Search

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal–to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

The development of non-invasive analytical methods is of widespread interest to the field of conservation science, providing chemical insight into the materials used to create painted works of art, which can, for example, inform decisions about their restoration and preservation, or help discern original works from forgeries. A key undertaking in this area is to develop practical methods for identifying and understanding the chemical processes that occur in paint films under ageing. Furthermore, whereas a number of scientific studies have focussed on model systems in which natural ageing processes are simulated in a short time by irradiation under ultraviolet (UV) light, it remains to be established to what extent natural and accelerated ageing induce similar chemical changes. In this work, we employ FT-Raman spectroscopy, together with a simple spectral-deconvolution algorithm, to study in detail the spectral changes accompanying the natural and UV-accelerated ageing of lead-based paint films. We find that the two processes differ significantly, and that spectroscopic signatures, principally in the fluorescence background, can thus be used to differentiate the two modes of ageing and hence possibly to identify attempted forgeries. Our studies also suggest that paints based on proteinaceous binders are more stable to ageing than lipid-bound ones. Finally, we investigate the possibility of using our chemometric deconvolution technique, in conjunction with multivariate analysis, for the semi-automated characterisation of the degree or extent of ageing in unknown samples.

Two-color microarrays are a powerful tool for genomic analysis, but have noise components that make inferences regarding gene expression inefficient and potentially misleading. Background fluorescence, whether attributable to nonspecific binding or other sources, is an important component of noise. The decision to subtract fluorescence surrounding spots of hybridization from spot fluorescence has been controversial, with no clear criteria for determining circumstances that may favor, or disfavor, background subtraction. While it is generally accepted that subtracting background reduces bias but increases variance in the estimates of the ratios of interest, no formal analysis of the bias-variance trade off of background subtraction has been undertaken. In this paper, we use simulation to systematically examine the bias-variance trade off under a variety of possible experimental conditions. Our simulation is based on data obtained from 2 self versus self microarray experiments and is free of distributional assumptions. Our results identify factors that are important for determining whether to background subtract, including the correlation of foreground to background intensity ratios. Using these results, we develop recommendations for diagnostic visualizations that can help decisions about background subtraction.

The π-shift method has been used to increase the sensitivity of lifetime-selective fluorescence imaging for different fluorophores. In this paper, to improve the sensitivity of a homodyne assisted phase-resolved fingerprint imaging system, an approach using an even-step-shift method to ease the application of the π-shift method is proposed and presented. By employing an even-step-shift method, the two phases used for the π-shift method can be determined directly by distinguishing the positions when the background fluorescence goes to zero. A detailed theoretical analysis of the proposed approach is presented. Experiments are carried out using fingerprint samples deposited on green fluorescent paper and black tape. The experimental results demonstrate the effectiveness and significance of the proposed approach.

We report a novel fluorescence imaging approach to imaging nonfluorescence-labeled biological tissue samples. The method was demonstrated by imaging neurons in Golgi-Cox-stained and epoxy-resin-embedded samples through the excitation of the background fluorescence of the specimens. The dark neurons stood out clearly against background fluorescence in the images, enabling the tracing of a single dendritic spine using both confocal and wide-field fluorescence microscopy. The results suggest that the reported fluorescence imaging method would provide an effective alternative solution to image nonfluorescence-labeled samples, and it allows tracing the dendritic spine structure of neurons.

Magnetically modulated optical nanoprobes (MagMOONs) have been synthesized for the detection of pH changes in intracellular environments. MagMOONs are nanosensors designed to emit fluorescent signals and to blink in response to rotating magnetic fields. Separating the blinking probe signal from the unmodulated background signal allows for simple and sensitive detection of low analyte concentrations in the presence of autofluoresence.

Polymers and plastics are receiving increased attention as materials for microfluidics and microTAS applications. Given the ubiquity of fluorescence detection techniques in micro-analytical systems, the fluorescence properties of polymers and plastics should not be overlooked. We survey some commonly available polymer thin-films for their fluorescence behaviour under standardized conditions to determine which materials are most suitable for high-sensitivity fluorescence detection lab chips. The initial fluorescence intensities of some of the materials surveyed were significantly higher than glass and fused silica controls, and decreased over the three hour period with complex kinetics. We then discuss how this has confounded fluorescence detection in our analytical context, and possible mechanisms for the decrease.

