High-resolution near-infrared imaging of DNA microarrays with time-resolved acquisition of fluorescence lifetimes

Ultrasensitive, near-infrared (NIR), time-resolved fluorescence is evaluated as a detection method for reading DNA hybridization events on solid surfaces for microarray applications. In addition, the potential of mulitiplexed analyses using time-resolved identification protocols is described. To carry out this work, a NIR time-resolved confocal imager was constructed to read fluorescence signatures from the arrays. The device utilized a 780-nm pulsed diode laser, a single-photon avalanche diode (SPAD), and a high-numerical-aperture microscope objective mounted in an epi-illumination format. Due to the small size of the components that are required to construct this imager, the entire detector could easily be mounted on high-resolution translational stages and scanned over the stationary arrays. The instrument response function of the device was determined to be 275 ps (fwhm), which is adequate for measuring fluorophores with subnanosecond lifetimes. To characterize the system, NIR dyes were deposited directly on different substrate materials typically used for DNA microarrays, and the fluorescence lifetimes of two representative dyes were measured. The fluorescence lifetime for aluminum tetrasulfonated naphthalocyanine was found to be 1.92 ns, and a value of 1.21 ns was determined for the tricarbocyanine dye, IRD800, when it was deposited onto poly(methyl methacrylate) (PMMA) and measured in the dry state. Finally, the imager was used to monitor hybridization events using probe oligonucleotides chemically tethered to a PMMA substrate via a glutardialdehyde linkage to an aminated-PMMA surface. The limit of detection for oligonucleotides containing a NIR fluorescent reporter was determined to be 0.38 molecules/microm2, with this detection limit improving by a factor of 10 when a time-gate was implemented. Fluorescence lifetime analysis of the hybridization events on PMMA indicated a lifetime value of 1.23 ns for the NIR-labeled oligonucleotides when using maximum-likelihood estimators.     

Fluorescence multiplexing with time-resolved and spectral discrimination using a near-IR detector

We report on the design and performance of a two-color, time-resolved detector for the acquisition of both steady-state and time-resolved fluorescence data acquired in real time during the capillary gel electrophoresis separation of DNA sequencing fragments. The detector consisted of a pair of pulsed laser diodes operating at 680 and 780 nm. The diode heads were coupled directly to single-mode fibers, which were terminated into a single fiber mounted via a FC/PC connector to the detector body. The detector contained a dichroic filter, which directed the dual-laser beams to an objective. The objective focused the laser light into a capillary gel column and also collected the resulting fluorescence emission. The dual-color emission was transmitted through the dichroic and focused onto a multimode fiber (core diameter 50 microm), which carried the luminescence to a pair of single-photon avalanche diodes (SPADs). The emission was sorted spectrally using a second dichroic onto one of two SPADs and isolated using appropriate interference filters (710- or 810-nm channel). The dual-color detector demonstrated a time response of 450 and 510 ps (fwhm) for the 710- and 810-nm channels, respectively. The mass detection limits for two near-IR dye-labeled sequencing primers electrophoresed in a capillary gel column were found to be 7.1 x 10(-21) and 3.2 x 10(-20) mol (SNR = 3) for the 710- and 810-nm detector channels, respectively. In addition, no leakage of luminescence excited at 680 nm was observed in the 810-nm channel or 780-nm excited luminescence into the 710-nm channel. An M13mp18 template was sequenced in a single capillary gel column using a two-color, two-lifetime format. The read length was found to be 650 base pairs for the test template at a calling accuracy of 95.1% using a linear poly(dimethylacrylamide) (POP6) gel column, with the read length determined primarily by the electrophoretic resolution produced by the sieving gel.     

