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The correct development of seeds is a pivotal requirement for species preservation. This process depends on the balance between sensing the environmental stimuli/stressors and hormone-mediated transduction, which results in physiological responses. The red and blue regions of the electromagnetic spectrum are known to influence seed dormancy and germination. Here, we report on the effects induced by the blue (peak at 430 nm) and red (peak at 650 nm) regions of the electromagnetic spectrum on seeds from photo- and skotomorphogenetic capsules developed under white, blue, or red light. Regardless of exposure, seeds from skotomorphogenetic capsules showed an almost absent dormancy in association with altered germination kinetics. Conversely, in seeds from photomorphogenetic capsules, the exposure to the blue region induced skotomorphogenetic-like effects, while the exposure to the whole visible range (350–750 nm), as well as to only the red region, showed a dose-related trend. The observed differences appeared to be dependent on the wavelengths in the red and to be based on transduction mechanisms taking place in fruits. While the molecular bases of this differential effect need to be clarified, the results hint at the role played by different light wavelengths and intensities in seed development and germination. These findings may be relevant for applications in crop production and species safeguarding.

Bio-optical imaging can noninvasively describe specific biochemical reaction events in small animals using endogenous or exogenous imaging reagents to label cells, proteins, or DNA. The fluorescence optical bio-imaging system excites the fluorescent group to a high energy state by excitation light and then generates emission light. However, many substances in the organism will also emit fluorescence after being excited by the excitation light, and the nonspecific fluorescence generated will affect the detection sensitivity. This paper designs and develops a set of high-level biosafety in vivo fluorescence imaging system for small animals suitable for virology research and proposes a target area extraction algorithm for fluorescence images. The fluorescence image target extraction algorithm first maps the nonlinear separation data in the low-dimensional space to the high-dimensional space. Then, based on the analysis of the characteristics of the fluorescent region, a method for discriminating the target fluorescent region based on the two-step entropy function is proposed, and the real target fluorescent region is obtained according to the set connected region. Based on the experiment of collecting and analyzing the in vivo fluorescent images of mice, it is verified that the proposed algorithm can automatically extract the target fluorescent region better than the classical linear model. It shows that the proposed algorithm is less affected by background fluorescence, and the estimated separated spectrum based on this method is closer to the real target spectrum.

Surveillance and detection of polioviruses (PV) remain crucial to monitoring eradication progress. Intratypic differentiation (ITD) using the real-time RT-PCR kit is key to the surveillance workflow, where viruses are screened after cell culture isolation before a subset are verified by sequencing. The ITD kit is a series of real-time RT-PCR assays that screens cytopathic effect (CPE)-positive cell cultures using the standard WHO method for virus isolation. Because ITD screening is a critical procedure in the poliovirus identification workflow, validation of performance of real-time PCR platforms is a core requirement for the detection of poliovirus using the ITD kit. In addition, the continual update and improvement of the ITD assays to simplify interpretation in all platforms is necessary to ensure that all real-time machines are capable of detecting positive real-time signals. Four platforms (ABI7500 real-time systems, Bio-Rad CFX96, Stratagene MX3000P, and the Qiagen Rotor-Gene Q) were validated with the ITD kit and a redesigned poliovirus probe. The poliovirus probe in the real-time RT-PCR pan-poliovirus (PanPV) assay was re-designed with a double-quencher (Zen™) to reduce background fluorescence and potential false negatives. The updated PanPV probe was evaluated with a panel consisting of 184 polioviruses and non-polio enteroviruses. To further validate the updated PanPV probe, the new assay was pilot tested in five Global Polio Laboratory Network (GPLN) laboratories (Madagascar, India, Philippines, Pakistan, and Democratic Republic of Congo). The updated PanPV probe performance was shown to reduce background fluorescence and decrease the number of false positives compared to the standard PanPV probe.

