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Biomedical subjects

Peter M Goodwin

Publications and source records attributed to Peter M Goodwin.

9 recordsLinked to original sources
PubMed2006

Single particle high resolution spectral analysis flow cytometry.

BACKGROUND: While conventional multiparameter flow cytometers have proven highly successful, there are several types of analytical measurements that would benefit from a more comprehensive and flexible approach to spectral analysis including, but certainly not limited to spectral deconvolution of overlapping emission spectra, fluorescence resonance energy transfer measurements, metachromic dye analysis, free versus bound dye resolution, and Raman spectroscopy. METHODS: Our system utilizes a diffraction grating to disperse the collected fluorescence and side-scattered light from cells or microspheres passing through the interrogation region over a rectangular charge-coupled-device image sensor. The flow cell and collection optics are taken from a conventional flow cytometer with minimal modifications to assure modularity of the system. RESULTS: Calibration of the prototype spectral analysis flow cytometer included wavelength characterization and calibration of the dispersive optics. Benchmarking of the system demonstrated a single particle/cell intensity sensitivity of 2160 MESF of R-Phycoerythrin. Single particle spectra taken with our instrument were validated against bulk solution fluorimeter and conventional flow cytometer measurements. Coefficients of variation of integrated spectral fluorescence intensity of several sets of standard fluorescent microspheres ranged from 1.4 to 4.8% on the spectral system. Spectral discrimination of free versus PI bound to cells is also demonstrated. CONCLUSIONS: It is demonstrated that the flow spectrometer has sufficient sensitivity and wavelength resolution to detect single cells and microspheres, including multi-fluorophore labeled microspheres. The capability to use both standard mathematical deconvolution techniques for data analysis, coupled with the feasibility of integration with existing flow cytometers, will improve the accuracy and precision of ratiometric measurements, enable the analysis of more discrete emission bands within a given wavelength range, and allow more precise resolution of the relative contribution of individual fluorophores in multiply-tagged samples, thereby enabling a range of new applications involving the spectral analysis of single cells and particles.

Calibration↗
PubMed2006

A two-dimensional view of the folding energy landscape of cytochrome c.

Time-correlated single photon counting (TCSPC) was combined with fluorescence correlation spectroscopy (FCS) to study the transition between acid-denatured states and the native structure of cytochrome c (Cyt c) from Saccharomyces cerevisiae. The use of these techniques in concert proved to be more powerful than either alone, yielding a two-dimensional picture of the folding energy landscape of Cyt c. TCSPC measured the distribution of distances between the heme of the protein and a covalently attached dye molecule at residue C102 (one folding reaction coordinate), whereas FCS measured the hydrodynamic radius (a second folding reaction coordinate) of the protein over a range of pH values. These two independent measurements provide complimentary information regarding protein conformation. We see evidence for a well defined folding intermediate in the acid renaturation folding pathway of this protein reflected in the distribution of lifetimes needed to fit the TCSPC data. Moreover, FCS studies revealed this intermediate state to be in dynamic equilibrium with unfolded structures, with conformational fluctuations into and out of this intermediate state occurring on an approximately 30-micros time scale.

Biophysics↗
PubMed2006

A comparison of the fluorescence dynamics of single molecules of a green fluorescent protein: one- versus two-photon excitation.

We report on the dynamics of fluorescence from individual molecules of a mutant of the wild-type green fluorescent protein (GFP) from Aequorea victoria, super folder GFP (SFGFP). SFGFP is a novel and robust variant designed for in vivo high-throughput screening of protein expression levels. It shows increased thermal stability and is able to retain its fluorescence when fused to poorly folding proteins. We use a recently developed single-molecule technique which combines fluorescence-fluctuation spectroscopy and time-correlated single photon counting in order to characterize the photophysical properties of SFGFP under one- (OPE) and two- (TPE) photon excitation conditions. We use Rhodamine 110 as a model chromophore to validate the methodology and to explain the single-molecule results of SFGFP. Under OPE, single SFGFP molecules undergo fluorescence flickering on the time scale of micros and tens of micros due to triplet formation and ground-state protonation-deprotonation, respectively, as demonstrated by excitation intensity- and pH-dependent experiments. OPE single-molecule fluorescence lifetimes indicate heterogeneity in the population of SFGFP, indicating the presence of the deprotonated I and B forms of the SFGFP chromophore. TPE of single SFGFP molecules results in the photoconversion of the chromophore. TPE of single SFGFP molecules show fluorescence flickering on the time scale of micros due to triplet formation. A flicker connected with protonation-deprotonation of the SFGFP chromophore is detected only at low pH. Our results show that SFGFP is a promising fusion reporter for intracellular applications using OPE and TPE microscopy.

