An automated microscope for quantitative cytology combining television image analysis and stage scanning microphotometry.

This paper describes an automated microscope developed for operation in conjunction with the Leyden Television Analysis System. It features automated control of magnification, illumination, movement of scanning stages, and fine focus. These functions are controlled by means of a microcomputer. This enables a flexible design and relieves the supervising computer of simple but time consuming tasks. The combination of an automated microscope and Leyden Television Analysis System provides a powerful tool in quantitative cytological research. The flexible design permits other microscopic functions to be added with relatively little effort.     

An automated microscale platform for evaluation and optimization of oxidative bioconversion processes

In this work an integrated robotic platform has been used for the development of a fully automated microscale process sequence comprising fermentation and bioconversion using E. coli TOP10 [pQR210] expressing cyclohexanone monooxygenase (CHMO). Ninety six-Deep Square Well (96-DSW) microtiter plates were used for microbial culture and enzyme-catalyzed conversion, where plate preparation, reagent addition, and sampling were all carried out without manual intervention. The adoption of automated robotic procedures has enabled the rapid collection of kinetic data for whole process optimization at the microscale. This high-throughput approach enabled a range of amino acid sources for media formulation and well fill volumes to be investigated highlighting when nutritional limitation and oxygen limitations took place. The automated process sequence has been applied to test six CHMO substrates including norcamphor and cycloheptanone all of which to the best of our knowledge have yet to be tested with E. coli TOP10 [pQR210]. Substrate specificity and product selectivity were effectively demonstrated and compared to both the natural substrate cyclohexanone and the model substrate bicyclo[3.2.0]hept-2-en-6-one used to demonstrate asymmetric synthesis. The results obtained using the developed process sequence could be reproduced at 75 L scale when a matched oxygen transfer coefficient k(L) a approach was used. The study demonstrates how automated microscale processing enables the rapid collection of kinetic quantitative data in a robust manner with clear implications for accelerating bioprocess development, optimization, and scale-up.     

An automated method for the fluorescamine reaction using a peristaltic pump.

An automated method is described for the measurement of amino acids and proteins by the fluorescamine reaction. Isopropanol is used as solvent for the fluorescamine, and all reagents are pumped by a peristaltic pump.     

Numerical evaluation of cytologic data. IV. Discrimination and classification

When observed data have to be assigned to one or another category, classification rules are needed. Linear discriminant functions provide easily computed rules; weighing the discriminat function according to the variances in the data sets helps reduce classification errors. Classification on the basis of a probability density involves nonlinear decision boundaries. Simple numerical examples for bivariate feature vectors are worked out to demonstrate these approaches to classification.     

An automated method for the evaluation of ram libido in real mating conditions

We investigated if sexual behaviour of rams can be assessed with an electronic Alpha-Detector (AD) which automatically records mounts of mating rams. To evaluate the rams’ libido (i.e. all sexual activities), we used six intact and six vasectomised rams in pen tests in three different seasons (late spring, autumn and early spring). The pen tests consisted of 30-min visual observations of each ram placed in a group of six Merino ewes (three ewes in oestrus and three ewes not in oestrus). In the pen tests, sexual behaviour was recorded and divided into two categories: pre-copulatory and copulatory. For validation purposes, during the pen tests the 12 rams were equipped with the AD and the number of times the 18 oestrous ewes were mounted were counted over a period of 3 days. Of the 1191 mounts visually identified in the six 30-min sessions, 1026 were recorded automatically by the AD (i.e. 94%). The paddock test is an automated method consisting of the same rams wearing an AD and placed in a flock of ~250 Merino ewes on two occasions (late spring (spring 1) and early spring of the following year (spring 2)), their copulatory activities were automatically recorded over a 5-day period. The results of the pen tests in the three seasons revealed no difference between the two types of rams (breeding v. detecting rams). Based on live observations high correlations (r=+0.81, P<0.003 for breeding and r=+0.76, P<0.02 for detecting rams) were found between pre-copulatory and copulatory behaviours. The libido of the two types of rams measured in pen tests showed high repeatability across the three seasons (83 and 75%, P<0.05 for copulatory and pre-copulatory behaviours, respectively). When measured automatically in paddock tests over two consecutive springs, even higher repeatability was observed in both breeding (94%; P<0.01) and detecting rams (97%; P<0.004) in the number of mounts. In addition, high correlations (+0.89<r<+0.94) between copulatory behaviours, as measured by live observations, and those measured by the AD were obtained. The automatic measurement of ram libido in paddock tests appears to be more reliable than pen tests and far less time consuming. We therefore recommend this automated method to estimate the libido of rams. In addition, this method can be used at any season of the year provided that ewes in oestrus are present in the flock.     

An automated rotisserie system for processing Western blots.

An apparatus to automate completely the processing of Western blots is described. The prototype is based on a popular rotisserie system design. The incubation chamber consists of an inner cylinder that rotates inside an outer cylinder (incubation chamber). The blot is contained in the inner cylinder. Two magnets are mounted at one end of the inner cylinder, and rotation of the inner cylinder is effected by two magnets mounted on a motor drive outside the incubation chamber. Movement of chemicals into and out of the incubation chamber is driven pneumatically, and the entire process is controlled by a computer. Processing a blot for chemiluminescent detection takes 7 h to complete without human intervention. The quality of the resulting image is comparable to or better than a blot using manual processing. In addition, the prototype is capable of re-collecting all three antisera for future use.     

An automated differential scanning dilatometer.

