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.     

Computer program for the IBM personal computer which searches for approximate matches to short oligonucleotide sequences in long target DNA sequences.

We describe a program which may be used to find approximate matches to a short predefined DNA sequence in a larger target DNA sequence. The program predicts the usefulness of specific DNA probes and sequencing primers and finds nearly identical sequences that might represent the same regulatory signal. The program is written in the C programming language and will run on virtually any computer system with a C compiler, such as the IBM/PC and other computers running under the MS/DOS and UNIX operating systems. The program has been integrated into an existing software package for the IBM personal computer (see article by Mount and Conrad, this volume). Some examples of its use are given.     

Use of 3D geometry modeling of osteochondrosis-like iatrogenic lesions as a template for press-and-fit scaffold seeded with mesenchymal stem cells

Computed tomography (CT) is an effective diagnostic modality for three-dimensional imaging of bone structures, including the geometry of their defects. The aim of the study was to create and optimize 3D geometrical and real plastic models of the distal femoral component of the knee with joint surface defects. Input data included CT images of stifle joints in twenty miniature pigs with iatrogenic osteochondrosis-like lesions in medial femoral condyle of the left knee. The animals were examined eight and sixteen weeks after surgery. Philips MX 8000 MX and View workstation were used for scanning parallel plane cross section slices and Cartesian discrete volume creation. On the average, 100 slices were performed in each stifle joint. Slice matrices size was 512 x 512 with slice thickness of 1 mm. Pixel (voxel) size in the slice plane was 0.5 mm (with average accuracy of +/-0.5 mm and typical volume size 512 x 512 x 100 voxels). Three-dimensional processing of CT data and 3D geometrical modelling, using interactive computer graphic system MediTools formerly developed here, consisted of tissue segmentation (raster based method combination and 5 % of manual correction), vectorization by the marching-cubes method, smoothing and decimation. Stifle- joint CT images of three individuals of different body size (small, medium and large) were selected to make the real plastic models of their distal femurs from plaster composite using rapid prototyping technology of Zcorporation. Accuracy of the modeling was +/- 0.5 mm. The real plastic models of distal femurs can be used as a template for developing custom made press and fit scaffold implants seeded with mesenchymal stem cells that might be subsequently implanted into iatrogenic joint surface defects for articular cartilage-repair enhancement.     

A microcomputer interface for a digital audio processor-based data recording system.

An inexpensive interface is described that performs direct transfer of digitized data from the digital audio processor and video cassette recorder based data acquisition system designed by Bezanilla (1985, Biophys. J., 47:437-441) to an IBM PC/XT microcomputer. The FORTRAN callable software that drives this interface is capable of controlling the video cassette recorder and starting data collection immediately after recognition of a segment of previously collected data. This permits piecewise analysis of long intervals of data that would otherwise exceed the memory capability of the microcomputer.     

森林资源模型仿真研究

一、引言森林是自然生态的主体,是人类赖以生存的重要条件。现在许多国家已把森林生态环境的好坏作为文明建设的标志之一。人们已经逐渐认识到,森林不仅是重要的木材资源,而且     

Two-dimensional spectrophotometry with high spatial and temporal resolution by digital video techniques and powerful computers

A two-dimensional spectrophotometer having a spatial raster resolution of 512 X 512 picture elements with 256 grey levels and a time resolution of 30 images per min is assembled by combination of digital video techniques and a powerful computer system. The instrument is applied to the analysis of pattern formation processes in cytoplasmic media.     

The frequency of recurrences and metastases of bladder cancer following a radical course of radiotherapy]

The authors compare the incidence of bladder carcinoma recurrences and dissemination after long-distance gamma-beam therapy, carried out according to a traditional method, and in an accelerated hyperfractionation mode combined with metronidazole therapy. The recurrences were most frequently detected at the site of the primary tumor (62-67% of cases) in 6 to 18 months after the treatment. They come to a conclusion that accelerated hyperfractionation and local metronidazole radio-modification were conducive to a reduction of the incidence of recurrences from 45 to 28% as against the traditional split radiotherapy course in the classical fractionation mode.     

