Classification of fluorescent anisotropy decay based on the distance approach in the frequency domain.

Frequency-domain (FD) fluorometry is a widely utilized tool to probe unique features of complex biological structures, which may serve medical diagnostic purposes. The conventional data analysis approaches used today to extract the fluorescence intensity or fluorescence anisotropy (FA) decay data suffer from several drawbacks and are inherently limited by the characteristics and complexity of the decay models. This paper presents the squared distance (D2) technique, which categorized samples based on the direct frequency response data (FRD) of the FA decay. As such, it improves the classification ability of the FD measurements of the FA decay as it avoids any distortion that results from the challenged translation into time domain data. This paper discusses the potential use of the D2 approach to classify biological systems. Mathematical formulation of D2 technique adjusted to the FRD of the FA decay is described. In addition, it validates the D2 approach using 2 simulated data sets of 6 groups with similar widely and closely spaced FA decay data as well as in experimental data of 4 samples of a fluorophore-solvent (fluorescein-glycerol) system. In the simulations, the classification accuracy was above 95% for all 6 groups. In the experimental data, the classification accuracy was 100%. The D2 approach can help classify samples whose FA decay data are difficult to extract making FA in the FD a realistic diagnostic tool. The D2 approach offers an advanced method for sorting biological samples with differences beyond the practical temporal resolution limit in a reliable and efficient manner based on the FRD of their time-resolved fluorescence measurements thereby achieving better diagnostic quality in a shorter time.

Characterization of Diabetic Retinopathy in Two Mouse Models and Response to a Single Injection of Anti-Vascular Endothelial Growth Factor.

In this study, we characterized diabetic retinopathy in two mouse models and the response to anti-vascular endothelial growth factor (VEGF) injection. The study was conducted in 58 transgenic, non-obese diabetic (NOD) mice with spontaneous type 1 diabetes (n = 30, DMT1-NOD) or chemically induced (n = 28, streptozotocin, STZ-NOD) type 1 diabetes and 20 transgenic db/db mice with type 2 diabetes (DMT2-db/db); 30 NOD and 8 wild-type mice served as controls. Mice were examined at 21 days for vasculopathy, retinal thickness, and expression of genes involved in oxidative stress, angiogenesis, gliosis, and diabetes. The right eye was histologically examined one week after injection of bevacizumab, ranibizumab, saline, or no treatment. Flat mounts revealed microaneurysms and one apparent area of tufts of neovascularization in the diabetic retina. Immunostaining revealed activation of Müller glia and prominent Müller cells. Mean retinal thickness was greater in diabetic mice. RAGE increased and GFAP decreased in DMT1-NOD mice; GFAP and SOX-9 mildly increased in db/db mice. Anti-VEGF treatment led to reduced retinal thickness. Retinas showed vasculopathy and edema in DMT1-NOD and DMT2-db/db mice and activation of Müller glia in DMT1-NOD mice, with some response to anti-VEGF treatment. Given the similarity of diabetic retinopathy in mice and humans, comparisons of type 1 and type 2 diabetic mouse models may assist in the development of new treatment modalities.

Effect of optical magnification on the detection of the reduced scattering coefficient in the blue regime: theory and experiments.

Imaging turbid media is range limited. In contrast, sensing the medium's optical properties is possible in larger depths using the iterative multi-plane optical properties extraction technique. It analyzes the reconstructed reemitted light phase image. The root mean square of the phase image yields two graphs with opposite behaviors that intersect at µ's,cp. These graphs enable the extraction of a certain range of the reduced scattering coefficient, µ's. Here, we aim to extend the range of µ's detection by optical magnification. We use a modified diffusion theory and show how µ's,cp shifts with the varying magnification. The theoretical results were tested experimentally, showing that the technique can be adapted to different ranges of µ's by changing the magnification.

Fluorophore spectroscopy in aqueous glycerol solution: the interactions of glycerol with the fluorophore.

