Multiple Fluorescence Signals Research Articles

Dual-modal fiber-optic sensor for simultaneous pH and temperature monitoring in tumor microenvironment

The tumor microenvironment (TME) is a complex system. Temperature, pH, oxygen content, and other biomarkers comprehensively reflect the growth state of the tumor, necessitating a multi-parameter detection strategy to garner the precise status of tumor for treatment guidance. Fluorescence imaging technology is a promising tool for realizing this goal due to its advantages of low cost, real-time responses, and high sensitivity. However, several limitations, including the shallow penetration depth of fluorescence signals in biological tissues and the potential crosstalk between multiple fluorescence signals, hinder the practical application of current fluorescence imaging technology. In this paper, a dual-mode optical fiber sensor consisting of a fiber fluorescence pH sensor and a fiber Bragg grating (FBG) temperature sensor is presented. The compact sensor uses different principles for pH and temperature monitoring, thus effectively eliminating potential crosstalk between different signals. In addition, its small size allows it to be inserted into deep tissues for detection. The sensor responds quickly to changes in pH.Moreover, the temperature sensitivity of FBG packaged with PDMS is 34 % higher than the uncoated FBG. In vivo experiments confirmed that the sensor can accurately distinguish normal tissues and tumor tissues. The proposed sensor provides a novel way to realize multi-parameter monitoring of tumor microenvironment and improve the specificity of tumor detection.

Multiplexed fluorescence and scatter detection with single cell resolution using on-chip fiber optics for droplet microfluidic applications

Droplet microfluidics has emerged as a critical component of several high-throughput single-cell analysis techniques in biomedical research and diagnostics. Despite significant progress in the development of individual assays, multiparametric optical sensing of droplets and their encapsulated contents has been challenging. The current approaches, most commonly involving microscopy-based high-speed imaging of droplets, are technically complex and require expensive instrumentation, limiting their widespread adoption. To address these limitations, we developed the OptiDrop platform; this platform is a novel optofluidic setup that leverages the principles of flow cytometry. Our platform enables on-chip detection of the scatter and multiple fluorescence signals from the microfluidic droplets and their contents using optical fibers. The highly customizable on-chip optical fiber-based signal detection system enables simplified, miniaturized, low-cost, multiparametric sensing of optical signals with high sensitivity and single-cell resolution within each droplet. To demonstrate the ability of the OptiDrop platform, we conducted a differential expression analysis of the major histocompatibility complex (MHC) protein in response to IFNγ stimulation. Our results showed the platform’s ability to sensitively detect cell surface biomarkers using fluorescently labeled antibodies. Thus, the OptiDrop platform combines the versatility of flow cytometry with the power of droplet microfluidics to provide wide-ranging, scalable optical sensing solutions for research and diagnostics.

3D microscaffolds with triple-marker sensitive nanoprobes for studying fatty liver disease in vitro.

Non-alcoholic fatty liver disease (NAFLD) is a heterogeneous condition that encompasses a wide range of liver diseases that progresses from simple hepatic steatosis to the life-threatening state of cirrhosis. However, due to the heterogeneity of this disease, comprehensive analysis of several physicochemical and biological factors that drive its progression is necessary. Therefore, an in vitro platform is required that would enable real-time monitoring of these changes to better understand the progression of these diseases. The earliest stage of NAFLD, i.e. hepatic steatosis, is characterised by triglyceride accumulation in the form of lipid vacuoles in the cytosol of hepatocytes. This fatty acid accumulation is usually accompanied by hepatic inflammation, leading to tissue acidification and dysregulated expression of certain proteases such as matrix metalloproteinases (MMPs). Taking cues from the biological parameters of the disease, we report here a 3D in vitro GelMA/alginate microscaffold platform encapsulating a triple-marker (pH, MMP-3 and MMP-9) sensitive fluorescent nanoprobe for monitoring, and hence, distinguishing the fatty liver disease (hepatic steatosis) from healthy livers on the basis of pH change and MMP expression. The nanoprobe consists of a carbon nanoparticle (CNP) core, which exhibits intrinsic pH-dependent fluorescence properties, decorated either with an MMP-3 (NpMMP3) or MMP-9 (NpMMP9) sensitive peptide substrate. These peptide substrates are flanked with a fluorophore-quencher pair that separates on enzymatic cleavage, resulting in fluorescence emission. The cocktail of these nanoprobes generated multiple fluorescence signals corresponding to slightly acidic pH (blue) and overexpression of MMP-3 (green) and MMP-9 (red) enzymes in a 3D in vitro fatty liver model, whereas no/negligible fluorescence signals were observed in a healthy liver model. Moreover, this platform enabled us to mimic fatty liver disease in a more realistic manner. Therefore, this 3D in vitro platform encapsulating triple-marker sensitive fluorescent nanoprobes would facilitate the monitoring of the changes in pH and MMP expression, thereby enabling us to distinguish a healthy liver from a diseased liver and to study liver disease stages on the basis of these markers.

