Framework for morphometric classification of cells in imaging flow cytometry

Imaging flow cytometry is an emerging technology that combines the statistical power of flow cytometry with spatial and quantitative morphology of digital microscopy. It allows high-throughput imaging of cells with good spatial resolution, while they are in flow. This paper proposes a general framework for the processing/classification of cells imaged using imaging flow cytometer. Each cell is localized by finding an accurate cell contour. Then, features reflecting cell size, circularity and complexity are extracted for the classification using SVM. Unlike the conventional iterative, semi-automatic segmentation algorithms such as active contour, we propose a noniterative, fully automatic graph-based cell localization. In order to evaluate the performance of the proposed framework, we have successfully classified unstained label-free leukaemia cell-lines MOLT, K562 and HL60 from video streams captured using custom fabricated cost-effective microfluidics-based imaging flow cytometer. The proposed system is a significant development in the direction of building a cost-effective cell analysis platform that would facilitate affordable mass screening camps looking cellular morphology for disease diagnosis. Lay description In this article, we propose a novel framework for processing the raw data generated using microfluidics based imaging flow cytometers. Microfluidics microscopy or microfluidics based imaging flow cytometry (mIFC) is a recent microscopy paradigm, that combines the statistical power of flow cytometry with spatial and quantitative morphology of digital microscopy, which allows us imaging cells while they are in flow. In comparison to the conventional slide-based imaging systems, mIFC is a nascent technology enabling high throughput imaging of cells and is yet to take the form of a clinical diagnostic tool. The proposed framework process the raw data generated by the mIFC systems. The framework incorporates several steps: beginning from pre-processing of the raw video frames to enhance the contents of the cell, localising the cell by a novel, fully automatic, non-iterative graph based algorithm, extraction of different quantitative morphological parameters and subsequent classification of cells. In order to evaluate the performance of the proposed framework, we have successfully classified unstained label-free leukaemia cell-lines MOLT, K562 and HL60 from video streams captured using cost-effective microfluidics based imaging flow cytometer. The cell lines of HL60, K562 and MOLT were obtained from ATCC (American Type Culture Collection) and are separately cultured in the lab. Thus, each culture contains cells from its own category alone and thereby provides the ground truth. Each cell is localised by finding a closed cell contour by defining a directed, weighted graph from the Canny edge images of the cell such that the closed contour lies along the shortest weighted path surrounding the centroid of the cell from a starting point on a good curve segment to an immediate endpoint. Once the cell is localised, morphological features reflecting size, shape and complexity of the cells are extracted and used to develop a support vector machine based classification system. We could classify the cell-lines with good accuracy and the results were quite consistent across different cross validation experiments. We hope that imaging flow cytometers equipped with the proposed framework for image processing would enable cost-effective, automated and reliable disease screening in over-loaded facilities, which cannot afford to hire skilled personnel in large numbers. Such platforms would potentially facilitate screening camps in low income group countries; thereby transforming the current health care paradigms by enabling rapid, automated diagnosis for diseases like cancer.

A semi-automated, field-portable microscopy platform for clinical diagnostic applications

Clinical microscopy is a versatile diagnostic platform used for diagnosis of a multitude of diseases. In the recent past, many microfluidics based point-of-care diagnostic devices have been developed, which serve as alternatives to microscopy. However, these point-of-care devices are not as multi-functional and versatile as clinical microscopy. With the use of custom designed optics and microfluidics, we have developed a versatile microscopy-based cellular diagnostic platform, which can be used at the point of care. The microscopy platform presented here is capable of detecting infections of very low parasitemia level (in a very small quantity of sample), without the use of any additional computational hardware. Such a cost-effective and portable diagnostic device, would greatly impact the quality of health care available to people living in rural locations of the world. Apart from clinical diagnostics, it's applicability to field research in environmental microbiology has also been outlined. (C) 2015 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License.