A common perception exists that glycerol provides an inert-like environment modifying viscosity and index of refraction by its various concentrations in aqueous solution. Said perception is herein challenged by investigating the effects of the glycerol environment on the spectroscopic properties of fluorescein, as a representative fluorophore, using steady-state and time-resolved techniques and computational chemistry. Results strongly suggest that the fluorescence quantum yield, measured fluorescence lifetime (FLT), natural lifetime and calculated fluorescence lifetime are all highly sensitive to the presence of glycerol. Glycerol was found to impact both the ground and first excited states of fluorescein, quenching and modifying both absorption and emission spectra, affecting the fundamental electrical dipoles of the ground and first excited singlet states, and lowering FLT and quantum yield. Furthermore, the Stern-Volmer, Lippert-Mataga, Perrin and Strickler-Berg relations indicate that glycerol acts upon fluorescein in aqueous solution as a quencher and alters the fluorescein geometry. Predictions made by computational chemistry impressively correspond to experimental results, both indicating changes in the properties of fluorescein at around 35% v/v aqueous glycerol, a clear indication that glycerol is not an innocent medium. This study proposes the Strickler-Berg relation as a means of detecting non-negligible effects of a hosting medium on its host fluorophore. These new insights on the molecular structures, the interactions between glycerol and its host fluorophore, and the effects of one on the other may be essential for understanding fundamental phenomena in chemistry and related fields.

The Scattering of Gold Nanorods Combined with Differential Uptake, Paving a New Detection Method for Macrophage Subtypes Using Flow Cytometery.

The strategy of identification for M1 and M2 macrophages both in vivo and in vitro would help to predict the health condition of the individual. Here, we introduced a solution to this problem with the advantage of both the phagocytic nature of macrophages and the scattering effect of gold nanorods (GNRs). The internalized GNRs, relating to their extent of intake, caused a conspicuous scattering profile at the red channel in flow cytometry, overruling the contribution of the cellular side scatters. This internalization is solely governed by the surface chemistry of GNRs. The PAH-GNRs showed maximum intake potency followed by Cit-, PSS-, and PEG-GNRs. On a substantial note, PAH-GNRs lead to differential uptake between M1 and M2 cells, with three times higher intake in M2 cells over M1. This is the first report of employing the scattering of unlabeled GNRs to discriminate M1 and M2 cell types using a flow cytometer.

Optical method to extract the reduced scattering coefficient from tissue: theory and experiments.

Tissues are considered challenging in terms of structure and composition analysis due to their tendency to multiple scatter the incident light. One of the most common theories for extracting optical properties of tissue is diffusion reflection (DR). In this Letter, we propose a new paradigm for estimating the reduced scattering coefficient of a medium from the reflected light phase. The technique is a modified DR theory wherein the phase is calculated by the product of the wavenumber and the average pathlength. This theory is supported by the reconstructed phase of tissue-like phantom experiments from an iterative algorithm.

[CONCISE NANOMEDICINE REVIEW].

INTRODUCTION: Nanomedicine is a rapidly evolving medical domain utilizing 1-100nm nanoscale particles to achieve medical goals in either one or more medical aspects - diagnosis, imaging and therapy. Nanomedicine employs a combination of methods stemming from life and exact sciences. This review deals briefly with the principles behind the scenes guiding the design, manufacture and employment of these nanoparticles. Some representative examples of the various applications are provided from the abundance of existing nanoparticles. The main topics discussed are those related to composition, characteristics of nanoparticles, usage in cancer, drug delivery and various central nervous system applications. Possible toxicity and future teratogenicity research pertaining to nanoparticles are also elaborated upon.

Gold nanorods reflectance discriminate benign from malignant oral lesions.

Nanoparticle-based contrast agents have been used as an imaging tool for selectively detecting cancerous processes. We aimed to evaluate the detection sensitivity of reflection measurements of gold nanorods (GNRs) bio-conjugated to anti-epidermal growth factor receptor (GNRs-EGFR) monoclonal antibodies in discriminating benign from premalignant and malignant human oral lesions. Tissue sections incubated with GNRs-EGFR and the reflectance spectrum was measured using hyperspectral microscopy. Reflectance intensity increased with the progression of the disease, lowest in the control group and increasing as the dysplastic changes increase (P<0.001 for linear trend of grade). Intensity was significantly higher in the moderate and severe dysplasias and cancer patients than in the controls and mild dysplasia (t test P=0.0003, Mann-Whitney P<0.0001). The GNRs reflection measurements can discriminate benign and mild dysplastic lesions from the more severe dysplasia and invasive cancer, suggesting an objective, not dependent on the qualification of a technician and with less interpretation errors.