A small-molecule probe for sulfatase is developed that shows a significant change in fluorescence upon reaction with sulfatase in an activity-based manner. As this probe is free from interference from background fluorescence caused by an unreacted probe, it could be a simple and efficient tool for the study of sulfatase activity.

The energy-transfer luminescence of complexes of Tb3+ with calix- (CAS), thiacalix- (TCAS), and sulfonylcalix[4]arene-p-tetrasulfonates (SO2CAS), in which four p-phenolsulfonates are jointed by –CH2–, –S–, and –SO2–, respectively, was applied to an ultratrace determination of the Tb3+ ion. Based on the complexation behavior of each calix ligand, a determination procedure was established. A time-resolved measurement was conveniently employed to separate the background fluorescence from the luminescence of the calix-Tb3+ complexes. An improved sensitivity was attained by sulfur-bridged calixes, TCAS and SO2CAS, as compared to CAS, owing to the photophysical properties of the Tb3+-complexes. The detection limits for Tb3+ ion by CAS, TCAS, and SO2CAS ligands were estimated to be 8.2×10−10 mol dm−3 (131 ppt), 2.0 × 10−10 mol dm−3 (32 ppt), and 2.3 × 10−10 mol dm−3 (37 ppt) at S/N = 3, respectively. In terms of the selectivity, the effect of diverse coexisting ions on the luminescence intensity of the Tb3+ comp...

Single molecule localization microscopy has become a standard tool for nanoscale imaging of biological systems. However, improper treatment of non-uniform background fluorescence can degrade image accuracy and precision. In particular this can be problematic in cases where the structure being imaged is correlated in space with high background fluorescence. Various methods have already been implemented for correction of background fluorescence. Here, we demonstrate that these background estimation and correction procedures lead to both systematic and random error in emitter localization, under experimentally relevant background conditions.

Visualization of microorganisms in soils and sediments using fluorescent dyes is a common method in microbial ecology studies, but is often ham- pered by strong nonspecific background fluorescence that can mask genu- ine cellular signals. The cyanine nucleic acid binding dyes TO-PRO-3 and TOTO-3 iodide enabled a clear detection of microbial cells in a mineral soil, while nonspecific background was greatly reduced compared with com- monly used dyes. When used as counterstains for fluorescence in situ hybrid- ization (FISH), both cyanine dyes allowed identification of microbial cells despite strong background from nonspecifically bound probes. TO-PRO-3 and TOTO-3 are easy to use and represent superior alternatives for detecting microorganisms in soil environments.

Förster resonance energy transfer (FRET) experiments are often used to study interactions between integral membrane proteins in cellular membranes. However, in addition to the FRET of sequence-specific interactions, these experiments invariably record a contribution due to proximity FRET, which occurs when a donor and an acceptor approach each other by chance within distances of ∼100 Å. This effect does not reflect specific interactions in the membrane and is frequently unappreciated, despite the fact that its magnitude can be significant. Here we develop a computational description of proximity FRET, simulating the cases of proximity FRET when fluorescent proteins are used to tag monomeric, dimeric, trimeric, and tetrameric membrane proteins, as well as membrane proteins existing in monomer-dimer equilibria. We also perform rigorous experimental measurements of this effect, by identifying membrane receptors that do not associate in mammalian membranes. We measure the FRET efficiencies between yellow fluorescent protein and mCherry-tagged versions of these receptors in plasma-membrane-derived vesicles as a function of receptor concentration. Finally, we demonstrate that the experimental measurements are well described by our predictions. The work presented here brings additional rigor to FRET-based studies of membrane protein interactions, and should have broad utility in membrane biophysics research.

Foodborne illnesses occur in both industrialized and developing countries, and may be increasing due to rapidly evolving food production practices. Yet some primary tools used to assess food safety are decades, if not centuries, old. To improve the time to result for food safety assessment a sensitive flow cytometer based system to detect microbial contamination was developed. By eliminating background fluorescence and improving signal to noise the assays accurately measure bacterial load or specifically identify pathogens. These assays provide results in minutes or, if sensitivity to one cell in a complex matrix is required, after several hours enrichment. Conventional assessments of food safety require 48 to 56 hours. The assays described within are linear over 5 orders of magnitude with results identical to culture plates, and report live and dead microorganisms. This system offers a powerful approach to real-time assessment of food safety, useful for industry self-monitoring and regulatory inspection.

