Two-color Fluorescence Detection Research Articles

Pulsed-Interleaved-Excitation Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy.

We report on pulsed-interleaved-excitation two-dimensional fluorescence lifetime correlation spectroscopy (PIE 2D FLCS) to study biomolecular structural dynamics with high sensitivity and high time resolution using Förster resonance energy transfer (FRET). PIE 2D FLCS is an extension of 2D FLCS, which is a unique single-molecule fluorescence method that uses fluorescence lifetime information to distinguish different fluorescence species in equilibrium and resolves their interconversion dynamics with a submicrosecond time resolution. Because 2D FLCS has used only a single-color excitation so far, it was difficult to distinguish a very low-FRET (or zero-FRET) species from only donor-labeled species. We overcome this difficulty by implementing the PIE scheme (i.e., alternate excitation of the donor and acceptor dyes using two temporally interleaved excitations with different colors) to 2D FLCS, realizing two-color excitation and two-color fluorescence detection in 2D FLCS. After proof-of-principle PIE 2D FLCS analysis on the photon data synthesized with Monte Carlo simulation, we apply PIE 2D FLCS to a DNA-hairpin sample and show that this method readily distinguishes four fluorescent species, i.e., high-FRET, low-FRET, and two single-dye-labeled species. In addition, we show that PIE 2D FLCS can also quantitatively evaluate the contributions of the donor-acceptor spectral crosstalk, which often appears as artifacts in FRET studies and degrades the information obtained.

Circuit Investigations With Open-Source Miniaturized Microscopes: Past, Present and Future.

The ability to simultaneously image the spatiotemporal activity signatures from many neurons during unrestrained vertebrate behaviors has become possible through the development of miniaturized fluorescence microscopes, or miniscopes, sufficiently light to be carried by small animals such as bats, birds and rodents. Miniscopes have permitted the study of circuits underlying song vocalization, action sequencing, head-direction tuning, spatial memory encoding and sleep to name a few. The foundation for these microscopes has been laid over the last two decades through academic research with some of this work resulting in commercialization. More recently, open-source initiatives have led to an even broader adoption of miniscopes in the neuroscience community. Open-source designs allow for rapid modification and extension of their function, which has resulted in a new generation of miniscopes that now permit wire-free or wireless recording, concurrent electrophysiology and imaging, two-color fluorescence detection, simultaneous optical actuation and read-out as well as wide-field and volumetric light-field imaging. These novel miniscopes will further expand the toolset of those seeking affordable methods to probe neural circuit function during naturalistic behaviors. Here, we will discuss the early development, present use and future potential of miniscopes.

Discrimination of Glycoproteins from Unglycosylated Proteins in Capillary Electrophoresis: Two-Color LIF Detection Coupled with Post-column Derivatization.

Glycosylation is one of the most important posttranslational modifications (PTMs) which lead to the functionalization of proteins. Here, we describe one method for discriminating glycosylated proteins from unglycosylated ones in their mixture sample by capillary electrophoretic separation and two-color laser-induced fluorescence detection coupled with post-column derivatization. Two lasers emitting at 450 and 532 nm permit the detection of amino groups of proteins derivatized by naphthalene-2,3-dicarboxaldehyde and a fluorescently labeled lectin, tetramethylrhodamine-labeled concanavalin A (Rh-Con A), respectively. When a protein mixture react with Rh-Con A, the glycoproteins bound with Rh-Con A exhibit signals at the same migration time in two electropherograms obtained by 450- and 532-nm lasers whereas unbound proteins show a signal only in the electropherogram of the 450-nm laser. So, when one protein is glycosylated it is detected at the same migration time in the electropherograms obtained by two lasers.

Manipulating ionic strength to improve single cell electrophoretic separations

A capillary electrophoresis system with ultrasensitive two-color laser-induced fluorescence detection was used to probe the effect of ionic strength on single cell separations of glycosphingolipids. Differentiated PC12 cells were incubated with two ganglioside substrates tagged with different fluorophores within the BODIPY family such that two distinct metabolic patterns could be simultaneously monitored. Aspiration of single differentiated PC12 cells suspended in a phosphate-buffered saline solution showed excessive peak dispersion, poor resolution, and peak efficiencies below 100,000 theoretical plates. Aspiration of single differentiated PC12 cells suspended in deionized water corrected peak dispersion. Average peak efficiencies ranged between 400,000 and 600,000 theoretical plates. Improved performance was due to the dilution of the high salt concentrations inside of single neuronal-like cells to produce field amplified sample stacking. Single cell separations showed the highest resolution when aspiration of single differentiated PC12 cells suspended in deionized water were separated using a running buffer of high ionic strength. The improvement in resolution allowed for the identification of analytes not previously detected in single cell metabolism studies.