Weak electromagnetic fields alter Ca(2+) handling and protect against hypoxia-mediated damage in primary newborn rat myotube cultures.

Weak electromagnetic fields (WEF) enhance Ca(2+) entry into cells via voltage-gated Ca(2+) channels and affect various aspects of metabolism, structure, and function. However, little information is available on the effect of WEF on skeletal muscle, which depends primarily on intracellular Ca(2+) stores for function and metabolism. Here, we examine the effects of 30 min exposure of rat primary myotube cultures to WEF (1.75 µT, 16 Hz) on Ca(2+) handling and creatine kinase (CK) release. Free myoplasmic Ca(2+) concentration ([Ca(2+) i]) was measured with the ratiometric dye indo-1. WEF did not affect basal [Ca(2+)]i but decreased the twitch [Ca(2+)]i transient in a time-dependent manner, and the twitch amplitude was decreased to ∼30 % after 30 min. WEF completely abolished the increase in [Ca(2+)]i induced by potassium chloride (∼60 mM) but had no effect on the increase induced by caffeine (∼6 mM). Hypoxia (2 h exposure to 100 % argon) resulted in a marked loss of CK into the medium (400 % of normoxic value), as well as a rapid (within 20 min) and sustained increase in basal [Ca(2+)]i (∼20 % above baseline). However, during exposure to WEF, basal [Ca(2+)]i remained constant during the initial 60 min of hypoxia and, thereafter, increased to levels similar to those observed in the absence of WEF. Finally, WEF blocked about 80 % of hypoxia-mediated CK release (P < 0.05). These data demonstrate that WEF inhibits increases in [Ca(2+)]i by interfering with muscle excitation and protects against muscle damage induced by hypoxia. Thus, WEF may have therapeutic/protective effects on skeletal muscle.

Utilizing fluorescent life time imaging microscopy technology for identify carriers of BRCA2 mutation.

Worldwide, more than one million women are diagnosed with breast cancer every year, making it the most common female malignancy in the developed world. Germline mutations in BRCA1 and BRCA2 genes are estimated to increase the risk for developing breast cancer by up to 87%. From a clinical point of view, identification of BRCA1 and BRCA2 mutation carriers offers an opportunity to early identify or prevent the development of malignancy; therefore the ability to determine which women are more likely to carry BRCA1 or BRCA2 mutations is of great importance. The available diagnostic tests for mutation analysis of BRCA1 and BRCA2 are time- and labor-intensive, expensive, and do not allow the identification of all the functional mutations. We utilized the Fluorescent lifetime (FLT) imaging microscopy method which allows recognizing different cell populations, in order to distinguish between lymphocytes from BRCA1 and BRCA2 mutation carriers and non-carrier women by using easily obtainable lymphocyte cells from peripheral blood. Our results demonstrate that cells originated from BRCA2-mutation carriers have significantly lower FLT values compared with BRCA1 mutation carriers and control cells. This simple, inexpensive and sensitive method may be utilized in the future to detect BRCA2 mutation carriers, particularly those bearing unknown functional mutations.

Fluorescence lifetime imaging of DAPI-stained nuclei as a novel diagnostic tool for the detection and classification of B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukaemia (B-CLL) and B-cell precursor acute lymphoblastic leukaemia (B-ALL) are the most common type of leukaemia in adults and children, respectively. Today, fluorescence in situ hybridization (FISH) is the standard for detecting chromosomal aberrations that reflect adverse and favorable outcome. This study revealed a new, simple, and fast diagnostic tool to detect pathological cells by measuring and imaging the fluorescence lifetime (FLT) using FLT imaging microscopy (FLIM) of the peripheral blood (PB) cells of B-CLL samples that were labeled with the DNA binder, DAPI. The FLT of DAPI in healthy individuals was found to be 2.66 ± 0.12 ns. In contrast, PB cells of B-CLL and BM cells of B-ALL patients were characterized by a specific group distribution of the FLT values. The FLT of DAPI was divided into four subgroups, relative to 2.66 ns: short+, normal, prolonged, and prolonged+. These alterations could be related to different chromatin arrangements of B-CLL and B-ALL interphase nuclei. Notably, extremely long FLT of nuclear DAPI correlate with the presence of extra chromosome 12, while moderate increases compared to normal characterize the deletion of p53. Such correlations potentially enable a FLT-based rapid automatic diagnosis and classification of B-CLL even when the frequency of genetic and chromosomal abnormalities is low. © 2016 International Society for Advancement of Cytometry.