We describe a technique for imaging single mRNAs in living cells based on fluorescent protein (FP) complementation. We employ the high affinity interaction between the bacterial phage MS2/PP7 coat proteins and their respective RNA binding motifs as an RNA scaffold to bring two halves of a split-FP together to image single reporter mRNAs without background fluorescence.

A method has been explored for assessing hydrophobic polypeptides in beer by measuring fluorescence developed upon interaction of the proteins with 1-anilino-8-naphthalenesulfonate. The method was initially applied to three separate purified proteins (bovine serum albumin, cytochrome c and catalase) and different response patterns were observed, suggesting that a simple picture would not be obtained with a complex polypeptide mixture such as beer. Furthermore substantial interference, including a high level of background fluorescence, was caused by beer components, especially in ales. Use of the method, after reducing interference by dilution or dialysis of the beer, does not appear to confer any additional value over the direct measurement of total protein in beer using Coomassie Blue-binding methods.

Photoluminescence detection of latent fingerprints has over the last quarter century brought about a new level of fingerprint detection sensitivity. The current state of the art is briefly reviewed to set the stage for upcoming new fingerprint processing strategies. These are designed for suppression of background fluorescence from articles holding latent prints, an often serious problem. The suppression of the background involves time-resolved imaging, which is dealt with from the perspective of instrumentation as well as the design of fingerprint treatment strategies. These focus on lanthanide chelates, nanocrystals, and nanocomposites functionalized to label fingerprints.

A series of aluminosilicate zeolites are characterized by UV Raman spectroscopy for the first time, and UV Raman spectra of various zeolites give strong and clear bands with high resolution, while conventional Raman spectra of these zeolites are difficult to obtain because of a strong background fluorescence. Additionally, these zeolites show several new bands in UV Raman spectroscopy. A summary of these UV Raman spectra over various zeolites suggests that the bands at 470–530, 370–430, 290–410, and 220–280 cm −1 can be assigned to the bending modes of 4-, 5-, 6-, and 8-membered rings of aluminosilicate zeolites, respectively. Furthermore, it is found that the band intensity of zeolites in UV Raman spectroscopy is dependent on the Si/Al ratio. Moreover, the UV Raman spectra of crystallization, for zeolite X at various times show that, in the initial stage of crystallization, the 4-membered rings (510 cm −1 ) interconnect each other to form β-cages with 6-membered rings (390 cm −1 ), which further crystallize to zeolite X.

For a 6-month period between May and September 1999, the staff and crew of the National Oceanic and Atmospheric Administration (NOAA) research ship R/V Ferrel augmented their activities to support a baseline fluorometry study for NOAA's Office of Response and Restoration (OR&R). During the study period, scientific data were collected at more than 50 stations in the Atlantic Ocean and Gulf of Mexico. The study was designed, in part, to assess the Special Monitoring of Applied Response Technologies (SMART) program. SMART is a joint U.S. Coast Guard (USCG), U.S. Environmental Protection Agency (EPA), Centers for Disease Control and Prevention (CDC), and NOAA monitoring program that provides near real-time feedback to the Unified Command during dispersant applications to mitigate marine oil spills. Using fluorometers to accurately measure dispersed oil concentrations is not a trivial task. Detector response values vary because of oil composition and oil weathering; dispersed oil is not in true solution; and natural waters contribute matrix effects and background fluorescence. It was the latter two that were investigated. Because seawater is a complex mixture of dissolved chemicals, particulates, and living plants and animals, archiving samples for future analysis relative to background fluorescence is problematic. Each contributes to background fluorescence and matrix effects when dispersed oil is present in the sample. Since water samples change with storage, only near real-time analyses are valid; therefore, using an at-sea laboratory like the NOAA's Ferrel was essential to the study design. Results suggest that the adverse flouorometry effects observed for open-ocean environments were within typical quality-objective goals. Therefore, the range of seawater composition changes observed in this investigation had very little effect on the ability to detect dispersed oil and meet the SMART mission objectives.

