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Biomedical subjects

B Hecht

Publications and source records attributed to B Hecht.

At least 19 recordsLinked to original sources
PubMed2006

Absorption and fluorescence of single molecules.

Simultaneous detection of single molecules by absorption and fluorescence is demonstrated using confocal microscopy at cryogenic temperature. Dynamical processes such as blinking and spectral jumping of single emitters are observed in both detection channels. The relative magnitude of fluorescence and absorption varies between molecules. In particular, we observe molecules that do not emit detectable Stokes-shifted fluorescence but show a strong absorption signal. The fact that coherent resonant scattering underlies the absorption process is demonstrated by a correlation between small linewidth and large absorption amplitude.

Journal Article↗
PubMed2005

Single quantum dot coupled to a scanning optical antenna: a tunable superemitter.

The interaction of a single quantum dot with a bowtie antenna is demonstrated for visible light. The antenna is generated at the apex of a Si3N4 atomic force microscopy tip by focused ion beam milling. When scanned over the quantum dot, its photoluminescence is enhanced while its excited-state lifetime is decreased. Our observations demonstrate that the relaxation channels of a single quantum emitter can be controlled by coupling to an efficiently radiating metallic nanoantenna.

Journal Article↗
PubMed2005

Resonant optical antennas.

We have fabricated nanometer-scale gold dipole antennas designed to be resonant at optical frequencies. On resonance, strong field enhancement in the antenna feed gap leads to white-light supercontinuum generation. The antenna length at resonance is considerably shorter than one-half the wavelength of the incident light. This is in contradiction to classical antenna theory but in qualitative accordance with computer simulations that take into account the finite metallic conductivity at optical frequencies. Because optical antennas link propagating radiation and confined/enhanced optical fields, they should find applications in optical characterization, manipulation of nanostructures, and optical information processing.

Journal Article↗
PubMed2003

Single-molecule near-field optical energy transfer microscopy with dielectric tips.

The fluorescence lifetime and the fluorescence rate of single molecules are recorded as a function of the position of a Si3N4 atomic force microscopy tip with respect to the molecule. We observe a decrease of the excited state lifetime and the fluorescence rate when the tip apex is in close proximity to the molecule. These effects are attributed to the fact that the dielectric tip converts non-propagating near-fields to propagating fields within the dielectric tip effectively quenching the fluorescence. The spatial extension of the quenching area is of subwavelength dimensions. The results are discussed in terms of molecular fluorescence in a system of stratified media. The experiment provides surprising new insights into the interactions between a fluorescent molecule and a dielectric tip. The methodology holds promise for applications in ultra high-resolution near-field optical imaging at the level of single fluorophores.

Carbocyanines↗
PubMed2001

Statistical analysis of single-molecule colocalization assays.

In chemical assays, specific molecular recognition events result in close physical proximity of two molecular species, e.g., ligands and receptors. Microscopy techniques that are able to image individual molecules allow for achieving a positional accuracy far beyond the resolution limit Therefore, independent position determination, e.g., by dual-color microscopy, becomes possible, permitting determination of intermolecular distances beyond the resolution limit. Nonzero measured distances occur due to experimental inaccuracies in case of a recognition event or due to accidental close proximity between ligand-receptor pairs. Using general statistical considerations, finite measured distances between single ligand-receptor pairs are directly translated into probabilities for true molecular recognition or mere accidental proximity. This enables a quantitative statistical analysis of single recognition events. It is demonstrated that in a general assay, even in the presence of strong unspecific background, the probability for a certain diagnosis and a measure for its reliability can be extracted from the observation of a few binding events. The power of the method is demonstrated at the example of a single-molecule DNA hybridization assay. Our findings are of major importance for future assay miniaturization and assaying with minute amounts of analyte.

Journal Article↗
PubMed2001

Probing confined fields with single molecules and vice versa.

Single dye molecules are used as local probes to map the spatial distribution of the squared electric field components in the focus of a high numerical aperture lens. Simulated field distributions are quantitatively verified by experimentally obtained fluorescence excitation maps. We show that annular illumination can be used to engineer the field distribution in the focus at a dielectric/air interface such that electric field components in all directions acquire comparable magnitudes. The 3D orientation of molecular absorption dipoles can be determined by comparing measured to simulated image patterns. The presence of longitudinal electric field components in a focus is of particular interest in tip-enhanced scanning near-field optical microscopy.