In historical paintings, the detection of low amounts of pigments and dyes by Raman spectroscopy can sometimes be challenging, in particular for fluorescent dyes. This issue can be overcome by using SERS (surface-enhanced Raman spectroscopy) which takes advantage of the properties of nanostructured metal surfaces to quench fluorescence and enhance Raman signals. In this work, silver nanostars (AgNSs) are applied for the first time to real art samples, in particular to painting cross-sections, exploiting their effective SERS properties for pigment identification. The case study is the Madonna della Misericordia of the National Gallery of Parma (Italy). Cross-sections were analyzed at first by optical microscopy, SEM-EDS, and micro-Raman spectroscopy. Unfortunately, in some cross-sections, the application of conventional Raman spectroscopy was hindered by an intense background fluorescence. Therefore, AgNSs were deposited and used as SERS-active agent. The experimentation was successful, allowing us to identify a modern dye, namely copper phthalocyanine. This result, together with the detection of other modern pigments (titanium white) and expert visual examination, allowed to reconstruct the painting history, postdating its realization from the 15th century (according to the Gallery inventory) to 19th century with a heavy role of recent (middle 20th century) restoration interventions.

Genetically-encoded indicators of neuronal activity enable the labeling of a genetically defined population of neurons to optically monitor their activities. However, researchers often find difficulties in identifying relevant signals from excessive background fluorescence. A photoactivatable version of a genetically encoded calcium indicator, sPA-GCaMP6f is a good example of circumventing such an obstacle by limiting the fluorescence to a region of interest defined by the user. Here, we apply this strategy to genetically encoded voltage (GEVI) and pH (GEPI) indicators. Three photoactivatable GEVI candidates were considered. The first one used a circularly-permuted fluorescent protein, the second design involved a Forster resonance energy transfer (FRET) pair, and the third approach employed a pH-sensitive variant of GFP, ecliptic pHluorin. The candidate with a variant of ecliptic pHluorin exhibited photoactivation and a voltage-dependent fluorescence change. This effort also yielded a pH-sensitive photoactivatable GFP that varies its brightness in response to intracellular pH changes.

Cellular processes take place over many timescales, prompting the development of precision measurement technologies that cover milliseconds to hours. Here we describe ArrayG, a bipartite fluorogenic system composed of a GFP-nanobody array and monomeric wtGFP binders. The free binders are initially dim but brighten 15 fold upon binding the array, suppressing background fluorescence. By balancing rates of intracellular binder production, photo-bleaching, and stochastic binder exchange on the array, we achieved temporally unlimited tracking of single molecules. Fast (20-180Hz) tracking of ArrayG tagged kinesins and integrins, for thousands of frames, revealed repeated state-switching and molecular heterogeneity. Slow (0.5 Hz) tracking of single histones for as long as 1 hour showed fractal dynamics of chromatin. We also report ArrayD, a DHFR-nanobody-array tag for dual color imaging. The arrays are aggregation resistant and combine high brightness, background suppression, fluorescence replenishment, and extended choice of fluorophores, opening new avenues for seeing and tracking single molecules in living cells.

Polymerase chain reaction (PCR) is a laboratory procedure to amplify and simultaneously quantify targeted DNA molecules, and then detect the product of the reaction at the end of all the amplification cycles. A more modern technique, real-time PCR, also known as quantitative PCR (qPCR), detects the product after each cycle of the progressing reaction by applying a specific fluorescence technique. The quantitative methods currently used to analyze qPCR data result in varying levels of estimation quality. This study compares the accuracy and precision of the estimation achieved by eight different models when applied to the same qPCR dataset. Also, the study evaluates a newly introduced data preprocessing approach, the taking-the-difference approach, and compares it to the currently used approach of subtracting the background fluorescence. The taking-the-difference method subtracts the fluorescence in the former cycle from that in the latter cycle to avoid estimating the background fluorescence. The results obtained from the eight models show that taking-the-difference is a better way to preprocess qPCR data compared to the original approach because of a reduction in the background estimation error. The results also show that weighted models are better than non-weighted models, and that the precision of the estimation achieved by the mixed models is slightly better than that achieved by the linear regression models.