DNA sequencing by capillary electrophoresis with four-decay fluorescence detection

A scheme for multiplex detection of dye-labeled DNA fragments in DNA sequencing is described in which on-the-fly, frequency-domain fluorescence lifetime detection is used to discriminate among the dye-labeled fragments of the four terminal bases in a single-lane CE separation. Two four-dye systems were evaluated, one excited at 488 nm and the other, at 514 nm. The 488 nm system proved successful for four-decay detection. Base calling was achieved either directly from on-the-fly lifetimes or from lifetime-resolved electropherograms recovered for each base from the electropherogram of the mixture of sequencing reaction products. The latter method was found to be more accurate (99% for two bases and 98.5% for three bases) and could achieve longer read lengths, but it was unsuccessful for sequencing of all four bases. The first method gave a base-calling accuracy of 96% for four-base sequencing over the fragment length range of 41-220 bases.     

Fast-gated intensified charge-coupled device camera to record time-resolved fluorescence spectra of tryptophan

The possibilities of a 200 ps gated intensified charge-coupled device (CCD) camera to record time-resolved fluorescence were explored using the fluorescing amino acid tryptophan and its derivative Nacetyl-tryptophan amide (NATA) as model compounds. The results were compared to complementary data from time-correlated single-photon counting (TCSPC) experiments. If a spectral resolution of 1-2 nm is desired, the fast-gated intensified CCD (ICCD) camera is the method of choice. For a 10(-5) M tryptophan solution, time-resolved emission spectra and intensity decays (measured over 12 ns at 25 ps resolution) could be obtained in typically 10 minutes, giving the well-known lifetimes of 0.5 and 3 ns. In addition, a longer lifetime of 7 ns was found at the red edge of the spectrum. The very short gate time of the ICCD camera allowed us to observe a shift in the emission maximum of tryptophan even within the first nanosecond of decay of the fluorescence emission. As expected from the tryptophan rotamer model, such a shift is not observed in NATA. Using amplitudes obtained by global analysis, decay-associated spectra of these lifetimes were constructed.     

Fluorescence lifetime for on-the-fly multiplex detection of DNA restriction fragments in capillary electrophoresis

This paper describes the first use of frequency-domain fluorescence lifetime for multiplex detection of DNA restriction fragments in capillary electrophoresis (CE). The fragments were labeled with monomeric intercalating dyes that can be excited by either the 488- or 514-nm line of an argon ion laser and have lifetimes in the range of 0.5-2.5 ns. We were able to achieve multiplex lifetime detection in the CE separation of a restriction fragment digest and a DNA size ladder in the same run, for fragments shorter than 700 bp. Different gel buffer systems, including a modified polyacrylamide gel and several tris-borate-EDTA/hydroxyethylcellulose (TBE/HEC) gels, were investigated for separation and detection of the dye-labeled DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a gel containing 1% high molecular weight (90,000-105,000) HEC and 0.3% low molecular weight (24,000-27,000) HEC in TBE buffer.     

Continuous flow thermal cycler microchip for DNA cycle sequencing

We report here on the use of a polymer-based continuous flow thermal cycler (CFTC) microchip for Sanger cycle sequencing using dye terminator chemistry. The CFTC chip consisted of a 20-loop spiral microfluidic channel hot-embossed into polycarbonate (PC) that had three well-defined temperature zones poised at 95, 55, and 60 degrees C for denaturation, renaturation, and DNA extension, respectively. The sequencing cocktail was hydrodynamically pumped through the microreactor channel at different linear velocities ranging from 1 to 12 mm/s. At a linear velocity of 4 mm/s resulting in a 36-s extension time, a read length of >600 bp could be obtained in a total reaction time of 14.6 min. Further increases in the flow rate resulted in a reduction in the total reaction time but also produced a decrease in the sequencing read length. The CFTC chip could be reused for subsequent sequencing runs (>30) with negligible amounts of carryover contamination or degradation in the sequencing read length. The CFTC microchip was subsequently coupled to a solid-phase reversible immobilization (SPRI) microchip made from PC for purification of the DNA sequencing ladders (i.e., removal of excess dye-labeled dideoxynucleotides, DNA template, and salts) prior to gel electrophoresis. Coupling of the CFTC chip to the SPRI microchip showed read lengths similar to that obtained from benchtop instruments but did not require manual manipulation of the cycle sequencing reactions following amplification.     

