Biomedical subjects

Michael B Braunfeld

Publications and source records attributed to Michael B Braunfeld.

6 recordsLinked to original sources

PubMed2006

Automated acquisition of electron microscopic random conical tilt sets.

Single particle reconstruction using the random conical tilt data collection geometry is a robust method for the initial determination of macromolecular structures by electron microscopy. Unfortunately, the broad adoption of this powerful approach has been limited by the practical challenges inherent in manual data collection of the required pairs of matching high and low tilt images (typically 60 degrees and 0 degrees). The microscopist is obliged to keep the imaging area centered during tilting as well as to maintain accurate focus in the tilted image while minimizing the overall electron dose, a challenging and time consuming process. To help solve these problems, we have developed an automated system for the rapid acquisition of accurately aligned and focused tilt pairs. The system has been designed to minimize the dose incurred during alignment and focusing, making it useful in both negative stain and cryo-electron microscopy. The system includes a feature for montaging untilted images to ensure that all of the particles in the tilted image may be used in the reconstruction.

Computational Biology↗

PubMed2006

Tunable instability mechanisms of polymer thin films by molecular self-assembly.

Incorporation of a block copolymer into a thin polymer film is observed to alter both the rate and mechanism by which the film dewets from an immiscible polymer substrate. Films with little or no copolymer dewet by classical nucleation and growth of circular holes, and the dewetting rate decreases with increasing copolymer concentration. Increasing the copolymer content at constant film thickness generates copolymer micelles that adsorb/aggregate along the polymer/polymer interface and promote nonclassical dewetting fluctuations similar in appearance to spinodal dewetting. At higher copolymer concentrations, dewetting proceeds after a lengthy induction period by the nucleation and growth of flower-shaped holes suggestive of film pinning or viscous fingering. Atomic force microscopy of the polymer/polymer interface after removal of the top film by selective dissolution reveals substantial structural development due to copolymer self-assembly.

Microscopy, Atomic Force↗

PubMed2006

Electron energy filtering significantly improves amplitude contrast of frozen-hydrated protein at 300kV.

The amplitude contrast of frozen-hydrated biological samples was measured using the bacterial flagellar filament embedded in vitreous ice at an accelerating voltage of 300kV. From the mean radial amplitude spectra of overfocused images, amplitude contrast was estimated to be 6.9+/-1.9% and 2.7+/-1.0% of the whole contrast at the low spatial frequency range with and without energy filtering, respectively, and that of the carbon film to be 9.5+/-2.0% and 5.8+/-1.8%. Energy filtering effectively doubled the signal-to-noise ratio in the images of frozen-hydrated filaments, and substantially improved intensity data statistics of layer lines up to at least approximately 25A resolution in their Fourier transforms. It also markedly improved inter-particle fitting phase residuals of averaged data at resolutions up to approximately 15A. Using the energy filtered data recorded on a new high-performance, lens-coupled CCD camera the three-dimensional map of the flagellar filament was calculated at 8A by applying the amplitude contrast of 6.9%. The map and its mean radial density distribution validated the obtained value of the amplitude contrast.

Algorithms↗

PubMed2006

UCSF tomography: an integrated software suite for real-time electron microscopic tomographic data collection, alignment, and reconstruction.

A real-time alignment and reconstruction scheme for electron microscopic tomography (EMT) has been developed and integrated within our UCSF tomography data collection software. This newly integrated software suite provides full automation from data collection to real-time reconstruction by which the three-dimensional (3D) reconstructed volume is immediately made available at the end of each data collection. Real-time reconstruction is achieved by calculating a weighted back-projection on a small Linux cluster (five dual-processor compute nodes) concurrently with the UCSF tomography data collection running on the microscope's computer, and using the fiducial-marker free alignment data generated during the data collection process. The real-time reconstructed 3D volume provides users with immediate feedback to fully asses all aspects of the experiment ranging from sample choice, ice thickness, experimental parameters to the quality of specimen preparation. This information can be used to guide subsequent data collections. Access to the reconstruction is especially useful in low-dose cryo EMT where such information is very difficult to obtain due to extraordinary low signal to noise ratio in each 2D image. In our environment, we generally collect 2048 x 2048 pixel images which are subsequently computationally binned four-fold for the on-line reconstruction. Based upon experiments performed with thick and cryo specimens at various CCD magnifications (50000x-80000x), alignment accuracy is sufficient to support this reduced resolution but should be refined before calculating a full resolution reconstruction. The reduced resolution has proven to be quite adequate to assess sample quality, or to screen for the best data set for full-resolution reconstruction, significantly improving both productivity and efficiency of system resources. The total time from start of data collection to a final reconstructed volume (512 x 512 x 256 pixels) is about 50 min for a +/-70 degrees 2k x 2k pixel tilt series acquired at every 1 degrees.

Algorithms↗

PubMed2004

Spindle pole body duplication in fission yeast occurs at the G1/S boundary but maturation is blocked until exit from S by an event downstream of cdc10+.

The regulation and timing of spindle pole body (SPB) duplication and maturation in fission yeast was examined by transmission electron microscopy. When cells are arrested at G1 by nitrogen starvation, the SPB is unduplicated. On release from G1, the SPBs were duplicated after 1-2 h. In cells arrested at S by hydroxyurea, SPBs are duplicated but not mature. In G1 arrest/release experiments with cdc2.33 cells at the restrictive temperature, SPBs remained single, whereas in cells at the permissive temperature, SPBs were duplicated. In cdc10 mutant cells, the SPBs seem not only to be duplicated but also to undergo partial maturation, including invagination of the nuclear envelope underneath the SPB. There may be an S-phase-specific inhibitor of SPB maturation whose expression is under control of cdc10(+). This model was examined by induction of overreplication of the genome by overexpression of rum1p or cdc18p. In cdc18p-overexpressing cells, the SPBs are duplicated but not mature, suggesting that cdc18p is one component of this feedback mechanism. In contrast, cells overexpressing rum1p have large, deformed SPBs accompanied by other features of maturation and duplication. We propose a feedback mechanism for maturation of the SPB that is coupled with exit from S to trigger morphological changes.

CDC2 Protein Kinase↗

PubMed2004

An improved strategy for automated electron microscopic tomography.

A prediction-based scheme is proposed and implemented for automated electron microscopic tomography. By assuming that the sample follows a simple geometric rotation and that the optical system can be characterized in terms of an offset between the optical and mechanical axes, it is found that the image movement in the x, y, and z directions due to stage tilt can be dynamically predicted with desired accuracy (15 nm in x-y position and 100 nm in focus). Thus, the microscope optical system (beam/image shift and focus) can be automatically adjusted to compensate for the predicted image movement prior to taking the projected image at each tilt angle. As a consequence, it is not necessary to either record additional images for tracking and focusing during the course of data collections or to spend valuable setup time in a lengthy pre-calibration of stage motions. Furthermore, this scheme is also found to tolerate a significant degree of non-eucentricity and to be quite robust in the collection of regular and cryo low-dose images on thin or thick samples even at magnifications greater than 62000x and angular step as large as 10 degrees. For interested users the software can be freely downloaded for non-profit use at http://www.msg.ucsf.edu/tomography.

Animals↗