Easily assembled digital data acquisition system for the analytical ultracentrifuge

A system for the acquisition of digital data from the analytical ultracentrifuge which uses a commercially available data acquisition board, a standard IBM compatible personal computer (PC), and an interface circuit has been developed. The system uses the signal from the standard Beckman scanner. Preliminary analysis and data reduction are performed at the PC within minutes of data acquisition using simple commercially available software, and final data fitting is performed with a mainframe computer. Procedures are described which allow approach to equilibrium to be followed and attainment of equilibrium to be demonstrated. Data density of 200 points per millimeter column height (500 points per 100 μ1 of sample) allows the use of short columns and hence short run times. Only 2 min are required to collect a complete scan, which is recorded in a format suitable for direct analysis by standard spreadsheet software. This allows multiple sequential scans to be quickly recorded at equilibrium and averaged to reduce noise prior to analysis. The combination of characteristics allows molecular weight determinations to be performed relatively quickly with only a few micrograms of protein. The system is inexpensive and easy to assemble given the centrifuge and a PC.     

A simulation support system for solving large physiological models on microcomputers

Although physiological modeling and computer simulation have become useful research tools to test new scientific theories and to design and analyze laboratory experiments, developing a new model can be a tedious process because the investigator must often write very complex and specific routines for data input and output. To facilitate the design of new models (as well as the use of existing models), we have developed MODSIM, a FORTRAN-based simulation support system for the IBM PC computer than can accommodate very large dynamic models having up to several thousand equations. It provides the investigator with utilities for continuous on-line graphical and/or tubular output, as well as facilities for dynamic interaction with the model. The user must only supply a model as a list of mathematical equations written in FORTRAN, along with the initial values of the model variables and parameters. The model is precompiled, compiled, and then linked to the MODSIM utilities. Without further programming, the user can then solve the model, select variables for graphical output, and stop the model at any time to analyze the data or to change a parameter before resuming the simulation. This simulation system makes it very easy to develop new models that actively interact with the experimental research of the investigator.     

Improved programs for DNA and protein sequence analysis on the IBM personal computer and other standard computer systems.

We have previously described programs for a variety of types of sequence analysis (1-4). These programs have now been integrated into a single package. They are written in the standard C programming language and run on virtually any computer system with a C compiler, such as the IBM/PC and other computers running under the MS/DOS and UNIX operating systems. The programs are widely distributed and may be obtained from the authors as described below.     

Automated Turbidimetric Bioassay Readout Instrument Using a Multiple Flow-Cell System

An instrument has been developed which permits the automatic quantitation of the turbidities of microbiological assay samples. Assay tubes were fed to the instrument at the end of the incubation period. The turbidity readings were automatically converted to digital data which were printed on International Business Machines (IBM) cards and from which potencies were calculated by an IBM computer. The instrument operated at a speed of over 240 tube readings per hour and was totally automatic in sample-mixing, readout, and data recording. The instrument is being used routinely at The Upjohn Co. for the turbidimetric bioassay of vitamins, with a coefficient of variation among repeated turbidity readings of 0.12 to 0.23%.     

The effect of sampling frequency on EMG measures of occupational mechanical exposure.

The purpose of the present study was to determine how much it is possible to downsample EMG without loosing the accuracy of some EMG measures of occupational mechanical exposure frequently used in the literature. The EMG signals of four muscles (two trapezius, right deltoid and right extensor digitorum) were collected (sampling frequency: 1024 Hz) from 20 subjects while performing a 20 min computer work task. The EMG RMS amplitude was computed from 0.125 s successive time-windows for the original (1024 Hz) and four additional digitally resampled (at 512, 256, 128 and 64 Hz) EMG signals. Three of the most frequently used data reduction methods (1. gaps analysis, 2. amplitude probability distribution function and 3. exposure variation analysis) were used to summarise the 20 min EMG activation profiles. The results from the exposure variation analysis were summarized into three variables using a new method detailed here. In general, the ANOVA for repeated measures demonstrated that a decrease of the sampling frequency significantly changed the EMG measures (relative to the 1024 Hz sampling condition) most of the time at 64 and 128 Hz, occasionally at 256 Hz, but practically never at 512 Hz. An analysis of the maximal errors (relative to the 1024 Hz condition) across all subjects supported these findings. Consequently, it was concluded that 512 Hz is quite conservative and should practically never lead to invalid EMG parameters estimations. Conversely, 256 Hz represents the lowest limit tolerable for some EMG parameters (gaps analysis, amplitude probability distribution function) while it is unacceptable for others (modified exposure variation analysis).     

Optimization of CSTRs in series by dynamic programming

This article concerns the development of a simple yet effective procedure for optimizing the design of a reactor system employing CSTRs in series. The basic approach used in this work was to translate the problem of reactor design to a mathematical programming model. The resulting model was then solved by dynamic programming. The procedure was tested on an IBM 3033 computer and an IBM PC-compatible machine, the CORONA PC-II microcomputer. The results of this study indicate that the optimization procedure developed is very effective.     