Background: Previous systems for dot (signal) counting in fluorescence in situ hybridization (FISH) images have relied on an auto-focusing method for obtaining a clearly defined image. Because signals are distributed in three dimensions within the nucleus and artifacts such as debris and background fluorescence can attract the focusing method, valid signals can be left unfocused or unseen. This leads to dot counting errors, which increase with the number of probes. Methods: The approach described here dispenses with auto-focusing, and instead relies on a neural network (NN) classifier that discriminates between in and out-of-focus images taken at different focal planes of the same field of view. Discrimination is performed by the NN, which classifies signals of each image as valid data or artifacts (due to out of focusing). The image that contains no artifacts is the in-focus image selected for dot count proportion estimation. Results: Using an NN classifier and a set of features to represent signals improves upon previous discrimination schemes that are based on nonadaptable decision boundaries and single-feature signal representation. Moreover, the classifier is not limited by the number of probes. Three classification strategies, two of them hierarchical, have been examined and found to achieve each between 83% and 87% accuracy on unseen data. Screening, while performing dot counting, of in and out-of-focus images based on signal classification suggests an accurate and efficient alternative to that obtained using an auto-focusing mechanism. Cytometry 43:87‐93, 2001. © 2001 Wiley-Liss, Inc.

1Departments of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA. 2Johns Hopkins Physical Sciences in Oncology Centre, Johns Hopkins University, Baltimore, MD 21218, USA. 3Departments of Medicine and Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA. 4BRIGHT Institute, Washington University School of Medicine, St. Louis, MO 63110, USA. 5Correspondence should be addressed to D.W. or G.D.L. (wirtz@jhu.edu or glongmor@dom.wustl.edu) Reply: reducing background fluorescence reveals adhesions in 3D matrices

Two enzyme-linked immunoassay (ELISA) systems for rapid screening of Listeria spp. were compared for their use in analysis of spiked foods regulated by the U.S. Food and Drug Administration. The Tecra Listeria kit is a 48 h visual ELISA that detects Listeria spp. through colorimetry. It has been approved for first action by AOAC INTERNATIONAL.The Vitek immunodiagnostic assay system for Listeria (\i\D AS LIS) is a fully automated 48 h ELISA that detects Listeria spp. by immunofluorescence. Fifty-two food samples were artificially contaminated with high (11-42 colony-forming units [cfu]/25 g food) and low (2-8 cfu/25 g food) levels of L monocytogenes and screened by the 2 protocols. Unspiked samples were also assayed as negative controls. Six unspiked samples werefound positive for Listeria spp. by both methods: 3 were identified as L. monocytogenes and 3 as L. innocua by official methods. Both ELISA methods detectedall spiked samples. One unspiked sample was assayed positive by Tecra and negative by VIDAS LIS. No Listeria spp. were recovered when the sample was tested by the conventional method. Nointerference due to background fluorescence of food matrixes was observed in the VIDAS LIS method. Results suggest a modified VIDAS LIS preenrichment medium may be used in place of the VIDAS standard medium in the protocol.

A cell permeable fluorescence turn-on probe, AcGQCy7, was developed to image β-galactosidase activity in living cells. Once internalized by β-galactosidase-expressing cells, the probe was hydrolyzed into a highly fluorescent molecule, and the fluorescent signal was retained in mitochondria for several days. This resulted in a long-lasting and strong β-galactosidase-dependent intracellular fluorescent signal with little background fluorescence in the culture media.