Automated estimation of fatigue crack length and closure/opening stress

An experimental technique is proposed for the estimation of crack length as well as crack closure/opening stress during fatigue crack growth. A specially designed, single cantilever, crack opening displacement gauge is used to monitor these variables during fatigue crack propagation testing. The technique was experimentally validated through electronfractography.

Reliable Means of Diagnosis and Serovar Determination of Blood-Borne Salmonella Strains: Quick PCR Amplification of Unique Genomic Loci by Novel Primer Sets

Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR-based diagnosis method by designing primers against a region that is unique to Salmonella enterica subsp. enterica serovar Typhi and Salmonella enterica subsp. enterica serovar Paratyphi A, corresponding to the STY0312 gene in S. Typhi and its homolog SPA2476 in S. Paratyphi A. An additional set of primers amplify another region in S. Typhi CT18 and S. Typhi Ty2 corresponding to the region between genes STY0313 to STY0316 but which is absent in S. Paratyphi A. The possibility of a false-negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella serovars as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying the region from clinical isolates of patients from various geographical locations in India, thereby showing that this region is potentially stable. These set of primers can also differentiate between S. Typhi CT18, S. Typhi Ty2, and S. Paratyphi A, which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivity of 95% compared to the Widal test which has a sensitivity of only 63%. As observed, in certain cases, the PCR assay was more sensitive than the blood culture test was, as the PCR-based detection could also detect dead bacteria.

Automated inspection of aircraft parts using a modified ICP algorithm

The application of computer-aided inspection integrated with the coordinate measuring machine and laser scanners to inspect manufactured aircraft parts using robust registration of two-point datasets is a subject of active research in computational metrology. This paper presents a novel approach to automated inspection by matching shapes based on the modified iterative closest point (ICP) method to define a criterion for the acceptance or rejection of a part. This procedure improves upon existing methods by doing away with the following, viz., the need for constructing either a tessellated or smooth representation of the inspected part and requirements for an a priori knowledge of approximate registration and correspondence between the points representing the computer-aided design datasets and the part to be inspected. In addition, this procedure establishes a better measure for error between the two matched datasets. The use of localized region-based triangulation is proposed for tracking the error. The approach described improves the convergence of the ICP technique with a dramatic decrease in computational effort. Experimental results obtained by implementing this proposed approach using both synthetic and practical data show that the present method is efficient and robust. This method thereby validates the algorithm, and the examples demonstrate its potential to be used in engineering applications.

Data mining approaches to software fault diagnosis

Automatic identification of software faults has enormous practical significance. This requires characterizing program execution behavior and the use of appropriate data mining techniques on the chosen representation. In this paper, we use the sequence of system calls to characterize program execution. The data mining tasks addressed are learning to map system call streams to fault labels and automatic identification of fault causes. Spectrum kernels and SVM are used for the former while latent semantic analysis is used for the latter The techniques are demonstrated for the intrusion dataset containing system call traces. The results show that kernel techniques are as accurate as the best available results but are faster by orders of magnitude. We also show that latent semantic indexing is capable of revealing fault-specific features.

An automated framework for understanding structural variations in the binding grooves of MHC class II molecules

Background: MHC/HLA class II molecules are important components of the immune system and play a critical role in processes such as phagocytosis. Understanding peptide recognition properties of the hundreds of MHC class II alleles is essential to appreciate determinants of antigenicity and ultimately to predict epitopes. While there are several methods for epitope prediction, each differing in their success rates, there are no reports so far in the literature to systematically characterize the binding sites at the structural level and infer recognition profiles from them. Results: Here we report a new approach to compare the binding sites of MHC class II molecules using their three dimensional structures. We use a specifically tuned version of our recent algorithm, PocketMatch. We show that our methodology is useful for classification of MHC class II molecules based on similarities or differences among their binding sites. A new module has been used to define binding sites in MHC molecules. Comparison of binding sites of 103 MHC molecules, both at the whole groove and individual sub-pocket levels has been carried out, and their clustering patterns analyzed. While clusters largely agree with serotypic classification, deviations from it and several new insights are obtained from our study. We also present how differences in sub-pockets of molecules associated with a pair of autoimmune diseases, narcolepsy and rheumatoid arthritis, were captured by PocketMatch(13). Conclusion: The systematic framework for understanding structuralvariations in MHC class II molecules enables large scale comparison of binding grooves and sub-pockets, which is likely to have direct implications towards predicting epitopes and understanding peptide binding preferences.