Multiplex fluorescence detection of single-cell droplet microfluidics and its application in quantifying protein expression levels.

This study presented a platform of multiplex fluorescence detection of single-cell droplet microfluidics with demonstrative applications in quantifying protein expression levels. The platform of multiplex fluorescence detection mainly included optical paths adopted from conventional microscopy enabling the generation of three optical spots from three laser sources for multiple fluorescence excitation and capture of multiple fluorescence signals by four photomultiplier tubes. As to platform characterization, microscopic images of three optical spots were obtained where clear Gaussian distributions of intensities without skewness confirmed the functionality of the scanning lens, while the controllable distances among three optical spots validated the functionality of fiber collimators and the reflector lens. As to demonstration, this platform was used to quantify single-cell protein expression within droplets where four-type protein expression of α-tubulin, Ras, c-Myc, and β-tubulin of CAL 27 (Ncell = 1921) vs WSU-HN6 (Ncell = 1881) were quantitatively estimated, which were (2.85 ± 0.72) × 105 vs (4.83 ± 1.58) × 105, (3.69 ± 1.41) × 104 vs (5.07 ± 2.13) × 104, (5.90 ± 1.45) × 104 vs (9.57 ± 2.85) × 104, and (3.84 ± 1.28) × 105 vs (3.30 ± 1.10) × 105, respectively. Neural pattern recognition was utilized for the classification of cell types, achieving successful rates of 69.0% (α-tubulin), 75.4% (Ras), 89.1% (c-Myc), 65.8% (β-tubulin), and 99.1% in combination, validating the capability of this platform of multiplex fluorescence detection to quantify various types of single-cell proteins, which could provide comprehensive evaluations on cell status.

Realization of a filter-free wavelength image sensor and imaging system for visualization of wavelength information

In this study, we fabricated a filter-free wavelength image sensor for the two-dimensional visualization of wavelength information, in addition to light intensity, and realized an imaging system. In this sensor, the electrons generated in silicon in different distributions at each wavelength are divided into two parts by the potential peaks. The wavelength can be identified from the current ratio, and the light intensity can be determined from the total current. In the proposed structure, the pixel current is sequentially output by controlling the rows and columns. The layout was designed based on the proposed structure and was fabricated at the Toyohashi University of Technology LSI factory. An imaging system was constructed using operational amplifiers, A/D converters, and a microcontroller. The ratio and sum of the two signals obtained from the sensor responded linearly to the wavelength and light intensity. The wavelength response was independent of changes in the light intensity. Even when the spot was narrowed down, the response was obtained only at the corresponding pixel and succeeded in the simultaneous imaging of the intensity and wavelength. The above results can be applied to cell analysis, such as multiple fluorescence staining, where multiple fluorescence signals are detected simultaneously.