Background Fluorescence in situ hybridization (FISH) analysis can provide important information in the management of patients with hematologic malignancies. However, FISH performed in addition to G-banded karyotype can be labor-intensive and expensive. The aim of this study was to evaluate whether FISH gives additional information in the setting of adequate conventional cytogenetics in cases of hematologic malignancies. Methods Bone marrow aspirates were obtained from 135 patients at diagnosis (56 AML, 32 MDS, 20 ALL, and 27 MM) between 2005 and 2010. Interphase FISH was performed using the following probes: BCR/ABL1, AML1/ETO, PML/RARA, CBFB, MLL, EGR1, CEP8, and D7S486 for AML; CEP8, D20S108, EGR1, and D7S486 for MDS; BCR/ABL1, MLL, CDKN2A (p16), ETV6, and 6q21/c-myc for ALL; IgH, TP53, D13S25, IgH/CCND1, IgH/MAF, IgH/FGFR3, and 1q21/8p21 for MM. We compared the results of FISH with the corresponding aberrations identified by G-banded karyotype. Results Additional genetic aberrations detected by FISH (which were not identified by G-banded karyotype) were 4%, 9%, 50%, and 67% in AML, MDS, ALL, and MM, respectively. In ALL, CDKN2A and ETV6 FISH revealed additional genetic aberrations in 33% and 28% of cases, respectively. In MM, FISH was of benefit in detecting IgH, D13S25, TP53, and 1q21 rearrangements, not detected by G-banded karyotype (31%, 36%, 20%, and 40%, respectively). Conclusion These results suggest that performing FISH in addition to G-banded karyotype may contribute little additional genetic information in AML and MDS, whereas routine FISH analysis appears to be an efficient screening method in ALL and MM.

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.

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.

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.

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.

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.

A procedure for quantification of distinct bacterial populations in aggre-gated ecosystems was developed. It is based on fluorescent in situ hybridization coupled with confocal-laser-scanning microscopy and surface measurement of hybridized bacteria by image analysis. The proportion of a targeted bacterial species was ob-tained by comparison between the surface hybridized by a specific probe and the surface hybridized with a general bacterial reference probe. The accuracy of the re-sults obtained was evaluated by direct visual counting on the same sets of images. The analytical uncertainty of the image analysis procedure was determined and de-pended on the threshold values selected by the operator for cutting between signal and background fluorescence. The number of fields to be analyzed for reliable quantification was also determined. This procedure allows a quantification of the proportions of bacterial species in aggregated bacterial ecosystems which gives more accurate re-sults than visual counting because of the large number of bacteria counted. Moreover, since floc structures are preserved, it also gives information about the distribution of bacterial populations in the floc material which might reveal bacterial interactions within the community.

The fluorescence method of Vurek and Pegram [1] is one of the most commonly used techniques to measure tubular fluid inulin concentration in micro-puncture studies [2–5]. This method has the advantages of relative simplicity and low cost. However, we recently discovered an inherent, systematic error in this method when employed in the setting of prolonged experiments. A consistent decrease in the background fluorescence of the dimedone-phosphoric acid blanks, inulin standards and tubular fluid samples was discovered when transferring into reagent-filled micro-cuvettes was carried out over several hours. The following report describes the manner in which the error was defined and outlines a technical modification which results in its correction.

Fluorescence lifetime of NADH had been used as an optical marker for monitoring cellular metabolism. In our pervious studies, we have demonstrated that NADH lifetime of hMSCs increase gradually with time of osteogenic differentiation. In this study, we measured NADH lifetime of hMSCs from a different donor as well as the corresponding metabolic indices such as ATP level, oxygen consumption and lactate release. We also measure the quantity of Complex I, III, IV and V. The results show that during differentiation more oxygen consumed, higher ATP level expressed and less lactate released, and the increase of NADH lifetime was associated with ATP level. Higher expression of the total Complex protein was observed at 3 and 4 weeks after differentiation than controls. However, Complex I expression did not show significant correlation with the increase of NADH fluorescence lifetime. In summary, we demonstrated that the change of NADH lifetime was associated with the metabolic change during osteogenic differentiation of hMSCs. The increase of NADH lifetime was in part due to the increased Complex protein interaction in mitochondria after differentiation.

A stacked plasmonic nanowell-nanopore biosensor strongly suppresses the background fluorescence from the bulk and yields net more than tenfold enhancement of the fluorescence intensity. The device offers extremely high signal-to-background (S/B) ratio for single-molecule detection at ultralow excitation laser intensities, while maintaining extremely high temporal bandwidth for single-DNA sensing.