The use of genetic engineering techniques allows researchers to combine functional proteins with fluorescent proteins (FPs) to produce fusion proteins that can be visualized in living cells, tissues, and animals. However, several limitations of FPs, such as slow maturation kinetics or issues with photostability under laser illumination, have led researchers to examine new technologies beyond FP-based imaging. Recently, new protein-labeling technologies using protein/peptide tags and tag-specific probes have attracted increasing attention. Although several protein-labeling systems are com mercially available, researchers continue to work on addressing some of the limitations of this technology. To reduce the level of background fluorescence from unlabeled probes, researchers have pursued fluorogenic labeling, in which the labeling probes do not fluoresce until the target proteins are labeled. In this Account, we review two different fluorogenic protein-labeling systems that we have recently developed. First we give a brief history of protein labeling technologies and describe the challenges involved in protein labeling. In the second section, we discuss a fluorogenic labeling system based on a noncatalytic mutant of β-lactamase, which forms specific covalent bonds with β-lactam antibiotics such as ampicillin or cephalosporin. Based on fluorescence (or Förster) resonance energy transfer and other physicochemical principles, we have developed several types of fluorogenic labeling probes. To extend the utility of this labeling system, we took advantage of a hydrophobic β-lactam prodrug structure to achieve intracellular protein labeling. We also describe a small protein tag, photoactive yellow protein (PYP)-tag, and its probes. By utilizing a quenching mechanism based on close intramolecular contact, we incorporated a turn-on switch into the probes for fluorogenic protein labeling. One of these probes allowed us to rapidly image a protein while avoiding washout. In the future, we expect that protein-labeling systems with finely designed probes will lead to novel methodologies that allow researchers to image biomolecules and to perturb protein functions.

A luminescent probe based on a europium complex is developed, which effectively distinguishes adenosine-5′-triphosphate (ATP) from adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in pure water at pH 6.8. With a longer lifetime (in ms range), the probe is prospectively applied to biological systems to monitor ATP levels by completely removing the background fluorescence of other molecules.

We propose a novel approach to background correction for Infinium HumanMethylation data to account for technical variation in background fluorescence signal. Our approach capitalizes on a new use for the Infinium I design bead types to measure non-specific fluorescence in the colour channel opposite of their design (Cy3/Cy5). This provides tens of thousands of features for measuring background instead of the much smaller number of negative control probes on the platforms (n = 32 for HumanMethylation27 and n = 614 for HumanMethylation450, respectively). We compare the performance of our methods with existing approaches, using technical replicates of both mixture samples and biological samples, and demonstrate that within- and between-platform artefacts can be substantially reduced, with concomitant improvement in sensitivity, by the proposed methods.

Background Fluorescence imaging at high spectral resolution allows the simultaneous recording of multiple fluorophores without switching optical filters, which is especially useful for time-lapse analysis of living cells. The collected emission spectra can be used to distinguish fluorophores by a computation analysis called linear unmixing. The availability of accurate reference spectra for different fluorophores is crucial for this type of analysis. The reference spectra used by plant cell biologists are in most cases derived from the analysis of fluorescent proteins in solution or produced in animal cells, although these spectra are influenced by both the cellular environment and the components of the optical system. For instance, plant cells contain various autofluorescent compounds, such as cell wall polymers and chlorophyll, that affect the spectral detection of some fluorophores. Therefore, it is important to acquire both reference and experimental spectra under the same biological conditions and through the same imaging systems. Results Entry clones (pENTR) of fluorescent proteins (FPs) were constructed in order to create C- or N-terminal protein fusions with the MultiSite Gateway recombination technology. The emission spectra for eight FPs, fused C-terminally to the A- or B-type cyclin dependent kinases (CDKA;1 and CDKB1;1) and transiently expressed in epidermal cells of tobacco (Nicotiana benthamiana), were determined by using the Olympus FluoView™ FV1000 Confocal Laser Scanning Microscope. These experimental spectra were then used in unmixing experiments in order to separate the emission of fluorophores with overlapping spectral properties in living plant cells. Conclusions Spectral imaging and linear unmixing have a great potential for efficient multicolor detection in living plant cells. The emission spectra for eight of the most commonly used FPs were obtained in epidermal cells of tobacco leaves and used in unmixing experiments. The generated set of FP Gateway entry vectors represents a valuable resource for plant cell biologists.

GelScann(1.0) is a user-friendly program that accurately quantitates DNA from CCD imaged agarose gels. The algorithm automatically locates lanes, locates bands within a given lane, and quantitates the intensity of each band. GelScann (1.0) uses a statistical method to discriminate DNA from local background fluorescence. The sensitivity of the LaneFinder and BandFinder can be adjusted by the user interacting with GelScann (1.0)'s option box. We demonstrate that GelScann(1.0) can accurately determine DNA that GelScann(1.0) can accurately determine DNA concentrations for routine molecular biology applications and determine the relative intensities of PCR amplified fragments for genetic testing.