Localization of multiple DNAzymes as a branchedzyme-powered nanodevice for the immunoassay of tumor biomarkers

Traditional immunoassay methods often face challenges due to the labeling procedure of protein enzymes, the use of multiple antibodies, and severe conditions. To address these limitations, we propose the concept of incorporating branchedzyme-powered nanodevices into immunoassays. In this strategy, multiple DNAzymes are localized onto gold nanoparticles (AuNPs) along with substrates. The localization format facilitates intramolecular reactions between DNAzymes and substrates, leading to accelerated kinetics of the nanodevice. Upon the formation of an immunocomplex with an antibody on a 96-well plate, the branchedzyme-powered nanodevice catalytically releases multiple fluorescent signals under ambient temperature, eliminating the need for secondary antibodies. The branched DNAzymes exhibit catalytic properties similar to those of protein enzymes, thus simplifying the assay procedure and achieving isothermal detection. Furthermore, the detection process can be controlled by the addition or deletion of cofactors. Additionally, the affinity ligand can be easily modified to construct nanodevices specific to different targets without requiring extensive redesign. This strategy has demonstrated successful quantification of tumor biomarkers such as alpha-fetoprotein (AFP) and prostate-specific antigen (PSA) at subpicomolar concentrations, showcasing its suitability for clinical applications. Consequently, the branchedzyme-powered nanodevice represents a valuable addition to the immunoassay toolbox, opening new possibilities for clinical diagnostics.

Reporter emission multiplexing in digital PCRs (REM-dPCRs): direct quantification of multiple target sequences per detection channel by population specific reporters.

Digital PCRs (dPCRs) are widely used methods for the detection and quantification of rare abundant sequences relevant to fields such as liquid biopsy or oncology. In order to increase the information content and save valuable sample materials, there is a significant need for digital multiplexing methods that are easy to establish, analyse, and interpret, and ideally allow the usage of existing lab equipment. Herein, we present a novel reporter emission multiplexing approach for the digital PCR method (REM-dPCR), which meets these requirements. It further increases the multiplexing capacity of commercial dPCR devices. For example, we present a stepwise increase in multiplexing degrees from a monochrome two-plex assay in one detection channel to a six-plex REM-dPCR assay in a three-color dPCR device for KRAS/BRAF single nucleotide polymorphism (SNP) target sequences. The guidelines for the REM-dPCR design are presented, and the process from duplex to six-plex assay establishment, taking into account the target sequence-dependent effects on assay performance, is discussed. Furthermore, the assay-specific, sensitive and precise quantification of different fractions of KRAS mutant and wild-type DNA sequences in different ratios is demonstrated. To increase the device capacitance and the degree of multiplexing, the REM-dPCR uses the advantage of n target-independent reporter molecules in combination with target sequence-specific mediator probes. Different reporter types are labelled with fluorophores of different signal intensities but not necessarily different emission spectra. This leads to the generation of n independent single-positive populations in the dataspace, created by k detection channels, whereby n > k and n ≥ 2. By usage of target-independent but population-specific reporter types, a fixed set of six optimized signalling molecules could be defined. This reporter set enables the robust generation and precise differentiation of multiple fluorescence signals in dPCRs and can be transferred to new target panels. The set which enables stable signal generation and differentiation in a specified device would allow easy transfer to new target panels.

Canonical, and Multistranded, Alternative and Transitional Helical (C‐MATH) Nucleic Acid Microarrays: Next Generation Double‐, and Four‐Stranded DNA and RNA Microarrays