Metabolic Cytometry: Capillary Electrophoresis with Two-Color Fluorescence Detection for the Simultaneous Study of Two Glycosphingolipid Metabolic Pathways in Single Primary Neurons

Metabolic cytometry is a form of chemical cytometry wherein metabolic cascades are monitored in single cells. We report the first example of metabolic cytometry where two different metabolic pathways are simultaneously monitored. Glycolipid catabolism in primary rat cerebella neurons was probed by incubation with tetramethylrhodamine-labeled GM1 (GM1-TMR). Simultaneously, both catabolism and anabolism were probed by coincubation with BODIPY-FL labeled LacCer (LacCer-BODIPY-FL). In a metabolic cytometry experiment, single cells were incubated with substrate, washed, aspirated into a capillary, and lysed. The components were separated by capillary electrophoresis equipped with a two-spectral channel laser-induced fluorescence detector. One channel monitored fluorescence generated by the metabolic products produced from GM1-TMR and the other monitored the metabolic products produced from LacCer-BODIPY-FL. The metabolic products were identified by comparison with the mobility of a set of standards. The detection system produced at least 6 orders of magnitude dynamic range in each spectral channel with negligible spectral crosstalk. Detection limits were 1 zmol for BODIPY-FL and 500 ymol for tetramethylrhodamine standard solutions.

Accurate DNA Fragment Sizing by Capillary Electrophoresis with Laser-Induced Fluorescence Array for Detection of Sequence Specificity of DNA Damage

Cancer has been linked to mutations within specific codons in genes that code for critical biomolecules such as tumor suppressor proteins (e.g., p53). Activated metabolites like benzo[a]pyrenediol epoxide act on preferred nucleotide sequences of DNA, and such mutations have been identified in cancers. DNA reaction site identification depends on accurate analysis of oligonucleotide fragment sizes produced by strand breakage at the damaged sites. Herein, we report a new method for DNA fragment sizing using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Absolute sizing accuracy and speed are achieved by utilizing a CE-LIF array with two-color fluorescence detection. Accuracy depends on correcting results with commercial standards by referring them to primary standards with the same sequences and identical labels as sample fragments. The method is demonstrated by detection of a [...GGCGCGCAG...] G reaction site for styrene oxide on an oligonucleotide representing the CYP1B1 gene. This approach avoids the need for radioactive isotopes and is less labor intensive and faster than the alternative PAGE with (32)P end labeling.

Axial Resolution Enhancement by 4Pi Confocal Fluorescence Microscopy with Two-Photon Excitation

Confocal fluorescence microscopy and two-photon microscopy have become important techniques for the three-dimensional imaging of intact cells. Their lateral resolution is about 200-300 nm for visible light, whereas their axial resolution is significantly worse. By superimposing the spherical wave fronts from two opposing objective lenses in a coherent fashion in 4Pi microscopy, the axial resolution is greatly improved to approximately 100 nm. In combination with specific tagging of proteins or other cellular structures, 4Pi microscopy enables a multitude of molecular interactions in cell biology to be studied. Here, we discuss the choice of appropriate fluorescent tags for dual-color 4Pi microscopy and present applications of this technique in cellular biophysics. We employ two-color fluorescence detection of actin and tubulin networks stained with fluorescent organic dyes; mitochondrial networks are imaged using the photoactivatable fluorescent protein EosFP. A further example concerns the interaction of nanoparticles with mammalian cells.