Programming for the DNA analysis of FCM data on an IBM microcomputer

The analysis of data generated on a flow cytometer (FCM) is often performed on a computer obtained especially for dedicated use with the flow cytometer. This computer component can be expensive and also presents the FCM user with the added burden of mastering specialized programming language or of accepting the secret analytical processes of protected proprietary program routines. We believe that the evolution of more accurate and efficient FCM analyses that have the power to consider complex signal distributions can be assisted by the availability of analysis programs written in languages common to many users. DNA analysis routines written for a relatively inexpensive microcomputer (IBM PC/XT) in Basic and Pascal are described here. The routines can automatically process multiple FCM data files and can provide high-resolution graphic hardcopy. A foreground/background utilization is also described that allows the computer to be available for other uses in the laboratory.     

Expression of human pyruvate carboxylase in insect cells using the baculovirus system

Constructs containing cDNA encoding human pyruvate carboxylase (PC) with and without a hexahistidine (6x His) tag at the N-terminal of the mature enzyme have been cloned under the control of the polyhedrin promoter. These two constructs were co-transfected with the baculovirus genome into Sf9 cells to produce recombinant baculoviruses harbouring human PC cDNA. The expression of human PC under the control of the polyhedrin promoter was found to be at its highest level at 4 days post-infection. The expressed material accounted for up to 70% of total cellular protein with 5% of this expressed material being found in the soluble fraction. The recombinant human 6x His-PC isolated with a purity of approximately 50% using a Ni-NTA agarose column was found to have the specific activity of 7U/mg, which was similar to that produced from a 293T stable line [Biochem. Biophys. Res. Commun. 266 (1999) 512]. This is the first report of a heterologous expression system for recombinant human PC.     

BioGraf 3D: an algorithm for optimization and graphical representation of nuclear structure spatial distributions

BioGraf 3D is a computer algorithm for rapid three dimensional visualization and analysis of digitized three dimensional biological data to aid in the subsequent generation of models of nuclei or cells. This graphically oriented program permits unlimited three dimensional perspective viewpoints of data in onscreen single frame, zoom or stereo forms. Analysis of data is enhanced by an optimization problem solution which determines viewpoint coordinates whereby maximum clustering of preselected structures is achieved. Mathematical representation of the transformations and optimization is presented in detail. Biograf 3D is written in the C programming language for IBM and compatible personal computers. In this study BioGraf 3D is utilized to examine the infrastructure of the nucleus and the spatial distribution of meiotic chromosomes in the hermaphroditic nematode Caenorhabditis elegans.     

Modular PC-based data acquisition and processing system for biological signals]

The data acquisition system described here is designed for biomedical research and permits the recording of up to eight biological signals simultaneously. A personal computer using the Windows 95 operating system is employed for data monitoring, data processing and analysis during experiments. The system has been designed for reliability, economy, flexibility and ease of handling, with the aim of achieving universal application. To avoid interface incompatibility, problems with transfer protocols and the data formats of commercially available products, analog signals are used for further processing. The individual input channels are electrically isolated from one another and the PC to avoid ground loops, and for reasons of safety. An isolated voltage supply is available for pre-amplifiers and bridges. A bandwidth of 0-5 kHz and the maximum sampling rate of 12.5 kHz suffice to pick up higher frequency signals such as EMG and ENG. The modular software and hardware concepts permit the use of almost any desktop or laptop PC as a central processing unit. The PC handless documentation, data acquisition, data analysis and the preparation of publications. If needed, further analytical functions can be added in modular form. Finally, the option of saving data in the ASCII format permits processing of results with such standard software packages as Excel, Access, Matlab and Origin.     

VOSTORG: a package of microcomputer programs for sequence analysis and construction of phylogenetic trees

VOSTORG is a new, versatile package of programs for the inference and presentation of phylogenetic trees, as well as an efficient tool for nucleotide (nt) and amino acid (aa) sequence analysis (sequence input, verification, alignment, construction of consensus, etc.). On appropriately equipped systems, these data can be displayed on a video monitor or printed as required. They are implemented on IBM PC/XT/AT/PS-2 or compatible computers and hardware graphic support is recommended. The package is designed to be easily handled by occasional computer users and yet it is powerful enough for experienced professionals.     

A computer program that simulates cloning of DNA

Easy Cloner is a computer program that manipulates DNA sequences as in cloning experiments and produces maps of the resulting plasmids. The program runs in the graphics mode of an IBM PC or compatible computer and is operated by using a mouse to point to the required actions. The program is available in the public domain.