Time-resolved fluorescence microspectroscopy for characterizing crude oils in bulk and hydrocarbon-bearing fluid inclusions

Time-resolved fluorescence data was collected from a series of 23 bulk crude petroleum oils and six microscopic hydrocarbon-bearing fluid inclusions (HCFI). The data was collected using a diode laser fluorescence lifetime microscope (DLFLM) over the 460-700 nm spectral range using a 405 nm excitation source. The correlation between intensity averaged lifetimes (tau) and chemical and physical parameters was examined with a view to developing a quantitative model for predicting the gross chemical composition of hydrocarbon liquids trapped in HCFI. It was found that tau is nonlinearly correlated with the measured polar and corrected alkane concentrations and that oils can be classified on this basis. However, these correlations all show a large degree of scatter, preventing accurate quantitative prediction of gross chemical composition of the oils. Other parameters such as API gravity and asphaltene, aromatic, and sulfur concentrations do not correlate well with tau measurements. Individual HCFI were analyzed using the DLFLM, and time-resolved fluorescence measurements were compared with tau data from the bulk oils. This enabled the fluid within the inclusions to be classified as either low alkane/high polar or high alkane/low polar. Within the high alkane/low polar group, it was possible to clearly discriminate HCFI from different locales and to see differences in the trapped hydrocarbon fluids from a single geological source. This methodology offers an alternative method for classifying the hydrocarbon content of HCFI and observing small variations in the trapped fluid composition that is less sensitive to fluctuations in the measurement method than fluorescence intensity based methods.     

Eight hundred-base sequencing in a microfabricated electrophoretic device

The human genome will be sequenced using capillary array electrophoresis technology. Although currently achieving only 550 base reads per run, capillary arrays have increased the efficiency and lowered the cost of sequencing by eliminating gel plate preparation, reducing sample volumes, and offering automation and speed. However, much higher throughput and greater cost reductions are needed. The next major advancement in sequencing technology is expected from the development of arrays of microfabricated channels in a plate or "chip" format. For de novo sequencing, the practical utility of the microdevice approach has been limited by device length to a read of 500-600 bases per run. We demonstrate a significant milestone for a microfabricated device by obtaining an average read length of 800 bases in 80 min (98% accuracy) for either M13 standards or DNA sequencing samples from the Whitehead Institute Center for Genomic Research (WICGR) production line. This result is achieved in 40-cm-long channels using a new class of large-scale microfabricated devices. Both microfabrication of extended structures and achievement of long reads are essential steps toward a 384-lane very-large-scale microfluidic (VLSMF) system for ultrahigh-throughput DNA sequencing analysis, currently under construction in our laboratory.     

DNA sequencing up to 1300 bases in two hours by capillary electrophoresis with mixed replaceable linear polyacrylamide solutions

This paper presents results on ultralong read DNA sequencing with relatively short separation times using capillary electrophoresis with replaceable polymer matrixes. In previous work, the effectiveness of mixed replaceable solutions of linear polyacrylamide (LPA) was demonstrated, and 1000 bases were routinely obtained in less than 1 h. Substantially longer read lengths have now been achieved by a combination of improved formulation of LPA mixtures, optimization of temperature and electric field, adjustment of the sequencing reaction, and refinement of the base-caller. The average molar masses of LPA used as DNA separation matrixes were measured by gel permeation chromatography and multiangle laser light scattering. Newly formulated matrixes comprising 0.5% (w/w) 270 kDa and 2% (w/w) 10 or 17 MDa LPA raised the optimum column temperature from 60 to 70 degrees C, increasing the selectivity for large DNA fragments, while maintaining high selectivity for small fragments as well. This improved resolution was further enhanced by reducing the electric field strength from 200 to 125 V/cm. In addition, because sequencing accuracy beyond 1000 bases was diminished by the low signal from G-terminated fragments when the standard reaction protocol for a commercial dye primer kit was used, the amount of these fragments was doubled. Augmenting the base-calling expert system with rules specific for low peak resolution also had a significant effect, contributing slightly less than half of the total increase in read length. With full optimization, this read length reached up to 1300 bases (average 1250) with 98.5% accuracy in 2 h for a single-stranded M13 template.     