Optimized Normalization for Antibody Microarrays and Application to Serum-Protein Profiling

The measurements of coordinated patterns of protein abundance using antibody microarrays could be used to gain insight into disease biology and to probe the use of combinations of proteins for disease classification. The correct use and interpretation of antibody microarray data requires proper normalization of the data, which has not yet been systematically studied. Therefore we undertook a study to determine the optimal normalization of data from antibody microarray profiling of proteins in human serum specimens. Forty-three serum samples collected from patients with pancreatic cancer and from control subjects were probed in triplicate on microarrays containing 48 different antibodies, using a direct labeling, two-color comparative fluorescence detection format. Seven different normalization methods representing major classes of normalization for antibody microarray data were compared by their effects on reproducibility, accuracy, and trends in the data set. Normalization with ELISA-determined concentrations of IgM resulted in the most accurate, reproducible, and reliable data. The other normalization methods were deficient in at least one of the criteria. Multiparametric classification of the samples based on the combined measurement of seven of the proteins demonstrated the potential for increased classification accuracy compared with the use of individual measurements. This study establishes reliable normalization for antibody microarray data, criteria for assessing normalization performance, and the capability of antibody microarrays for serum-protein profiling and multiparametric sample classification.

Competition on Nitrocellulose-immobilized Antibody Arrays: From Bacterial Protein Binding Assay to Protein Profiling in Breast Cancer Cells

Large scale comparative evaluation of protein expression requires miniaturized techniques to provide sensitive and accurate measurements of the abundance of molecules present as individual and/or assembled protein complexes in cells. The principle of competition between target molecules for binding to arrayed antibodies has recently been proposed to assess differential expression of numerous proteins with one-color or two-color fluorescence detection methods. To establish the limiting factors and to validate the use of alternative detection for protein profiling, we performed competitive binding assays under different conditions. A model experimental protocol was developed whereby the competitive displacement of multi-subunit bacterial RNA polymerase and/or its subunits was evaluated through binding to subunit-specific immobilized monoclonal antibodies. We show that the difference in physico-chemical properties of unlabeled and labeled molecules significantly affects the performance of one-color detection, whereas epitope inaccessibility in the protein complex can prohibit the assessment of competition by both detection methods. Our data also demonstrate that antibody cross-reactivity, target protein truncation and abundance, as well as the cellular compartment of origin are major factors that affect protein profiling on antibody arrays. The experimental conditions established for prokaryotic proteins were adopted to compare protein profiles in the breast tumor-derived cell lines MDA MB-231 and SKBR3. Competitive displacement was detected and confirmed for a number of proteins using both detection methods; however, we show that overall the two-color method is better suited for accurate expression profile evaluation of a large, complex set of proteins. Antibody array data confirm the functional linkage between the ErbB2 receptor and AP-2 transcription factors in these cell lines and highlight unexpected differences in G1 cyclin expression.

The utility of a two-color fluorescence electrophoretic mobility shift assay procedure for the analysis of DNA replication complexes.

Protein-DNA and protein-protein interactions play essential roles in many biologic processes such as transcription, replication, recombination, and DNA repair. One of the most popular approaches to investigate specific protein-nucleic acid interactions is the electrophoretic mobility shift assay (EMSA). We have developed a new nonradioactive method to detect both nucleotides and protein in EMSA. Nucleic acids are detected with SYBR Green EMSA dye, while proteins are subsequently detected with SYPRO Ruby EMSA dye. All fluorescent staining steps are performed after the entire gel-shift experiment is completed, so there is no need to prelabel either the nucleic acids or the protein. The two-color fluorescence dye staining procedure is fast, simple, and allows independent quantitative determination of either free or bound nucleic acids and protein. The interactions between lac repressor-operator, and among the T4 primase-helicase, primase-DNA, helicase-DNA, and within T4 [ssDNA-primase-helicase6] primosome, were used to demonstrate the advantages of this two-color fluorescence detection EMSA method.

Single-molecule spectroscopy for nucleic acid analysis: a new approach for disease detection and genomic analysis.

Recently developed single-molecule spectroscopy (SMS) permits the analysis of fluorescent mixtures one molecule at a time. SMS methods provide the means to make rapid measurements on small, complex samples without the need for separations and target amplification enabling a new class of ultrasensitive nucleic acid assays. Here we give a brief overview of the current state of the art of SMS nucleic acid analysis and discuss ongoing work in our laboratory on two-color single-molecule fluorescence detection of specific nucleic acid sequences. In the future, two-color SMS nucleic acid assays will be used for a variety of applications including: gene expression analysis, disease detection and genomics.

Human genomic DNA analysis using a semi-automated sample preparation, amplification, and electrophoresis separation platform.