Fluorescence angiography with indocyanine green (ICG) provides real-time information regarding the patency of vessels. To enhance the capability to delineate flow direction, flow velocity and sequence of dye filling in different components of complex spinal vascular lesions such as perimedullary arteriovenous fistulas (AVFs), we tried selective intraarterial injection of ICG with catheterization in the proximity of the AVFs.Prior to taking the position for surgery, a metallic introducer sheath was placed into the femoral artery, and placed over the patient's lateral buttock. After the exposure of the AVFs, a standard angiographic catheter was advanced into the proximal portion of the feeding artery and a small volume of diluted ICG (0.06 mg in 5 ml saline for one examination) injected repeatedly. To avoid the thromboembolism, heparinized saline was perfused continuously thorough the catheter and sheath.The small injection volume and the close proximity of the injection site to the lesions resulted in quick rise and fall of the fluorescence without any background. Time and spatial resolution of analysis were enhanced; flow dynamics such as direction, velocity and alteration after temporary occlusion were well visualized. The feeders and drainers were clearly distinguished, and the shunts could be precisely identified.Selective intraarterial injection ICG fluorescence angiography was very useful for perimedullary AVFs. Albeit that it requires intraoperative selective catheterization, this repeatable technique has an advantage to improve temporary resolution and provides accurate information of the flow dynamics through the complex anatomy of vascular lesions.

Trypanothione (TSH2), a metabolite unique to trypanosomal parasites, was evaluated as a potential biomarker for trypanosomal infection using fluorescence as the means of detection. Fluoroescein arsenical helix binder (FLASH) was prepared and used to detect TSH2. Since it has low background fluorescence and forms a highly emissive complex with TSH2, it can be used to detect low micromolar concentrations of TSH2 in serum. The large dynamic range of FLASH and its selectivity for detection of the dithiol metabolite indicate that arsenical probes may offer a promising new platform for the diagnosis of trypanosomal infection.

INTRODUCTIONThe activity of ß-glucuronidase (GUS) can be accurately determined in extracts of plant tissue using 4-methylumbelliferyl ß-D-glucuronide (4-MUG) as a substrate. Upon hydrolysis by GUS, the fluorochrome 4-methyl umbelliferone (4-MU) is produced. Using excitation at 365 nm and measuring emission at 455 nm, the amount of 4-MU produced can be quantified. Under these conditions, background fluorescence from the substrate is negligible, especially if the appropriate filter is selected.

Problems encountered in field applications of water tracing dyes include background fluorescence and interference between dyes, where the complexity of hydrological systems, particularly in karst areas, makes multiple dye tracing experiments desirable. The fluorescence of water tracing dyes may vary dramatically according to the pH of the water, and this property can be used to address these problems. The response of a number of commonly available water tracing dyes to pH is investigated; the distinctive curves provide a means of distinguishing between dyes, and between dyes and background fluorescence caused by algae. It is not necessary to reproduce the full pH response curve to utilize these properties. Recommendations are made in the use of pH buffering to improve sensitivity in detecting dyes, and for quantitative laboratory analysis. Buffering to a small number of predetermined pH values can be used effectively to distinguish both between dyes which fluoresce at the same wavelength, and between natural and induced fluorescence. It is particularly useful in the case of the green dyes, both for detection when there are high natural backgrounds and because it allows more than one green dye to be used simultaneously. If Rhodamine is used together with, for example, Fluorescein and Pyranine, triple dye tracing experiments can be executed.

In protein microarray performance, the choice of an appropriate surface is a crucial factor. Three-dimensional substrates like nitrocellulose are known to have higher binding capacities than planar surfaces. Furthermore, they can enable the immobilization of proteins in a functional manner. One disadvantage of today's nitrocellulose-based microarrays is the high background fluorescence, which can interfere with the detection of low-abundance proteins. We have developed an innovative black nitrocellulose membrane-based protein microarray that exhibits low autofluorescence in combination with increased sensitivity and improved LOD (limit of detection). The applicability of the novel material was demonstrated with main focus on reversed-phase microarray experiments. In comparison to various commercially available microarrays, a higher sensitivity in regard to the spotted protein was achieved. In contrast to other porous nitrocellulose-based microarrays, the black nitrocellulose provides a significant lower autofluorescence and background intensity.