Journal Article↗
PubMed2000

Orientational imaging of single molecules by annular illumination

The absorption dipole orientation of single fluorescent molecules is determined by mapping the spatial distribution of the squared electric field components in a high-numerical-aperture laser focus. Annular illumination geometry and the vicinity of a plane dielectric/air interface strongly enhance the longitudinal field component and the transverse fields perpendicular to the polarization direction. As a result, all three excitation field components in the focus are of comparable magnitude. The scheme holds promise to monitor rotational diffusion of single molecules in complex environments.

Journal Article↗
PubMed2000

Nonclassical photon statistics in single-molecule fluorescence at room temperature

The fluorescence of single terrylene molecules in a crystalline host is investigated at room temperature by scanning confocal optical microscopy. Photon arrival times are analyzed in terms of interphoton time distributions, second order correlation functions, and the variance of the photon number probability distribution. Antibunching at short times and bunching behavior for longer times is observed, associated with sub- and super-Poissonian statistics, respectively. A rate-equation analysis of the molecular level populations indicates an accelerated reverse intersystem crossing.

Journal Article↗
PubMed2000

Single-molecule identification by spectrally and time-resolved fluorescence detection

A method to identify single molecules rapidly and with high efficiency based on simple probability considerations is proposed. In principle, any property of a detected photon in a single-molecule fluorescence experiment, e.g., emission wavelength, arrival time after pulsed excitation, and polarization, can be analyzed within the framework of the outlined methodology. Monte Carlo simulations show that less than 500 photons are needed to assign an observed single molecule to one out of four species with a confidence level higher than 99.9%. We show that single dye molecules of four different dyes embedded in a polymer film can be identified with time-correlated single-photon counting spectrally resolved in two channels.

Journal Article↗
PubMed1995

The effect of interposition membrane on the outcome of lumbar laminectomy and discectomy.

STUDY DESIGN: This study evaluated clinical and magnetic resonance imaging differences of patients treated surgically for lumbar disc herniation. Clinical follow-up and magnetic resonance imaging evaluation of epidural fibrosis were used to assess patient outcome. OBJECTIVES: The purpose of this study was to evaluate the difference in clinical outcome with either free-fat graft, Gelfoam, or no interposition membrane placed in the laminectomy defect after nerve root decompression. SUMMARY OF BACKGROUND DATA: Epidural fibrosis has been considered a cause of recurrent symptoms after lumbar laminectomy, and numerous materials have been evaluated for prophylaxis of the "laminectomy membrane." These have been mainly histologic and animal studies with no data correlating clinical symptoms and postoperative epidural scar formation. METHODS: One hundred fifty-six patients who were treated surgically for lumbar disc herniation were randomly assigned to one of three groups and followed prospectively for at least 1 year. Thirty-three of these patients were received magnetic resonance imaging evaluations after 6 months by an independent radiologist who graded the amount of epidural scar formation. The patients were assessed at 1 year and given a rating of excellent, good, fair, or poor, and the scar was graded as none, minimal, or moderate. RESULTS: Although 97% of all patients improved, 83% were rated excellent or good. There were no statistical differences between the three groups clinically and radiographically. Patients with workers compensation had a statistically significant lower success rate (P < 0.001). CONCLUSIONS: Clinical outcome after lumbar disc surgery does not correlate with the use or type of interposition membrane used to prevent epidural fibrosis.

Adolescent↗
PubMed1995

Characterization of non-inducible Tet repressor mutants suggests conformational changes necessary for induction.

Non-inducible tetracycline repressor (TetR) mutants were grouped in three structurally distinct classes. We quantitated in vivo operator binding, inducibility, and in vitro tetracycline binding of mutants from each class. Mutation of residues close to tetracycline (class 1) leads to reduced affinity for the drug. Mutation of residues located at the connection of the DNA-reading head with the protein core (class 2) and at the dimerization interface (class 3) bind inducer with the same affinity as wild-type TetR. These mutations interfere with the induced, but not the operator-binding conformation of TetR. The affinity of some class 1 mutants for tetracycline is less affected than their inducibility, suggesting that the mutated residues are important for triggering those conformational changes necessary for induction.

Amino Acid Sequence↗
PubMed1993

Comparison of the methods of artificial insemination on the incidence of conception in single unmarried women.