Many different types of nucleic acids, with unique structures, play important roles in prokaryotic and eukaryotic regulation of an organisms genome, e.g., canonical double‐stranded (ds‐) right‐handed B‐DNA, alternative left‐handed ds‐Z‐DNA, triplex DNA, G4‐quadruplex DNA, i‐motif DNA, slipped‐strand DNA and cruciform DNA. These canonical and exotic (e.g., Z‐DNA, and four‐stranded G4‐quadruplex DNA) nucleic acid structures also regulate many different pathological conditions. The application of exotic nucleic acids, based on its structure, has not been applied to commercial, conventional DNA microarrays. Conventional DNA microarrays are powerful tools that allow for gene expression studies, however, they have severe limitations. The basic principle behind conventional DNA microarrays is that complementary sequences of DNA molecules will bind to each other. Commercial DNA microarrays measure the relative abundance of mRNA sequences isolated from cells by taking advantage of complementary DNA base pairing. Researchers employ DNA microarrays to quantify the different levels of gene expression involving very large numbers of genes simultaneously, or to genotype numerous areas of an organisms genomic makeup. DNA microarrays needs to evolve beyond these limitations. The novel, next generation DNA and RNA microarrays, i.e., Canonical, and Multistranded, Alternative and Transitional Helical (C‐MATH) Nucleic Acid Microarrays, allow for a completely different approach to studying gene expression [e.g., genes (B‐DNA, Z‐DNA) and telomers (G4‐quadruplex DNA)], based on the different structure of ds‐DNA and RNA, and multi‐stranded DNA and RNA molecules. It also allows for the characterization of different drugs and biologics that can directly bind to nucleic acids and inhibit gene expression (or enhance it). C‐MATH DNA microarrays can enhance the “drug discovery” part of “drug discovery and development”, resulting in new drugs, new uses for old drugs and lower coast for drug‐based research. Recent advances in the novel C‐MATH nucleic acid microarrays allow for more precise quantification of multiple fluorescent signals coming from a single immobilized DNA molecule (fluorescently labelled), i.e., B‐DNA, Z‐DNA and G4‐quadruplex DNA. Fluorescent and colorimetric DNA microarrays scanners were employed. The C‐MATH microarrays allow for superior analysis of colocalization, and permits competitive binding assays involving two or more fluorescently labelled drugs or biologicals binding to a gene sequence. Positive and negative controls involve direct binding by DNA‐binding chemicals and anti‐DNA antibodies. Conventional image analysis software was used, along with the development of novel algorithms to differentiate multiple fluorescence signals and intensity. The C‐MATH DNA and RNA microarrays will allow for development of new classes of DNA and RNA structure‐based drugs, and biologics (e.g., antibody, transcription factors), i.e., sequence‐specific and non‐sequence‐specific DNA and RNA binding therapeutics. Additionally, it will also allow for characterization of nucleic acids under different environmental conditions similar to a cell.

Identification of Water-Soluble Polymers through Discrimination of Multiple Optical Signals from a Single Peptide Sensor.

The pollution of water environments is a worldwide concern. Not only marine pollution by plastic litter, including microplastics, but also the spillage of water-soluble synthetic polymers in wastewater have recently gained increasing attention due to their potential risks to soil and water environments. However, conventional methods to identify polymers dissolved in water are laborious and time-consuming. Here, we propose a simple approach to identify synthetic polymers dissolved in water using a peptide-based molecular sensor with a fluorophore unit. Supervised machine learning of multiple fluorescence signals from the sensor, which specifically or nonspecifically interacted with the polymers, was applied for polymer classification as a proof of principle demonstration. Aqueous solutions containing different polymers or multiple polymer species with different mixture ratios were identified successfully. We found that fluorophore-introduced biomolecular sensors have great potential to provide discriminative information regarding water-soluble polymers. Our approach based on the discrimination of multiple optical signals of water-soluble polymers from peptide-based molecular sensors through machine learning will be applicable to next-generation sensing systems for polymers in wastewater or natural environments.

Construction of a dual-response fluorescent probe for copper (II) ions and hydrogen sulfide (H2S) detection in cells and its application in exploring the increased copper-dependent cytotoxicity in present of H2S

Multiple types of metal ions and active small molecules (reactive nitrogen species, reactive oxygen species, reactive sulfur species, etc.) exist in living organisms. They have connections to each other and can interact and/or interfere with each other. To investigate the relationship of metal ions and active small molecules in living cells, it is necessary and critical to develop molecular tools that can track two kinds of associated certain metal ions and reactive molecules with multiple fluorescence signals. However, this is a challenging task that requires an ingenious molecular design to achieve this goal. Here, we present a fluorescent probe (D-CN) that can offer fluorescence imaging of H2S and copper (II) ions with different response signals. Recognition of H2S and Cu (II) by the new probe can result in green and red emissions, respectively, providing different signal responses to the two substances in living cells and zebrafish. In addition, we used this probe to visually prove that the cytotoxicity of copper ions in living cells increases in the presence of hydrogen sulfide and could lead to cell apoptosis.

