturation; these results show that the finale region can be expected to behave much like Gaussian random noise.