Probes for detection of specific DNA sequences at the single-molecule level

A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe-target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules.     

Concentration quenching of a red emitting electroluminescent dye tetraphenylporphyrin: A time-resolved photoluminescence study

This work deals with the steady-state and time-resolved photoluminescence study of a well-known red emitting electroluminescent dye, tetraphenylporphyrin (TPP), doped in poly(methylmethacrylate). From the time-resolved photoluminescence (PL) study, exponential decay of TPP emission was observed for these doped films in the studied concentrations region (10~mol/L). At lower doping concentrations below 10 mol/L, PL lifetimes of TPP were relatively constant (∼ 10 ns) and equal to that of TPP in dilute solution. A decrease in PL lifetime was observed at high doping concentration, down to 8 ns at a concentration of 10 mol/L. This observed decrease in PL lifetime of TPP with the increase of TPP concentration was due to the concentration quenching of TPP monomer emission by the TPP dimer/aggregate sites formed at such higher concentrations. The results of concentration quenching in TPP system were compared to that of tetraphenylchlorin system reported earlier.     

Calculation of focal positions in an optical head for parallel data processing with a monolithic four-beam laser diode

A method for calculating focal positions in a multibeam optical head by use of a multibeam laser diode, in which conditions for misalignment of the light source are taken into consideration, is introduced. One calculates the focal positions by using the practical characteristics of a monolithic four-beam laser diode and the practical specifications of the optics in an optical head. The results show that each focal position is defocused mainly as a result of curvature of the fields of the lenses. The adaptability of focal positions for various calculated conditions is discussed from the standpoint of depth of focus.     

Two-Dimensional Visualization of Fluorescence Lifetimes by use of a Picosecond Laser and a Streak Camera

Two-dimensional distributions of the effective lifetime of the fluorescence emission induced by short-pulsed laser radiation are obtained from two-dimensional images recorded with a streak camera and a charge-coupled device by means of a separation algorithm method (SAM). In theory, the best response with respect to noise is obtained for lifetimes corresponding to a range of pixels of 5-50 in the CCD, that is, 5-50 ps at the fastest streak speed. In experiments the SAM is compared with pure time-resolved measurements, and it is used for two-dimensional lifetime evaluation. The laser-pulse duration is 25 ps, and the lower limit of the lifetime resolution as used in the experiments is estimated to be 200-250 ps. The results demonstrate the possibility of performing pattern recognition independently of the relative distribution of emission intensity between regions of different fluorescence lifetimes. The technique is demonstrated for static objects but can in principle be extended to nonstationary objects if two detectors are used.     

Spatially correlated fluorescence/AFM of individual nanosized particles and biomolecules

Individual fluorescent polystyrene nanospheres (<10-100-nm diameter) and individual fluorescently labeled DNA molecules were dispersed on mica and analyzed using time-resolved fluorescence spectroscopy and atomic force microscopy (AFM). Spatial correlation of the fluorescence and AFM measurements was accomplished by (1) positioning a single fluorescent particle into the near diffraction-limited confocal excitation region of the optical microscope, (2) recording the time-resolved fluorescence emission, and (3) measuring the intensity of the excitation laser light scattered from the apex of an AFM probe tip and the AFM topography as a function of the lateral position of the tip relative to the sample substrate. The latter measurements resulted in concurrent high-resolution (approximately 10-20 nm laterally) images of the laser excitation profile of the confocal microscope and the topography of the sample. Superposition of these optical and topographical images enabled unambiguous identification of the sample topography residing within the excitation region of the optical microscope, facilitating the identification and structural characterization of the nanoparticle(s) or biomolecule(s) responsible for the fluorescence signal observed in step 2. These measurements also provided the lateral position of the particles relative to the laser excitation profile and the surrounding topography with nanometer-scale precision and the relationship between the spectroscopic and structural properties of the particles. Extension of these methods to the study of other types of nanostructured materials is discussed.     