OBJECTIVE: To compare pregnancy rates after intrauterine insemination (IUI) versus pericervical insemination in absolute male factor infertility using each patient as her own control. DESIGN: Ovulatory women with patent fallopian tubes without male partners were alternately inseminated with cryopreserved donor semen using either IUI or pericervical insemination techniques. A total of 81 cycles, which included up to 4 cycles per patient were performed. In this manner a comparison between the efficacy of each method could be evaluated. SETTING: The donor insemination program at the Center For Assisted Reproduction at Northwestern University Medical School. PATIENTS: Twenty-six single, healthy, unmarried women with patent fallopian tubes and < 40 years of age without male partners (absolute male factor infertility). MAIN OUTCOME MEASURES: Positive quantitative serum subunit of human chorionic gonadotropin followed by the presence of an intrauterine gestational sac seen by transvaginal ultrasonography. RESULTS: Fourteen (54%) of 26 patients conceived including two (14%) miscarriages within four insemination cycles. Seven (17.5%) patients after IUI, and 7 (17.1%) patients after pericervical insemination conceived. The pregnancy rates were similar regardless of the order of insemination method. CONCLUSION: These findings reveal that there is no statistical difference in the pregnancy outcome between these two methods of insemination in absolute male factor infertility.

Adult↗
PubMed1993

Noninducible Tet repressor mutations map from the operator binding motif to the C terminus.

We have developed a new genetic selection system for Tet repressor mutations with a noninducible phenotype for tetracycline (TetRs). Extensive chemical mutagenesis of tetR yielded 93 single-site Tet repressor mutations. They map from residue 23 preceding the alpha-helix-turn-alpha-helix operator binding motif to residue 196 close to the C terminus of the repressor. Thirty-three of the mutations are clustered between residues 95 and 117, and another 27 are clustered between residues 131 to 158. Several of the mutants were characterized quantitatively in vivo for induction by tetracycline and anhydrotetracycline. While all of these are severely reduced in tetracycline-mediated induction, only some of them are affected for anhydrotetracycline-mediated induction.

Amino Acid Sequence↗
PubMed1993

Salpingitis or oophoritis: what causes fever following oocyte aspiration and embryo transfer?

BACKGROUND: Febrile morbidity following in vitro fertilization and embryo transfer (IVF-ET) is a rare but possibly serious complication. This report describes a case of salpingitis after IVF-ET and discusses the possible reasons for febrile morbidity following this common procedure. CASE: A 37-year-old woman undergoing IVF-ET for tubal factor infertility developed sudden, severe pelvic pain, fever, and leukocytosis 24 hours after ET. Laparoscopy revealed bilateral suppurative pyosalpinges with cystic, hemorrhagic ovaries. Pain, fever, and leukocytosis resolved with conservative surgery and intravenous antibiotic therapy. CONCLUSIONS: This case presents laparoscopic documentation of a rare complication of oocyte aspiration and/or ET, namely, salpingitis. Possibilities for the development of salpingitis following IVF-ET include activation of quiescent bacteria within the fallopian tubes from a previous pelvic infection, puncture of the bowel during oocyte aspiration, inoculation of the pelvis with cervicovaginal flora during oocyte aspiration, and introduction of bacteria-laden secretions or air into the fallopian tubes during ET. Although rare, the possibility of severe pelvic infection following IVF-ET warrants consideration of prophylactic antibiotic coverage.

Adult↗
PubMed1992

Functional roles of amino acid residues involved in forming the alpha-helix-turn-alpha-helix operator DNA binding motif of Tet repressor from Tn10.

The Tn10 derived Tet repressor contains an amino acid segment with high homology to the alpha-helix-turn-alpha-helix motif (HTH) of other DNA binding proteins. The five most conserved amino acids in HTH are probably involved in structural formation of the motif. Their functional role was probed by saturation mutagenesis yielding 95 single amino acid replacement mutants of Tet repressor. Their binding efficiencies to tet operator were quantitatively determined in vivo. All functional mutants contain amino acid substitutions consistent with their proposed role in a HTH. In particular, only the two smallest amino acids (serine, glycine) can substitute a conserved alanine in the proposed first alpha-helix without loss of activity. The last position of the first alpha-helix, the second position in the turn, and the fourth position in the second alpha-helix require mostly hydrophobic residues. The proposed C-terminus of the first alpha-helix is supported by a more active asparagine compared to glutamine replacement mutant of the wt leucine residue. The turn is located close to the protein surface as indicated by functional lysine and arginine replacements for valine. A glycine residue at the first position in the turn can be replaced by any amino acid yielding mutants with at least residual tet operator affinity. A structural model of the HTH of Tet repressor is presented.

Amino Acid Sequence↗