Multiplexed intracellular detection based on dual-excitation/dual-emission upconversion nanoprobes

Multiplexed intracellular detection is desirable in biomedical sciences for its higher efficiency and accuracy compared to the single-analyte detection. However, it is very challenging to construct nanoprobes that possess multiple fluorescent signals to recognize the different intracellular species synchronously. Herein, we proposed a novel dual-excitation/dual-emission upconversion strategy for multiplexed detection through the design of upconversion nanoparticles (UCNP) loaded with two dyes for sensitization and quenching of the upconversion luminescence (UCL), respectively. Based on the two independent energy transfer processes of near-infrared (NIR) dye IR845 to UCNP and UCNP to visible dye PAPS-Zn, ClO− and Zn2+ were simultaneously detected with a limit of detection (LOD) of 41.4 and 10.5 nM, respectively. By utilizing a purpose-built 830/980 nm dual-laser confocal microscope, both intrinsic and exogenous ClO− and Zn2+ in live MCF-7 cells have been accurately quantified. Such dual-excitation/dual-emission ratiometric UCL detection mode enables not only monitoring multiple intracellular analytes but also eliminating the detection deviation caused by inhomogeneous probe distribution in cells. Through modulation of NIR dye and visible dye with other reactive groups, the nanoprobes can be extended to analyze various intracellular species, which provides a promising tool to study the biological activities in live cells and diagnose diseases.

Abstract 5115: Enhancing data quality for clinical studies of investigational cancer immunotherapy drug candidates by a new data analytics tool

Abstract Cancer immunotherapy is rapidly becoming a treatment of choice in fighting deadly cancers, especially late stage or metastatic cancers. Humanized antibodies such as alemtuzumab and nivolumab alter the dynamics of regulatory T cells, cytotoxic T cells, B cells, NK cells and dendritic cells. All of these cells can be analyzed for a wide array of intracellular and surface biomarkers with flow cytometric technology. The biggest challenges with the use of flow cytometry in the clinical trial setting are the assessment of the amount of data produced and the lack of replicable standards for each set of biomarkers as it relates to the particular characteristics and functions of leukocytes. Previously, we reported a new method to improve data quality in clinical research using big data analytics and data visualization (AACR 2018 Bioinformatics and System Biology, Abstract #2605). By statistical analysis and pattern recognition, we can simultaneously monitor unlimited data points related to numerous biomarkers. Here we describe widening the application of the new analytic method for use in the field of flow cytometry and cancer immunotherapy. A flow cytometer measures multiple fluorescence signals at once, and antibodies directed against different cellular proteins can be conjugated with a variety of fluorochromes. When an antibody, such as anti-PD-L1 Alexa Fluor 647, has a quality issue, caused either by the manufacturing process or a technical error within the clinical laboratory, an error signal can be detected by running data analytic. Through consistently monitoring each reagent used, we are working toward improving the reliability of the process itself and the quality of data. Human-machine interaction and automation are some of the most profound changes in the clinical research setting. The entire process relies heavily on the high volume of sample processing and data generated by advanced machine systems, and intermittent human intervention between each cycle of data collection, analysis, reporting and finalization. Every step in flow cytometric analyses, such as antibody quality and data validation, as well as improvement in staff training and administration of proficiency testing may be improved by this new data analytic tool. In the near future clinical research combining with artificial intelligence and machine learning will be more efficient. This will substantially reduce the time, cost and failure rate of research and development in creating new drugs. Citation Format: Chengsen Xue, Joanne Cuomo, Melissa L. Baptiste, Kai Chen, Danielle L. Kurkowski, Thomas W. Mc Closkey. Enhancing data quality for clinical studies of investigational cancer immunotherapy drug candidates by a new data analytics tool [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5115.

Single Fluorescent Probe Separately and Continuously Visualize H2S and HClO in Lysosomes with Different Fluorescence Signals.

