Biomedical subjects

Thomas T Perkins

Publications and source records attributed to Thomas T Perkins.

5 recordsLinked to original sources

PubMed2007

Stabilization of an optical microscope to 0.1 nm in three dimensions.

Mechanical drift is a long-standing problem in optical microscopy that occurs in all three dimensions. This drift increasingly limits the resolution of advanced surface-coupled, single-molecule experiments. We overcame this drift and achieved atomic-scale stabilization (0.1 nm) of an optical microscope in 3D. This was accomplished by measuring the position of a fiducial mark coupled to the microscope cover slip using back-focal-plane (BFP) detection and correcting for the drift using a piezoelectric stage. Several significant factors contributed to this experimental realization, including (i) dramatically reducing the low frequency noise in BFP detection, (ii) increasing the sensitivity of BFP detection to vertical motion, and (iii) fabricating a regular array of nanometer-sized fiducial marks that were firmly coupled to the cover slip. With these improvements, we achieved short-term (1 s) stabilities of 0.11, 0.10, and 0.09 nm (rms) and long-term (100 s) stabilities of 0.17, 0.12, and 0.35 nm (rms) in x, y, and z, respectively, as measured by an independent detection laser.

Algorithms↗

PubMed2006

Gold nanoparticles: enhanced optical trapping and sensitivity coupled with significant heating.

Gold nanoparticles appear to be superior handles in optical trapping assays. We demonstrate that relatively large gold particles (R(b)=50 nm) indeed yield a sixfold enhancement in trapping efficiency and detection sensitivity as compared to similar-sized polystyrene particles. However, optical absorption by gold at the most common trapping wavelength (1064 nm) induces dramatic heating (266 degrees C/W). We determined this heating by comparing trap stiffness from three different methods in conjunction with detailed modeling. Due to this heating, gold nanoparticles are not useful for temperature-sensitive optical-trapping experiments, but may serve as local molecular heaters. Also, such particles, with their increased detection sensitivity, make excellent probes for certain zero-force biophysical assays.

Energy Transfer↗

PubMed2004

Measuring 0.1-nm motion in 1 ms in an optical microscope with differential back-focal-plane detection.

Back-focal-plane detection of micrometer-sized beads offers subnanometer resolution for single-molecule, optical trapping experiments. However, laser beam-pointing instability and mechanical drift of the microscope limit the resolution of optical-trapping experiments. By combining two infrared lasers with improved differential beam-pointing stability (< or = 0.05 microrad), we simultaneously measure and subtract the motion of the microscope stage, leading to a resolution of <0.1 nm in 1 ms and stability of 0.5 nm over 60 s. Repeated steps of 0.4 nm at 1 Hz are resolved with a signal-to-noise ratio of 25.

Equipment Design↗

PubMed2004

Forward and reverse motion of single RecBCD molecules on DNA.

RecBCD is a processive, DNA-based motor enzyme with both helicase and nuclease activities. We used high-resolution optical trapping to study individual RecBCD molecules moving against applied forces up to 8 pN. Fine-scale motion was smooth down to a detection limit of 2 nm, implying a unitary step size below six basepairs (bp). Episodes of constant-velocity motion over hundreds to thousands of basepairs were punctuated by abrupt switches to a different speed or by spontaneous pauses of mean length 3 s. RecBCD occasionally reversed direction, sliding backward along DNA. Backsliding could be halted by reducing the force, after which forward motion sometimes resumed, often after a delay. Elasticity measurements showed that the DNA substrate was partially denatured during backsliding events, but reannealed concomitant with the resumption of forward movement. Our observations show that RecBCD-DNA complexes can exist in multiple, functionally distinct states that persist for many catalytic turnovers: such states may help tune enzyme activity for various biological functions.

Binding Sites↗

PubMed2003

Sequence-dependent pausing of single lambda exonuclease molecules.

Lambda exonuclease processively degrades one strand of duplex DNA, moving 5'-to-3' in an ATP-independent fashion. When examined at the single-molecule level, the speeds of digestion were nearly constant at 4 nanometers per second (12 nucleotides per second), interspersed with pauses of variable duration. Long pauses, occurring at stereotypical locations, were strand-specific and sequence-dependent. Pause duration and probability varied widely. The strongest pause, GGCGAT TCT, was identified by gel electrophoresis. Correlating single-molecule dwell positions with sequence independently identified the motif GGCGA. This sequence is found in the left lambda cohesive end, where exonuclease inhibition may contribute to the reduced recombination efficiency at that end.

Bacteriophage lambda↗