PubMed Health⌕ Search

Biomedical subjects

T French

Publications and source records attributed to T French.

10 recordsLinked to original sources
PubMed1997

Two-photon fluorescence lifetime imaging microscopy of macrophage-mediated antigen processing.

Two-photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage-mediated endocytosis, intracellular vacuolar encapsulation, and protease-dependent processing. Fluorescein-conjugated bovine serum albumin (FITC-BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time-dependent changes in fluorescence properties. Using two-photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0.5 ns increased to approximately 3.0 ns. Control experiments using fluorescein conjugated poly-L-lysine and poly-D-lysine demonstrated that the increase in fluorescence parameters observed with FITC-BSA were due to intracellular proteolysis since addition of the inert D-isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC-dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two-photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.

Animals↗
PubMed1996

Fluorescence generalized polarization of cell membranes: a two-photon scanning microscopy approach.

We use the lipophilic fluorescence probe Laurdan to study cell membranes. The generalized polarization (GP) of Laurdan-labeled cells contains useful information about membrane fluidity and polarity. A high GP is usually associated with low fluidity, low polarity, or high cholesterol content of the membranes, and a low GP is the opposite. We have combined the GP method and two-photon fluorescence microscopy to provide an alternative approach to study cell membranes. Using two-photon excitation in a conventional microscope offers great advantages for studying biological samples. These advantages include efficient background rejection, low photodamage, and improved depth discrimination. We performed GP measurements on mouse fibroblast cells and observed that both intensity and GP images are not spatially uniform. We tested for possible GP artifacts arising from cellular autofluorescence and lifetime quenching, using a procedure for background fluorescence subtraction and by direct lifetime measurements in the microscope. GP measured in a single cell displays a broad distribution, and the GP of 40 different cells grown on the same cover glass is also statistically distributed. The correlations between intensity and GP images were analyzed, and no monotonic dependence between the two was found. By digitally separating high and low GP values, we found that high GP values often associate with the regions of the plasma membrane and low GP values link with the nuclear membranes. Our results also show local GP variations within the plasma and nuclear membranes.

2-Naphthylamine↗
PubMed1995

Fluorescence lifetime imaging by asynchronous pump-probe microscopy.

We report the development of a scanning lifetime fluorescence microscope using the asynchronous, pump-probe (stimulated emission) approach. There are two significant advantages of this technique. First, the cross-correlation signal produced by overlapping the pump and probe lasers results in i) an axial sectioning effect similar to that in confocal and two-photon excitation microscopy, and ii) improved spatial resolution compared to conventional one-photon fluorescence microscopy. Second, the low-frequency, cross-correlation signal generated allows lifetime-resolved imaging without using fast photodetectors. The data presented here include 1) determination of laser sources' threshold powers for linearity in the pump-probe signal; 2) characterization of the pump-probe intensity profile using 0.28 microns fluorescent latex spheres; 3) high frequency (up to 6.7 GHz) lifetime measurement of rhodamine B in water; and 4) lifetime-resolved images of fluorescent latex spheres, human erythrocytes and a mouse fibroblast cell stained by rhodamine DHPE, and a mouse fibroblast labeled with ethidium bromide and rhodamine DHPE.

Animals↗
PubMed1992

The expressed VP4 protein of bluetongue virus binds GTP and is the candidate guanylyl transferase of the virus.

A minor core protein, VP4, of bluetongue virus serotype 10 (BTV-10) has been synthesized in insect cells infected with a genetically manipulated recombinant baculovirus. When insect cells were coinfected by this recombinant virus and a recombinant baculovirus expressing the two major core proteins (VP3 and VP7) of the virus, core-like particles (CLPs) consisting of all three proteins were formed. Purified CLPs reacted with [32P]GTP which was covalently bound to VP4 only. Similarly reconstituted CLPs with VP1 or VP6 did not form covalent complexes with [32P]GTP. The virion-derived VP4 was also shown to have GTP-binding activity. The covalent binding of GTP indicates that expressed VP4 not only is biologically active but also is the candidate guanylyl transferase of the virus. The optimum reaction conditions for GTP binding by VP4 have been investigated.

Bluetongue virus↗
PubMed1992

Protective efficacy of virus-like particles for bluetongue disease.

Bluetongue virus-like particles (VLPs) derived from multiple baculovirus expression vectors have been administered in the presence of various adjuvants to sheep, a vertebrate host susceptible to the virus, and the neutralizing antibody responses are measured. Vaccinated sheep are challenged after 4 months of inoculation, and clinical reaction indices and viraemia determined. The results indicate that these multiprotein virus-like particles lacking the genetic material are highly immunogenic and as little as 10 micrograms of VLPs in conjunction with appropriate adjuvant elicit an immune response which protects against infectious virus challenge. The formation of virus-like particles using this new technology offers a novel approach in vaccinology.

Animals↗
PubMed1990

Diagnosis of group A beta-hemolytic Streptococcus using clinical scoring criteria, Directigen 1-2-3 group A streptococcal test, and culture.

Cultures for group A beta-hemolytic Streptococcus were performed on 806 patients presenting with a sore throat to five urgent care centers. The accuracies of a clinical scoring system and of a liposomal in-office direct test for Streptococcus were compared with culture results. The Directigen 1-2-3 group A streptococcal test had a sensitivity of 67%, a specificity of 85%, a positive predictive value of 61%, and a negative predictive value of 89% compared with culture. The scoring system had a sensitivity of 26%, a specificity of 94%, a positive predictive value of 58%, and a negative predictive value of 79%. Using a combination of the direct test results and the clinical score did not improve the accuracy significantly over the use of either alone. The rates of delayed treatment, unnecessary treatment, and increased costs were compared using different combinations of the clinical scoring system, the in-office streptococcal test, and culture. Neither the Directigen 1-2-3 group A streptococcal test nor the clinical score can replace culture in the diagnosis of group A beta-hemolytic streptococcal pharyngitis.

Adolescent↗
PubMed1989

Comparison of the Directigen 1-2-3 Group A Strep Test with culture for detection of group A beta-hemolytic streptococci.

The Directigen 1-2-3 Group A Strep Test (DGAST; BBL Microbiology Systems, Cockeysville, Md.) was compared with conventional culture procedures on Trypticase soy agar with 5% sheep blood (BBL) and Selective Streptococcal Agar (ssA; BBL) for detection of group A beta-hemolytic streptococci (GABHS) for 1,006 patients complaining of sore throat. The DGAST was performed at five acute-care clinics according to the instructions of the manufacturer; interpretation of the cultures was done at the central microbiology laboratory. Of 924 patients with complete data, 243 (26.3%) were positive for GABHS on culture when both sheep blood agar and ssA were used. Of the patients with positive cultures, 159 were detected by the DGAST, yielding a sensitivity of 65.4%, a specificity of 84.7%, a positive predictive value of 60.5%, and a negative predictive value of 87.3%. The greater the number of colonies on culture, the greater the sensitivity of the DGAST, and the more intense the positive reaction on the DGAST, the higher the positive predictive value of the test. For the identification of GABHS, sheep blood agar was superior to ssA by 12.9% at 24 h and by 3.4% at 48 h of incubation.

Acute Disease↗