A complicated relationship between the active small molecules exists in cells. On the organelle level, active small molecules also play an important role in the maintenance of organelle functions and roles. To investigate the relationship of biomolecules in subcellular, it is necessary and critical to develop molecular tools that can track two kinds of associated biomolecules within organelles with multiple fluorescence signals. However, this is still an unmet challenge up to date. Herein, we present the first single-fluorescent probe (Lyso-HA-HS) that can detect oxidative (HOCl) and reductive (H2S) substances within an organelle (lysosomes) with multiresponse signals. The reactions of the new probe with H2S and HOCl simultaneously result in the blue and red channels emissions, respectively, providing different signal responses to the oxidative and reductive substances in the cellular lysosomes. Using a single fluorescent probe, we first achieved dual-channel imaging of the endogenous hypochlorous acid and hydrogen sulfide, respectively, in the lysosomes in the living cells. Moreover, the highly desirable attributes of the probe Lyso-HA-HS (such as high selectivity, good membrane-permeability, and lysosome enrichment ability) may enable it to be used in revealing the relationship of HOCl and H2S in lysosomes.

Multi-Spectral Fluorescence Imaging of Colon Dysplasia InVivo Using a Multi-Spectral Endoscopy System

BACKGROUND AND STUDY AIM: To develop a molecular imaging endoscopic system that eliminates tissue autofluorescence and distinguishes multiple fluorescent markers specifically on the cancerous lesions. METHODS: Newly developed multi-spectral fluorescence endoscope device has the potential to eliminate signal interference due to autofluorescence and multiplex fluorophores in fluorescent probes. The multiplexing capability of the multi-spectral endoscope device was demonstrated in the phantom studies and multi-spectral imaging with endoscopy and macroscopy was performed to analyze fluorescence signals after administration of fluorescent probe that targets cancer in the colon. Because of the limitations in the clinical application using rigid-type small animal endoscope, we developed a flexible channel insert-type fluorescence endoscope, which was validated on the colonoscopy of dummy and porcine model. RESULTS: We measured multiple fluorescent signals simultaneously, and the fluorescence spectra were unmixed to separate the fluorescent signals of each probe, in which multiple fluorescent probes clearly revealed spectral deconvolution at the specific targeting area in the mouse colon. The positive area of fluorescence signal for each probe over the whole polyp was segmented with analyzing software, and showed distinctive patterns and significantly distinguishable values: 0.46 ± 0.04, 0.39 ± 0.08 and 0.73 ± 0.12 for HMRG, CET-553 and TRA-675 probes, respectively. The spectral unmixing was finally demonstrated in the dummy and porcine model, corroborating the targeted multi-spectral fluorescence imaging of colon dysplasia. CONCLUSION: The multi-spectral endoscopy system may allow endoscopists to clearly identify cancerous lesion that has different patterns of various target expression using multiple fluorescent probes.

Spectrally Resolved Fiber Photometry for Multi-component Analysis of Brain Circuits

To achieve simultaneous measurement of multiple cellular events in molecularly defined groups of neurons invivo, we designed a spectrometer-based fiber photometry system that allows for spectral unmixing of multiple fluorescence signals recorded from deep brain structures in behaving animals. Using green and red Ca2+ indicators differentially expressed in striatal direct- and indirect-pathway neurons, we were able to simultaneously monitor the neural activity in these two pathways in freely moving animals. We found that the activities were highly synchronized between the direct and indirect pathways within one hemisphere and were desynchronized between the two hemispheres. We further analyzed the relationship between the movement patterns and the magnitude of activation in direct- and indirect-pathway neurons and found that the striatal direct and indirect pathways coordinately control the dynamics and fate of movement. VIDEO ABSTRACT.