The growing importance of analyzing the human genome to detect hereditary and infectious diseases associated with specific DNA sequences has motivated us to develop automated devices to integrate sample preparation, real-time PCR, and microchannel electrophoresis (MCE). In this report, we present results from an optimized compact system capable of processing a raw sample of blood, extracting the DNA, and performing a multiplexed PCR reaction. Finally, an innovative electrophoretic separation was performed on the post-PCR products using a unique MCE system. The sample preparation system extracted and lysed white blood cells (WBC) from whole blood, producing DNA of sufficient quantity and quality for a polymerase chain reaction (PCR). Separation of multiple amplicons was achieved in a microfabricated channel 30 microm x 100 microm in cross section and 85 mm in length filled with a replaceable methyl cellulose matrix operated under denaturing conditions at 50 degrees C. By incorporating fluorescent-labeled primers in the PCR, the amplicons were identified by a two-color (multiplexed) fluorescence detection system. Two base-pair resolution of single-stranded DNA (PCR products) was achieved. We believe that this integrated system provides a unique solution for DNA analysis.

Simultaneous red/green dual fluorescence detection on electroblots using BODIPY TR-X succinimidyl ester and ELF 39 phosphate.

A two-color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY TR-X dye to proteins immobilized on polyvinylidene difluoride membranes, followed by detection of target proteins using the fluorogenic, precipitating substrate ELF 39-phosphate in combination with alkaline phosphatase conjugated reporter molecules. This results in all proteins in the profile being visualized as fluorescent red signal while those detected specifically with the alkaline phosphatase conjugate appear as fluorescent green signal. The dichromatic detection system is broadly compatible with ultraviolet epi- or trans-illuminators combined with photographic or charge-coupled device cameras, and xenon-arc sources equipped with appropriate excitation/emission filters. The dichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of target proteins relative to the entire protein profile on a single electroblot, obviating the need to run replicate gels that would otherwise require visualization of total proteins by silver staining and subsequent alignment with chemiluminescent or colorimetric signals generated on electroblots. Combining the detection approach with an Alexa Fluor 350 dye conjugated monoclonal antibody permits simultaneous fluorescence detection of two antigens and the total protein profile on the same electroblot.

Quantitation of viral DNA by real-time PCR applying duplex amplification, internal standardization, and two-color fluorescence detection.

A real-time PCR method was developed to quantitate viral DNA that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve. Applied to human parvovirus B19 DNA, the linear range was from 10(2) to at least 5 x 10(6) copies per ml of sample. The coefficient of variation was 0.29 using a run control of 2,876 copies per ml. The method reduces the risk of false-negative results, yields high precision, and is applicable for other DNA targets.

Simultaneous, two-color fluorescence detection of total protein profiles and beta-glucuronidase activity in polyacrylamide gel.

A dichromatic method for measuring the specific activity of beta-glucuronidase from complex cell homogenates or partially purified protein fractions is presented. Dual fluorescence is achieved by using the green emitting fluorogenic substrate ELF 97 beta-D-glucuronide to detect beta-glucuronidase activity, followed by the red emitting SYPRO Ruby protein gel stain or SYPRO Ruby IEF gel stain to detect the remaining proteins in the electrophoretic profile. Both ELF 97 alcohol, the highly fluorescent hydrolytic product generated from the enzyme substrate, and the SYPRO Ruby total protein stains are maximally excited by ultraviolet illumination. ELF 97 alcohol emits maximally at 525 nm while the SYPRO Ruby dyes emit maximally at 610 nm. Since ELF 97 beta-glucuronide is a precipitating substrate, it allows precise localization of beta-glucuronidase activity with minimal band diffusion. The staining method is simple and direct, without the requirement for ancillary coupling reactions. Dichromatic protein detection is demonstrated after sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis, carrier ampholyte-mediated isoelectric focusing or two-dimensional gel electrophoresis.