Simulation of oxygen saturation measurement in a single blood vein.

The value of oxygen saturation in venous blood, SvO2, has important clinical significance since it is related to the tissue oxygen utilization, which is related to the blood flow to the tissue and to its metabolism rate. However, existing pulse oximetry techniques are not suitable for blood in veins. In the current study we examine the feasibility of difference oximetry to assess SvO2 by using two near-infrared wavelengths and collecting the backscattered light from two photodetectors located at different distances from the light source.

Gold nanorods as absorption contrast agents for the noninvasive detection of arterial vascular disorders based on diffusion reflection measurements.

In this study we report the use of gold nanorods (GNRs) as absorption contrast agents in the diffusion reflection (DR) method for the in vivo detection of atherosclerotic injury. The early detection and characterization of atherosclerotic vascular disease is considered to be one of the greatest medical challenges today. We show that macrophage cells, which are major components of unstable active atherosclerotic plaques, uptake gold nanoparticles, resulting in a change in the optical properties of tissue-like phantoms and a unique DR profile. In vivo DR measurements of rats that underwent injury of the carotid artery showed a clear difference between the DR profiles of the injured compared with healthy arteries. The results suggest that DR measurements following GNRs administration represent a potential novel method for the early detection of atherosclerotic vascular disease.

In vivo tumor detection using polarization and wavelength reflection characteristics of gold nanorods.

This paper presents a novel concept involving sensing the back-reflected and absorbed light at two polarization states and at several wavelengths from gold nanorods (GNRs). While the GNRs are flowing in the bloodstream the reflected light has a high degree of polarization and only one resonance wavelength. When the GNRs are located in a tumor the reflected light has a low degree of polarization and two resonance wavelengths are detected. Such characteristics can assist in detecting a tumor in passive targeting and without labeling it.

Optical Method for Detection and Classification of Heavy Metal Contaminants in Water Using Iso-pathlength Point Characterization.

Water pollution caused by hazardous substances, particularly heavy metal (HM) ions, poses a threat to human health and the environment. Traditional methods for measuring HM in water are expensive and time-consuming and require extensive sample preparation. Therefore, developing robust, simple, and sensitive techniques for the detection and classification of HM is needed. We propose an optical approach that exploits the full scattering profile, meaning the angular intensity distribution, and utilizes the iso-pathlength (IPL) point. This point appears where the intensity is constant for different scattering coefficients, while the absorption coefficient is set. The absorption does not affect the IPL point position, it only reduces its intensity. In this paper, we explore the wavelength influence on the IPL point both in Monte Carlo simulations and experimentally. Next, we present the characterization of ferric chloride (FeCl2) by this phenomenon. Eventually, we exhibit the detection of FeCl2 and intralipid mixed in concentrations of 50-100 and 20-30 ppm, respectively. These findings endorse the idea that the IPL point is an intrinsic parameter of a system serving as an absolute calibration point. The method provides an efficient way of differentiating contamination in water. Its characterization technique is easy, precise, and versatile making it preferable for water monitoring.

Extracting Superficial Scattering by Q-Sensing Technique.

Optical properties determine how light interacts with biological tissues. The current methods for measuring these optical properties are influenced by both deep and superficial skin layers. Polarization-based methods have been proposed in order to determine the influence of deep layer scattering. Polarized light allows for the separation of ballistic photons from diffuse ones, enhancing image contrast and resolution while providing additional tissue information. The Q-sensing technique captures co-polarized I ∥ $$ \left({I}_{\parallel}\right) $$ and cross-polarized I ⊥ $$ \left({I}_{\perp}\right) $$ signals, making it possible to isolate the superficial scattering. However, the random structure of tissues leads to rapid depolarization of the polarized light. Detecting where the light becomes depolarized aids in sensing abnormalities within the tissues. Hence, this research focuses on identifying where depolarization occurs within the tissue. Tissue-mimicking phantoms, simulating the optical properties of biological tissues, are created to measure depolarization at various thicknesses. Experimental findings are validated with a Monte Carlo simulation, modeling polarized light behavior through the polydisperse tissue (as the tissue scatterers are heterogeneous in size). Additionally, the research demonstrates how polarized light can extract the optical properties of the medium.