Hyperspectral Imaging Approaches for Measuring Three‐Dimensional FRET

Förster Resonance energy transfer (FRET) imaging has become a standard method for assessing protein‐protein interactions as well as changes in the concentration of signaling molecules, such as cAMP. However, many FRET probes or pairs have low signal‐to‐noise ratios and limited dynamic range. Our previous work suggested that hyperspectral imaging and analysis allows quantitative measurement of FRET efficiency in 3D (x, y, and t) within single cell. Here, we present data demonstrating that hyperspectral imaging approaches allow 4D (x, y, z, and t) assessment of FRET efficiency in living cells. In these studies, we have utilized a FRET biosensor consisting of a cAMP binding domain from Epac sandwiched between donor and acceptor fluorophores, Turquoise and Venus. Binding of cAMP induces a conformational change that reduces FRET efficiency. The FRET probe was transfected into HEK293 or pulmonary microvascular endothelial cells. To further demonstrate the potential of 4D hyperspectral approaches to simultaneously assess multiple fluorescence signals in single cells, we co‐transfected the calcium probe RCaMP into cells. 4D hyperspectral images were acquired using a Nikon A1R confocal microscope as follows: Excitation was provided using 405 and 561 nm laser lines. Spectral emission was detected from 424 to 724 nm in increments of 10 nm using a 32‐wavelength spectral detector on a Nikon A1R confocal microscope. A 3D (x, y, z) hyperspectral image was acquired every 15 seconds for 15 minutes. Following one minute measurement of baseline signals, we treated the cells with either 50 μM forskolin (an adenylyl cyclase activator), 0.1 μM or 1 μM isoproterenol (a beta adrenergic agonist), 10 μM rolipram (a PDE4 inhibitor), 1 μM prostaglandin, and/or calcium modulating agents such as 0.1 or 1 μM thapsigargin. Spectral images were processed using custom MATLAB scripts. Briefly, spectral images were unmixed to individual endmembers including Turquoise, Venus, DRAQ5 (nuclear label), WGA‐TRITC (plasma membrane label), and RCaMP. 4D FRET images were calculated from unmixed images. Data were re‐sliced to visualize fluorescence signals in different orientations/projections through each cell. This overall approach allowed assessment of changes in FRET signals in 4D. We observed little or no gradients in the lateral direction with real time 2D (x, y, and t) imaging. However, with 3D imaging (x, y, and z), we found axial gradients (from apical to basolateral) in the cell. With 4D (x, y, z, and t) imaging, we observed that cAMP gradients move from apical to basolateral side over time. Our data suggest that 3D (x, y, and t) imaging approaches may provide only a limited perspective of cAMP gradients and that 4D (x, y, z and t) studies are required to study cAMP gradients and signaling mechanisms in a cell.Support or Funding InformationThis work is supported by P01HL066299, S10RR027535, S10OD020149, R01HL058506, and the Abraham Mitchell Cancer Research Fund.

Establishment, Validation, and Application of Multispectral Imaging Technology to Studies of Bovine Reproductive Tract Development.

Development of the bovine uterus begins prenatally and is completed postnatally with formation of endometrial glands. Disruption of this process during neonatal life can reduce adult uterine capacity to support pregnancy. However, little is known about mechanisms regulating bovine endometrial development. With a goal of defining molecular and cellular events associated with bovine endometrial histogenesis and cytodifferentiation from birth (postnatal day 0 = PND 0), objectives here were to establish methods for immunohistochemical (IHC) localization of progesterone receptor (PR-A, -B forms) and estrogen receptor (ESR1), as well as two markers of cell proliferation, Ki-67 and bromodeoxyuridine (BrdU), in developing bovine endometrium using multispectral imaging (MSI). For purposes of technical validation, uterine tissue was obtained from a single Holstein heifer treated with BrdU (5mg/kg BW/day, i.v.) for five days prior to euthanasia on PND 40. Tissue was fixed in 4% paraformaldehyde, embedded in Paraplast-plus, sectioned (6μm) and subjected to antigen retrieval in citrate buffer (pH 6). An indirect, non-amplified, multi-label IHC method was employed using primary antibodies chosen based upon target specificity, host species and immunoglobulin subtype. Matched, AlexaFluor-labeled secondary antibodies (Invitrogen Corporation; Carlsbad, CA) were used to produce target-specific fluorescent signals. All tissue sections were counterstained with POPO-1 to reveal nuclear DNA. Fluorescent staining for cytokeratin 8 was used to distinguish epithelium from stroma in image analyses. The Nuance FX MSI system (Caliper Life Sciences; Hopkinton, MA) was used to capture quantifiable image data at specific wavelengths from as many as four unique fluorescent signals simultaneously. Fluorescent signals were analyzed using CellProfiler (www.cellprofiler.com). Spectrally unmixed target signal intensities were compared between stromal and epithelial cell compartments on a within tissue basis after extraction of compartment-specific regions of interest from single- and multi-labeled tissue sections. Data were subjected to analysis of variance to evaluate technical sources of variation. Images generated using multi-label IHC and MSI procedures were equivalent, qualitatively and quantitatively, to those obtained with single labeling procedures. Using MSI, quantifiable, wavelength-specific data were extracted from spectrally unmixed images generated from multi-labeled tissues. Automated analysis procedures permitted rapid generation of unique and colocalized fluorescent signal intensities and/or labeling indices for ESR1, PR, Ki-67 and BrDU in stromal and epithelial cell compartments. Relative signal intensity for ESR1 and PR was higher (P<0.001) in epithelium than stroma. However, labeling indices for these targets were greater (P<0.0001) in stroma than in epithelium. Labeling idices for both cell proliferation markers, Ki-67 and BrdU, were greater (P<0.001) for epithelium than for stroma. Establishment and validation of multi-label IHC and MSI procedures for qualitative and quantitative analysis of multiple fluorescent signals in single tissue sections sets the stage for studies designed to evaluate patterns of neonatal bovine endometrial development at cellular and molecular levels simultaneously. [Support: AU-CVM AHDR and NSF-EPS 0814103.]