A sensitive scanning dilatometer has been constructed which continuously records the apparent partial specific volume of small biological samples (10–100 mg) as a function of temperature. Changes in volume of 0.02 μl can be resolved. Tedious calibrations with their inherent errors are eliminated by employing a differential approach in which a recording electrobalance responds to the difference in buoyancy of identical sample and reference tubes. Agreement with the literature values for the specific volume of n-eicosane and the apparent partial specific volume of KCl was excellent. Apparent partial specific volumes obtained for dipalmitoyl lecithin, egg albumin, and bovine serum albumin agreed well with published values.     

Boosting accuracy of automated classification of fluorescence microscope images for location proteomics

Background

Detailed knowledge of the subcellular location of each expressed protein is critical to a full understanding of its function. Fluorescence microscopy, in combination with methods for fluorescent tagging, is the most suitable current method for proteome-wide determination of subcellular location. Previous work has shown that neural network classifiers can distinguish all major protein subcellular location patterns in both 2D and 3D fluorescence microscope images. Building on these results, we evaluate here new classifiers and features to improve the recognition of protein subcellular location patterns in both 2D and 3D fluorescence microscope images.

Results

We report here a thorough comparison of the performance on this problem of eight different state-of-the-art classification methods, including neural networks, support vector machines with linear, polynomial, radial basis, and exponential radial basis kernel functions, and ensemble methods such as AdaBoost, Bagging, and Mixtures-of-Experts. Ten-fold cross validation was used to evaluate each classifier with various parameters on different Subcellular Location Feature sets representing both 2D and 3D fluorescence microscope images, including new feature sets incorporating features derived from Gabor and Daubechies wavelet transforms. After optimal parameters were chosen for each of the eight classifiers, optimal majority-voting ensemble classifiers were formed for each feature set. Comparison of results for each image for all eight classifiers permits estimation of the lower bound classification error rate for each subcellular pattern, which we interpret to reflect the fraction of cells whose patterns are distorted by mitosis, cell death or acquisition errors. Overall, we obtained statistically significant improvements in classification accuracy over the best previously published results, with the overall error rate being reduced by one-third to one-half and with the average accuracy for single 2D images being higher than 90% for the first time. In particular, the classification accuracy for the easily confused endomembrane compartments (endoplasmic reticulum, Golgi, endosomes, lysosomes) was improved by 5–15%. We achieved further improvements when classification was conducted on image sets rather than on individual cell images.

Conclusions

The availability of accurate, fast, automated classification systems for protein location patterns in conjunction with high throughput fluorescence microscope imaging techniques enables a new subfield of proteomics, location proteomics. The accuracy and sensitivity of this approach represents an important alternative to low-resolution assignments by curation or sequence-based prediction.

     

An automated evaluation of cineangiograms of patients with interatrial septal defects

Curves of contrast medium dilution based on cineangiograms of patients with congenital heart disease, allow the calculation of a relative shunt value, the assessment of topographic anatomical features of an interatrial septal defect, and the detection of phasic manifestations of change in a contrast medium concentration.     

An automated method for acetylcholinesterase kinetics

An automated assay for acetylcholinesterase (EC 3.1.1.7.) has been developed based on the manual spectrophotometric method of Ellmanet al. (1). This method was used to determine (a) the enzyme activity of an unknown sample and (b) the dependence of initial rates given by a fixed enzyme concentration on the substrate concentration. Methods to minimize possible enzyme modification by DTNB (2) are described. Finally a modification of the conventional autoanalyser procedure permitted rapid and reproducible enzyme kinetic analysis under various conditions. This helped to minimize the effects of possible enzyme inactivation at high dilutions especially when using crude enzyme preparations.     

An automated coordinate recorder for biometry

Many measuring devices used in micropaleontology are unsatisfactory because observations must be recorded manually. Moreover, most use a linear scale that depends on the presence of predetermined morphological loci. An x, y recorder, largely adapted from instruments available in geological laboratories, is discussed. It obviates manual recording, frees data collection from the constraint of homologous loci and. in some applications, permits more shape information to be collected.     

An automated assay for phosphorylase kinase

A fully automated assay of phosphorylase kinase capable of processing 40 samples/hr is described. Problems due to enzyme adsorbed to the incubation coil and thereby producing a continuous rise of the base line were overcome by washing with a solution containing 0.05% Triton X-100, 0.03% SDS, and 5 mm EDTA. Under these conditions, a linear relationship between phosphorylase kinase concentration in the incubation mixture and absorbancy at pH's 6.8 and 8.2 is obtained when 1% glycogen is present in the reaction mixture.Inclusion of glycogen allows reduction of the concentration of phosphorylase in the incubation mixture. Due to the presence of Triton X-100 in this test, the carbohydrate acts like an allosteric activator of phosphorylase kinase. The standard deviation of the automated kinase test is 1.3% lower than that of the manual test.     

An automated assay for brain serotonin

This communication describes an automated assay for brain serotonin. The assay is an adaptation of a commonly used manual assay which utilizes the reaction of o-phthalaldehyde with serotonin to develop fluorescence (2). In the automated assay the samples to be analyzed were mixed with 7.5 N HCl and o-phthalaldehyde in the presence of reduced glutathione, heated at 80°C, cooled and their fluorescence recorded. Linearity with respect to serotonin was demonstrated from 0 to at least 5 μM, and the lower limit of sensitivity was 0.7 ng serotonin (0.4 ml of 0.01 μM). The % error (standard deviation times 100 divided by the mean) was always 2% or less. The specificity was studied, and the assay was applied to the measurement of rat brain serotonin levels by demonstrating an increase in brain serotonin levels after pargyline treatment and a decrease after reserpine treatment.