Green/red dual fluorescence detection of total protein and alkaline phosphate-conjugated probes on blotting membranes

A two-color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY FL-X to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl(DDAO)-phosphate in combination with alkaline-phosphatase-conjugated reporter molecules. This results in all proteins in the profile being visualized as green signal while those detected specifically with the alkaline-phosphatase conjugate appear as red signal. The dichromatic detection system is broadly compatible with a wide range of analytical imaging devices including UV epi- or transilluminators combined with photographic or charge-coupled device (CCD) cameras, xenon-arc sources equipped with appropriate excitation/emission filters, and dual laser gel scanners outfitted with a 473 nm second-harmonic generation or 488 nm argon-ion laser as well as a 633 nm helium-neon or 635 nm diode laser. The dichromatic detection method permits detection of low nanogram amounts of protein and allows for unambiguous identification of target proteins relative to the entire protein profile on a single electroblot, obviating the need to run replicate gels that would otherwise require visualization of total proteins by silver staining and subsequent alignment with chemiluminescent or colorimetric signals generated on electroblots.

A simple, two-color fluorescence detection method for membrane blotting analysis using alkaline phosphatase and horseradish peroxidase.

We have developed a one-step, two-color fluorescence detection method using simultaneously two fluorogenic substrates for both Southern and Western blots on nylon membranes. For this enzyme-mediated reporter system, a mixture of (i) 3-hydroxy-N-2'-biphenyl-2-naphthalenecarboxamide phosphate ester (HNPP), a substrate for alkaline phosphatase and (ii) N-(4-amino-5-methoxy-2-methylphenyl)benzamide (AMMB), a fluorogenic substrate for horseradish peroxidase was used. The reaction with these substrates produces blue (HNPP) and yellow (AMMB) fluorescent signals under ultraviolet light (302 nm). Therefore, this simple method allows the simultaneous visualization of two different targets on a single nylon membrane, e.g. nucleic acids or proteins.

High-speed DNA genotyping using microfabricated capillary array electrophoresis chips.

Capillary array electrophoresis (CAE) chips have been designed and fabricated with the capacity to rapidly (< 160 s) analyze 12 different samples in parallel. Detection of all lanes with 0.3 s temporal resolution was achieved using a laser-excited confocal-fluorescence scanner. The operation and capabilities of these CAE microdevices were first determined by performing electrophoretic separations of pBR322 MspI DNA samples. Genotyping of HLA-H, a candidate gene for the diagnosis of hereditary hemochromatosis, was then performed to demonstrate the rapid analysis of biologically relevant samples. Two-color multiplex fluorescence detection of HLA-H genotypes was accomplished by prelabeling the standard pBR322 MspI DNA ladder with a red emitting bis-intercalation dye (butyl TOTIN) and on-column labeling of the HLA-H DNA with thiazole orange. This work establishes the feasibility of using CAE chips for high speed, high-throughput genotyping.

Genome analysis with gene expression microarrays.

Advances in biochemistry, chemistry and engineering have enabled the development of a new gene expression assay. This 'chip-based' approach utilizes microscopic arrays of cDNAs printed on glass as high-density hybridization targets. Fluorescent probe mixtures derived from total cellular messenger RNA (mRNA) hybridize to cognate elements on the array, allowing accurate measurement of the expression of the corresponding genes. Array densities of > 1,000 cDNAs per cm2 enable quantitative expression monitoring of a large number of genes in a single hybridization. A two-color fluorescence detection scheme allows rapid and simultaneous differential expression analysis of independent biological samples. Mass-produced microarrays provide a new tool for genome expression analysis that may revolutionize genetic dissection, drug discovery and human disease diagnostics.

Volumetric capillary cytometry: a new method for absolute cell enumeration.

This article describes a novel cytometry system that is capable of imaging, identifying, and counting populations of fluorescently labeled cells within a precisely known volume of unlysed whole blood contained in a precision capillary. The whole blood is reacted with dye-labeled antibodies directed at cell surface antigens. A red excitation source (helium-neon laser) is used to minimize auto-fluorescence and interference by blood components. A two-color fluorescence detection system is described, using the dyes Cy5 and Cy5.5, allowing the simultaneous detection and quantitation of two different types of surface antigens. Image processing techniques are used to identify cellular events in the image and to quantitate the fluorescent intensities in both color planes. The utility of this technique is demonstrated by performing absolute CD3+/CD4+ lymphocyte counts in whole blood, with minimal sample preparation, and with the blood sample completely contained in a disposable cartridge. A correlation study comparing this technique to flow cytometry gives a correlation coefficient of 0.98. In addition, the ability of this system to monitor reaction kinetics is demonstrated with a measurement of the rate of association of the anti-CD4 antibody onto the surface of CD4+ lymphocytes.