During fluorescence cystoscopy, it is observed that the acquired images are sometimes blurred by a greenish background originating from the bladder washout fluid. Several fluorophores are involved in this overall liquid fluorescence, and their exact origin and relative contributions remain unknown. In this study, the bladder washout fluid is sampled at different times during fluorescence cystoscopy examinations. In total, 32 samples from 12 patients are analyzed with a spectrofluorimeter (excitation range: 350-445 nm, emission range 380-700 nm). This study shows clearly that the position of the fluorescence peaks (excitation/emission wavelengths: 450/525 nm, 405/625 nm) and shoulder (440/525 nm) is reproducible between different patients. It also suggests that an excitation at wavelengths higher than 400 nm helps to suppress this solution background fluorescence. Additionally, the pH of the solution seems to influence the position of the fluorescence peaks, and this suggests that changing the pH of the examination liquid could help in avoiding the greenish background.

Anti-fluorescein antibodies were found to prevent the fading of emitted fluorescence from fibroblasts stained with fluorescein-labelled fibronectin antibodies. The prevention of fading is the result of specific binding of the fluorochromes present on the stained cells by the anti-fluorescein antibodies. The sheep anti-FITC antibody used in this study was equally effective in preventing the fading of both FITC- and carboxyfluorescein-labelled fibronectin antibodies. The method is simple, effective, does not interfere with the primary immune reaction, and in addition to preventing the fading of fluorescence it reduced the background fluorescence of the specimens. The procedure is expected to make an important contribution to improving the quality of fluorescence immunohistochemical techniques used in diagnosis.

蛍光顕微鏡と画像処理を用いた,蛍光性カルシウム指示薬Fura-2による細胞内遊離カルシウムイオン濃度分布測定で,細胞周辺部のカルシウムイオン濃度が異常に高濃度に推定される場合が多い.これは,蛍光顕微鏡でしばしば見られる蛍光像のハレーションが原因である.このため,細胞の透過光像と,カルシウムイオン分布測定時の蛍光像を重ね合わせて表示して,バックグラウンド蛍光強度と共に,ハレーション強度を指定できるようにした.更にハレーション強度以下の蛍光強度の場合,カルシウムイオン分布を表示しないようにすることで,細胞像とよく一致した分布像を得ることが容易になった.このバックグラウンド蛍光推定法は,カルシウムイオン以外に,同様な蛍光性指示薬が用いられるマグネシウムやナトリウムイオン,pHなどでも利用可能である.

In this presentation we describe a novel methodology for ultra-sensitive fluorescence immunoassays based on a new class of fluorescent biomarkers, which are strongly enhanced by nano-size metallic particles. Specifically, we discuss development of the immunoassay on the surfaces coated with metallic particles for high sensitivity detection of cardiac markers. This technology will allow detection of the biomarkers in serum and blood without separation and amplification steps. We present an experimental platform that uses front-face excitation in total internal reflection mode for efficient rejection of background fluorescence.

The rapidly emerging field of optical imaging of tumors is an exciting area of translational research. A search in PubMed for “clinical optical imaging in cancer” reveals that the first article published was in 1964 and almost half of all the articles have been published in the last 5 years. Fluorescent probes and mechanisms to target them to cancer cells were developed for in vitro experiments and rodent models of cancer. However, with improvements in hardware, targeting agents, and fluorescent agents, optical imaging is rapidly gaining new and expanded applications in preclinical and clinical research. At the forefront of this field is the discovery of monoclonal antibodies as targeting agents and fluorescent markers that can reliably be conjugated to proteins, while minimizing interference from background fluorescence. Further sophistication and flexibility of imaging systems have and will allow these techniques to move further into the clinical realm. In this chapter, we will review the basic aspects of cancer optical imaging, including therapeutic, diagnostic, and surgical applications.