A Self-Calibrated Single Wavelength Biosensor for Measuring Oxygen Saturation.

Traditional methods for measuring blood oxygen use multiple wavelengths, which produce an intrinsic error due to ratiometric measurements. These methods assume that the absorption changes with the wavelength, but in fact the scattering changes as well and cannot be neglected. We found that if one measures in a specific angle around a cylindrical tissue, called the iso-pathlength (IPL) point, the reemitted light intensity is unaffected by the tissue's scattering. Therefore, the absorption can be isolated from the scattering, which allows the extraction of the subject's oxygen saturation. In this work, we designed an optical biosensor for reading the light intensity reemitted from the tissue, using a single light source and multiple photodetectors (PDs), with one of them in the IPL point's location. Using this bio-device, we developed a methodology to extract the arterial oxygen saturation using a single wavelength light source. We proved this method is not dependent on the light source and is applicable to different measurement locations on the body, with an error of 0.5%. Moreover, we tested thirty-eight males and females with the biosensor under normal conditions. Finally, we show the results of measuring subjects in a hypoxic chamber that simulates extreme conditions with low oxygen.

Dependency of crossover point on absorption changes in bilayer diffusion reflection measurements.

Significance: A better understanding of diffusion reflection (DR) behavior may allow it to be used for more noninvasive applications, including the development of in vivo non-damaging techniques, especially for medical topical diagnosis and treatments. Aim: For a bilayer opaque substance where the attenuation of the upper layer is larger than the attenuation of the lower layer, the DR crossover point ( C p ) is location where the photons coming from the bottom layer start affecting the DR. We aim to study the dependency of the C p on absorption changes in different layers for constant scattering and top layer thickness. Approach: Monolayer and bilayer optical tissue-like phantoms were prepared and measured using a DR system. The results were compared with Monte Carlo simulations. Results: There is an agreement between the experiments and the simulations. C p correlates with the square root of the absorption coefficient ratio of the lower layer to the top layer. Conclusion: The experimental findings support and validate the theoretical prediction describing the dependency of the C p on the square root of the ratio of the layers' absorption coefficients. In addition, a secondary breaking point is suggested to be observed experimentally at the entrance to the noise area.

Recent Advances in the Spintronic Application of Carbon-Based Nanomaterials.

The term "carbon-based spintronics" mostly refers to the spin applications in carbon materials such as graphene, fullerene, carbon nitride, and carbon nanotubes. Carbon-based spintronics and their devices have undergone extraordinary development recently. The causes of spin relaxation and the characteristics of spin transport in carbon materials, namely for graphene and carbon nanotubes, have been the subject of several theoretical and experimental studies. This article gives a summary of the present state of research and technological advancements for spintronic applications in carbon-based materials. We discuss the benefits and challenges of several spin-enabled, carbon-based applications. The advantages include the fact that they are significantly less volatile than charge-based electronics. The challenge is in being able to scale up to mass production.

Detecting Contaminants in Water Based on Full Scattering Profiles within the Single Scattering Regime.

Clean water is essential for maintaining human health. To ensure clean water, it is important to use sensitive detection methods that can identify contaminants in real time. Most techniques do not rely on optical properties and require calibrating the system for each level of contamination. Therefore, we suggest a new technique to measure water contamination using the full scattering profile, which is the angular intensity distribution. From this, we extracted the iso-pathlength (IPL) point which minimizes the effects of scattering. The IPL point is an angle where the intensity values remain constant for different scattering coefficients while the absorption coefficient is set. The absorption coefficient does not affect the IPL point but only attenuates its intensity. In this paper, we show the appearance of the IPL in single scattering regimes for small concentrations of Intralipid. We extracted a unique point for each sample diameter wherein light intensity remained constant. The results describe a linear dependency between the angular position of the IPL point and the sample diameter. In addition, we show that the IPL point separates the absorption from the scattering, which allows the absorption coefficient to be extracted. Eventually, we present how we used the IPL point to detect the contamination levels of Intralipid and India ink in concentrations of 30-46 and 0-4 ppm, respectively. These findings suggest that the IPL point is an intrinsic parameter of a system that can be used as an absolute calibration point. This method provides a new and efficient way of measuring and differentiating between various types of contaminants in water.