Biophysical Phenomena↗
PubMed2005

Site-specific dimensions across a highly denatured protein; a single molecule study.

Do highly denatured proteins adopt random coil configurations? Here, we address this question by measuring residue-to-residue separations across the denatured FynSH3 domain. Using single-molecule Forster resonance energy transfer techniques, we have collected transfer efficiency probability distributions for dye-labeled, denatured protein. Applying maximum likelihood analysis to the interpretation of these distributions, we have determined the through-space distance between five residue pairs in the protein's guanidine hydrochloride-unfolded and trifluoroethanol-unfolded states. We find that, while the dimensions of the guanidine hydrochloride -unfolded molecule generally coincide with the dimensions predicted for a random coil ensemble, potentially statistically significant deviations from random coil behavior are also evident. These small, site-specific deviations may provide a means of reconciling earlier, scattering-based evidence for the random coil nature of the unfolded state with more site-specific spectroscopic evidence suggesting residual structure. We have also studied the unfolded ensemble populated in 50% trifluoroethanol, a denaturant that induces a highly helical unfolded state. We find that the size and shape of the unfolded ensemble under these conditions is effectively indistinguishable from that populated in guanidinium hydrochloride solutions, suggesting that the gross structure of the denatured state is, perhaps surprisingly, independent of the chemistry of the cosolvent.

Escherichia coli Proteins↗
PubMed2004

Exonuclease I hydrolyzes DNA with a distribution of rates.

We report heterogeneity in the time necessary for Exonuclease I to hydrolyze identical DNA fragments. A real-time fluorescence method measured the time required by molecules of Exonuclease I to hydrolyze single-stranded DNA that was synthesized to have two fluorescently labeled nucleotides. One fluorescently labeled nucleotide was located near the 3' end of the DNA and the other near the 5' end. Heterogeneity in the hydrolysis rate of the exonuclease population was inferred from the distribution of times necessary to cleave these DNA fragments. In particular, we found simple first-order kinetics, using a single hydrolysis rate, did not result in a good fit to the data. Better fits to the data were obtained if one assumed a distribution of hydrolysis rates for the exonuclease population. Under our experimental conditions, this broad distribution of rates was centered near 100 nt/s.

DNA↗
PubMed2004

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.

DNA↗
PubMed2003

A simple quenching method for fluorescence background reduction and its application to the direct, quantitative detection of specific mRNA.

New genome sequence information is rapidly increasing the number of nucleic acid (NA) targets of use for characterizing and treating diseases. Detection of these targets by fluorescence-based assays is often limited by fluorescence background from unincorporated or unbound probes that are present in large excess over the target. To solve this problem, energy transfer-based probes have been developed and used to reduce the fluorescence from unbound probes. Although these probes have revolutionized NA target detection, their use requires scrupulous attention to design constraints, extensive probe quality control, and individually optimized experimental conditions. Here, we describe a simpler background reduction approach using singly labeled quencher oligomers to suppress excess unbound probe fluorescence following probe-target hybridization. A second limitation of most fluorescence-based NA target detection and quantification assays is the requirement for enzymatic amplification of target or signal for sensitivity. Amplification steps make quantification of original target copy number problematic because of variations in amplification efficiencies between the sequence targets and the experimental conditions. To avoid amplification, we coupled our quenching approach to a two-color NA assay with correlated, two-color, single-molecule fluorescence detection. We demonstrate a >100-fold background reduction and detection of targets present at concentrations as low as 100 fM using the two-color assay. The application of this technique to the detection and quantification of specific mRNA sequences enabled us to estimate beta-actin copy numbers in cell-derived total RNA without an amplification step.

Actins↗
PubMed2003

Progress towards single-molecule DNA sequencing: a one color demonstration.

Single molecules of fluorescently labeled nucleotides were detected during the cleavage of individual DNA fragments by a processive exonuclease. In these experiments, multiple (10-100) strands of DNA with tetramethyl rhodamine labeled dUMP (TMR-dUMP) incorporated into the sequence were anchored in flow upstream of the detection region of an ultra sensitive flow cytometer. A dilute solution of Exonuclease I passed over the microspheres. When an exonuclease attached to a strand, processive digestion of that strand began. The liberated, labeled bases flowed through the detection region and were detected at high efficiency at the single-molecule level by laser-induced fluorescence. The digestion of a single strand of DNA by a single exonuclease was discernable in these experiments. This result demonstrates the feasibility of single-molecule DNA sequencing. In addition, these experiments point to a new and practical means of arriving at a consensus sequence by individually reading out identical sequences on multiple fragments.

Color↗