Single-molecule spectroscopic study of enhanced intrinsic phycoerythrin fluorescence on silver nanostructured surfaces

In this paper, we report on steady-state and time-resolved single-molecule fluorescence measurements performed on a phycobiliprotein, R-phycoerythrin (RPE), assembled on silver nanostructures. Single-molecule measurements clearly show that RPE molecules display a 10-fold increase in fluorescence intensity, with a 7-fold decrease in lifetime when they are assembled on silver nanostructured surfaces, as compared to control glass slides. The emission spectrum of individual RPE molecules also displays a significant fluorescence enhancement on silver nanostructures as compared to glass. From intensity and lifetime histograms, it is clear that the intensities as well as lifetimes of individual RPE molecules on silver nanostructures are more heterogeneously distributed than that on glass. This single-molecule study provides further insight on the heterogeneity in the fluorescence intensity and lifetimes of the RPE molecules on both glass and SiFs surfaces, which is otherwise not possible to observe using ensemble measurements. Finite-difference time-domain calculations have been performed to study the enhanced near-fields induced around silver nanoparticles by a radiating excited-state fluorophore, and the effect of such enhanced fields on the fluorescence enhancement observed is discussed.     

Time-resolved step-scan infrared imaging system utilizing a linear array detector

A time-resolved infrared (IR) imaging system combined with a multichannel IR microscope, which utilizes a 16 channel linear array (LA) detector, and step-scan Fourier transform infrared (FT-IR) microscope was developed. The LA detector integrates a readout circuit on each detector element, so the detected signals can be read simultaneously. Thus, this system can perform high speed imaging using the step-scan method, similar to a single channel detector. To verify the capabilities of this system, a reflective sample was examined whose position was altered using a piezo actuator activated by an alternating voltage. In addition, the localization of relaxation dynamics for the liquid crystal (LC) molecules in an LC cell under oscillating electric field conditions was detected by this system.     

Recombination dynamics of localized excitons in self-formed InGaN quantum dots

The dynamic behavior of radiative recombination has been assessed in the InGaN-based purple, blue and green light emitting diodes (LEDs), as well as in purple laser diode (LD) components by means of time-resolved electroluminescence (TREL) and time-resolved photoluminescence (TRPL) spectroscopy. It was found that excitons localized at deep trap centers play an important role in the recombination process. Microstructural analysis suggests that these centers originate from the In-rich regions, acting as quantum dots which are self-formed within the wells. The radiative lifetime of localized excitons in the LD structure was almost constant at 6 ns in the temperature range between 20 and 200 K, indicating the zero-dimensional feature of excitons.     

Label-free detection of single protein molecules using deep UV fluorescence lifetime microscopy

We present the detection of single beta-galactosidase molecules from Escherichia coli (Ecbeta Gal) using deep UV laser-based fluorescence lifetime microscopy. The native fluorescence from intrinsic tryptophan emission has been observed after one-photon excitation at 266 nm. Applying the time-resolved single-photon counting method, we investigated the fluorescence lifetime distribution and the bursts of autofluorescence photons from tryptophan residues in Ecbeta Gal protein as well as fluorescence correlation spectroscopy of Ecbeta Gal. The results demonstrate that deep UV laser-based fluorescence lifetime microscopy is useful for identification of biological macromolecules at the single-molecule level using intrinsic fluorescence.