Multiplex Imaging of an Intracellular Proteolytic Cascade by using a Broad‐Spectrum Nanoquencher

Turn off the lights! A universal nanoquencher that quenches a broad range of visible to near-infrared dyes by using a series of dark quenchers incorporated into a cell-permeable mesoporous silica nanoparticle has been developed. In combination with dye-labeled substrates, this nanoquencher boosts multiple fluorescence signals upon specific proteolysis, which allows real-time imaging of proteolytic cascades (see scheme).

Digitally synchronized LCD projector for multi-color fluorescence excitation in parallel capillary electrophoresis detection

A simple method is proposed for modulating the excitation light used for multi-color fluorescence detection in a single capillary electrophoresis (CE) channel. In the proposed approach, a low-cost commercial liquid crystal device (LCD) projector with digitally-modulated LCD switches is used to provide the illumination light source and the fluorescence emitted from the CE chip is synchronously detected using an ultraviolet–visible–near infrared (UV–vis–NIR) spectrometer. The modulated light source enables the detection of multiple fluorescence signals within a single CE channel without the need of mechanically switching optical components. In order to enhance the sensing performance of the proposed system, two short-pass filters and one band-pass filter are inserted into the LCD projector to modify the wavelength spectra for fluorescence excitation. With this simple approach, the signal-to-noise (SN) ratio of the fluorescence detection signals is greatly improved by a factor of approximately 22 when detecting Atto647N fluorescent dye. The feasibility of the proposed multi-color CE detection approach is demonstrated by detecting two different samples including a mixed sample comprising FITC, Rhodamine B and Atto647N fluorescent dyes and a bio-sample composed of two ssDNAs labeled with FITC and Cy3, respectively. Results confirm that the digitally-modulated excitation system proposed in this study has significant potential for the parallel analysis of fluorescently-labeled bio-samples using a multi-color detection scheme.

Isothermal Target and Signaling Probe Amplification Method, Based on a Combination of an Isothermal Chain Amplification Technique and a Fluorescence Resonance Energy Transfer Cycling Probe Technology

An iTPA (isothermal target and signaling probe amplification) method for the quantitative detection of nucleic acids, based on a combination of novel ICA (isothermal chain amplification) and fluorescence resonance energy transfer cycling probe technology (FRET CPT), is described. In the new ICA method, which relies on the strand displacement activity of DNA polymerase and the RNA degrading activity of RNase H, two displacement events occur in the presence of four specially designed primers. This phenomenon leads to powerful amplification of target DNA. Since the amplification is initiated only after hybridization of the four primers, the ICA method leads to high specificity for the target sequence. As part of the new ICA method, iTPA is achieved by incorporating FRET CPT to generate multiple fluorescence signals from a single target molecule. Using the resulting dual target and signaling probe amplification system, even a single copy level of a target gene can be successfully detected and quantified under isothermal conditions.