Accurate measurement of antigen-positive cells by flow cytometry can be hampered by background fluorescence of antigen-negative cells and other particles (e.g., debris). This article focuses on three major causes of background (autofluorescence, spectral overlap, and undesirable antibody binding) by reviewing individual aspects of flow cytometric measurements that contribute to these causes. The appropriate use of controls facilitates a thorough understanding of these contributing factors as well as the development of robust cell labeling protocols intended for routine flow cytometric analysis. We present a set of recommendations that enables the user to develop an optimized cell labeling protocol that minimizes background and maximizes the ability to reliably distinguish between a positive and a negative population of cells. These recommendations are also intended to augment existing guidelines designed to aid in the formulation of a consensus regarding the utility of flow cytometry for the analysis of clinical samples. (C) 2009 Clinical Cytometry Society

Analysis of the errors caused by a drift in background fluorescence is performed for a fluorescence probe operated in a backscatter configuration. In some cases, significant errors can result if changes in background fluorophores during the course of fermentation are not accounted for. It is shown that the probe's sensitivity to background fluorophores must be considered to calibrate a fluorescence probe properly.

QDs may offer significant advantages in environmental and bead-based applications where the target cells need to be discriminated above background fluorescence. We have examined the possible applications of QDs for flow cytometric measurements (FCM) by studying their excitation - emission spectra and their binding to paramagnetic beads. We labelled beads with either QDs or a commonly-used fluorochrome (FITC) and studied their fluorescence intensity by FCM. Flow cytometric comparisons indicated that the minimum fluorophore concentration required for detection of QDs above autofluorescent background was 100-fold less than for FITC.

Time-lapse imaging of fluorescent proteins in living cells has become an indispensable tool in biological sciences. However, its application at the organismal level still faces a number of obstacles, such as large specimen sizes preventing illumination of internal tissues, high background fluorescence and uncontrollable movement of target tissues or embryos. Here we describe our solutions for these issues to obtain 4-D fluorescent images from living Drosophila embryos using confocal microscopes. A computational procedure that detects and corrects the shift of moving objects to virtually stabilize them in time-lapse movies (iSEMS) is presented. We discuss the importance of postimaging treatment of raw image stacks for the discovery of novel phenotypes that have previously escaped attention from the analyses of fixed specimens.

m-Benziporphodimethene is presented here as a long-wavelength Zn2+ specific chemosensor; this sensor shows fluorescence switch-on upon Zn2+ binding with no apparent background fluorescence.

Fluorescence tomography problems can be ill-posed, particularly when background fluorescence is high. We propose methods to improve robustness and present a new algorithm which reduces the intensity of artifacts.

Fourier Transform Raman spectroscopy has been developed in recent years. The application to industrial type problems provides advantages which are a major advance on conventional Raman spectroscopy. The reduction in background fluorescence permits the analysis of many samples previously not amenable to Raman spectroscopy. This article details experimental considerations of the method and provides a variety of areas in which it is making an impact in the industrial laboratory.

Fluorescence cystoscopy (FC) efficiently enhances the detection and improves the therapeutic management of early bladder cancer. During an FC, about 150 ml of water is needed to inflate the bladder. The water is quickly diluted by urine which can be fluorescent. If this bladder washout fluid (BWF) becomes fluorescent, the FC images are frequently degraded. Unfortunately, it is unclear which elements of the diet may contribute to this background fluorescence. We propose to start this exploration with over-the-counter (OTC) vitamin supplements. To this end, we measured excitation-emission matrices of urine samples and the kinetics of modifications of urine fluorescence obtained from nine healthy volunteers before, during, and after intake of a commercially available OTC vitamin supplement. The pharmacokinetics shows that the BWF fluorescence values reach a maximum 8 to 10 h after vitamin intake. They decrease in the half-day that follows and reach values close to baseline similar to 1 day afterward. Based on these results, we conclude that, in order to avoid degradations of fluorescence images, it is likely best that the intake of OTC vitamin supplements be avoided during the week preceding an FC. (C) 2015 Society of Photo-Optical Instrumentation Engineers (SPIE)

Fluorescence molecular tomography is an emerging imaging technique that resolves the bio-distribution of engineered fluorescent probes developed for in vivo reporting of specific cellular and sub-cellular targets. The method can detect fluorochromes in picomole amounts or less, imaged through entire animals, but the detection sensitivity and imaging performance drop in the presence of background, non-specific fluorescence. In this study, we carried out a theoretical and an experimental investigation on the effect of background fluorescence on the measured signal and on the tomographic reconstruction. We further examined the performance of three subtraction methods based on physical models of photon propagation, using experimental data on phantoms and small animals. We show that the data pre-processing with subtraction schemes can improve image quality and quantification when non-specific background florescence is present.