Thomas M. Yoshida

Flow cytometer for resolving signals from heterogeneous fluorescence emissions and quantifying lifetime in fluorochrome‐labeled cells/particles by phase‐sensitive detection

A phase‐sensitive flow cytometer has been developed that combines flow cytometry and fluorescence lifetime spectroscopy measurement principles to provide unique capabilities for making phase‐resolved measurements on fluorochrome‐labeled cells and particles. Stained cells are analyzed as they intersect a high‐frequency intensity‐modulated (sinusoid) laser excitation beam. Fluorescence is measured orthogonally using only a single‐channel optical detector. The detector output signals, which are phase shifted from a reference signal and amplitude demodulated, are processed by phase‐sensitive detection electronics to resolve signals from heterogeneous fluorescence emissions and quantify single‐component decay times. Results show signal phase shift and amplitude demodulation on fluorospheres; a detection limit threshold of 300–500 fluorescein molecules equivalence for excitation frequencies 1–30 MHz; a measurement precision (coefficient of variation) of 1.8% on alignment fluorospheres and 3.6% on cells stained ...

Differentiation of Bacillus anthracis, B. cereus, and B. thuringiensis by Using Pulsed-Field Gel Electrophoresis

ABSTRACT A pulsed-field gel electrophoresis (PFGE) method was developed for discriminating Bacillus anthracis from B. cereus and B. thuringiensis. A worldwide collection of 25 B. anthracis isolates showed high-profile homology, and these isolates were unambiguously distinguished from B. cereus and B. thuringiensis isolates by cluster analysis of the whole-genome macrorestriction enzyme digestion patterns generated by NotI.

Statistics of single-molecule measurements: Applications in flow-cytometry sizing of DNA fragments

The measurement of physical properties from single molecules has been demonstrated. However, the majority of single‐molecule studies report values based on relatively large data sets (e.g., N > 50). While there are studies that report physical quantities based on small sample sets, there has not been a detailed statistical analysis relating sample size to the reliability of derived parameters.

New flow cytometric capabilities at the national flow cytometry resource

The authors provide a brief review of flow cytometry and a broad view of new flow cytometry technologies. A brief introduction to flow cytometry and cell sorting is provided along with a summary of current commercial capabilities. New developments designed to overcome current limitations are described in terms of capabilities and a characterization of their performance. New capabilities include Fourier transform flow cytometry, phase sensitive detection, digital data acquisition, data clustering algorithms, high speed sorting, and slit scanning. >

Performance assessment of DNA fragment sizing by high-sensitivity flow cytometry and pulsed-field gel electrophoresis

ABSTRACT The sizing of restriction fragments is the chief analytical technique utilized in the production of DNA fingerprints. Few techniques have been able to compete with pulsed-field gel electrophoresis (PFGE), which is capable of discriminating among bacteria at species and strain levels by resolving restriction fragments. However, an ultrasensitive flow cytometer (FCM) developed in our lab has also demonstrated the ability to discriminate bacteria at species and strain levels. The abilities of FCM warrant a quantitative parallel comparison with PFGE to assess and evaluate the accuracy and precision of DNA fragment sizing by both techniques. Replicate samples of Staphylococcus aureus Mu50 were analyzed along with two clinical S. aureus isolates. The absolute fragment sizing accuracy was determined for PFGE (5% ± 2%) and FCM (4% ± 4%), with sequence-predicted Mu50 SmaI fragment sizes used as a reference. Precision was determined by simple arithmetic methods (relative standard deviation for PFGE [RSDPFGE ] = 3% ± 2% and RSDFCM = 1.2% ± 0.8%) as well as by the use of dendrograms derived from Dice coefficient-unweighted pair group method with arithmetic averages (UPGMA) and Pearson-UPGMA analyses. All quantitative measures of PFGE and FCM precision were equivalent, within error. The precision of both methods was not limited by any single sample preparation or analysis step that was tracked in this study. Additionally, we determined that the curve-based clustering of fingerprint data provided a more informative and useful assessment than did traditional band-based methods.

Evaluation of different nucleic acid stains for sensitive double-stranded DNA analysis with capillary electrophoretic separation

This paper outlines the first use of SYTOX Orange, SYTO 82 and SYTO 25 nucleic acid stains for on-column staining of double-stranded DNA (dsDNA) fragments separated by capillary electrophoresis (CE). Low-viscosity, replaceable poly(vinylpyrrolidone) (PVP) polymer solution was used as the sieving matrix on an uncoated fused-silica capillary. The effects of PVP concentration, electric field strength, and incorporated nucleic acid stain concentrations on separation efficiency were examined for a wide range of DNA fragment sizes. Our study was focused on using nucleic acid stains efficiently excitable at a wavelength of 532 nm. Among the five tested nucleic acid stains, SYTOX Orange stain was shown to have the best sensitivity for dsDNA detection by CE. About a 500-fold lower detection limit was obtained compared to commonly used ethidium bromide and propidium iodide. SYTOX Orange stain also provided a wide linear dynamic range for direct DNA quantitation with on-line CE detection. Use of SYTOX Orange stain can greatly improve the measurement of DNA fragments by CE, which will enable an expanded set of applications in genomics and diagnostics.

Evaluation of Bacillus anthracis and Yersinia pestis sample collection from nonporous surfaces by quantitative real-time PCR.

Aim:  We will validate sample collection methods for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment.

Market Research Survey of Commercial Off-the-Shelf Mass Spectrometers for In-Field Analysis. FY 15 Update

This report is an update of the 2013 Market Research Survey1-3 of field-deployable commercial off-the-shelf (COTS) mass spectrometry instruments for safeguards application.

NGSI FY15 Final Report. Innovative Sample Preparation for in-Field Uranium Isotopic Determinations

Our FY14 Final Report included an introduction to the project, background, literature search of uranium dissolution methods, assessment of commercial off the shelf (COTS) automated sample preparation systems, as well as data and results for dissolution of bulk quantities of uranium oxides, and dissolution of uranium oxides from swipe filter materials using ammonium bifluoride (ABF). Also, discussed were reaction studies of solid ABF with uranium oxide that provided a basis for determining the ABF/uranium oxide dissolution mechanism. This report details the final experiments for optimizing dissolution of U3O8 and UO2 using ABF and steps leading to development of a Standard Operating Procedure (SOP) for dissolution of uranium oxides on swipe filters.