An automated framework for understanding structural variations in the binding grooves of MHC class II molecules

Background: MHC/HLA class II molecules are important components of the immune system and play a critical role in processes such as phagocytosis. Understanding peptide recognition properties of the hundreds of MHC class II alleles is essential to appreciate determinants of antigenicity and ultimately to predict epitopes. While there are several methods for epitope prediction, each differing in their success rates, there are no reports so far in the literature to systematically characterize the binding sites at the structural level and infer recognition profiles from them. Results: Here we report a new approach to compare the binding sites of MHC class II molecules using their three dimensional structures. We use a specifically tuned version of our recent algorithm, PocketMatch. We show that our methodology is useful for classification of MHC class II molecules based on similarities or differences among their binding sites. A new module has been used to define binding sites in MHC molecules. Comparison of binding sites of 103 MHC molecules, both at the whole groove and individual sub-pocket levels has been carried out, and their clustering patterns analyzed. While clusters largely agree with serotypic classification, deviations from it and several new insights are obtained from our study. We also present how differences in sub-pockets of molecules associated with a pair of autoimmune diseases, narcolepsy and rheumatoid arthritis, were captured by PocketMatch(13). Conclusion: The systematic framework for understanding structural variations in MHC class II molecules enables large scale comparison of binding grooves and sub-pockets, which is likely to have direct implications towards predicting epitopes and understanding peptide binding preferences.

Advanced transmission electron microscope triboprobe with automated closed-loop nanopositioning

Here the design and operation of a novel transmission electron microscope (TEM) triboprobe instrument with real-time vision control for advanced in situ electron microscopy is demonstrated. The NanoLAB triboprobe incorporates a new high stiffness coarse slider design for increased stability and positioning performance. This is linked with an advanced software control system which introduces both new and flexible in situ experimental functional testing modes, plus an automated vision control feedback system. This advancement in instrumentation design unlocks new possibilities of performing a range of new dynamical nanoscale materials tests, including novel friction and fatigue experiments inside the electron microscope.

Comparative study of filter-bank mean-energy distance for automated segmentation of speech signals

This paper describes a method of automated segmentation of speech assuming the signal is continuously time varying rather than the traditional short time stationary model. It has been shown that this representation gives comparable if not marginally better results than the other techniques for automated segmentation. A formulation of the 'Bach' (music semitonal) frequency scale filter-bank is proposed. A comparative study has been made of the performances using Mel, Bark and Bach scale filter banks considering this model. The preliminary results show up to 80 % matches within 20 ms of the manually segmented data, without any information of the content of the text and without any language dependence. 'Bach' filters are seen to marginally outperform the other filters.

Language independent automated segmentation of speech using Bach scale filter-banks

This correspondence describes a method for automated segmentation of speech. The method proposed in this paper uses a specially designed filter-bank called Bach filter-bank which makes use of 'music' related perception criteria. The speech signal is treated as continuously time varying signal as against a short time stationary model. A comparative study has been made of the performances using Mel, Bark and Bach scale filter banks. The preliminary results show up to 80 % matches within 20 ms of the manually segmented data, without any information of the content of the text and without any language dependence. The Bach filters are seen to marginally outperform the other filters.

Design and fabrication of an automated temperature programmed reaction system to evaluate 3-way catalysts Ce1-x-y(La/Y)(x)PtyO2-delta

A completely automated temperature-programmed reaction (TPR) system for carrying out gas-solid catalytic reactions under atmospheric flow conditions is fabricated to study CO and hydrocarbon oxidation, and NO reduction. The system consists of an all-stainless steel UHV system, quadrupole mass spectrometer SX200 (VG Scientific), a tubular furnace and micro-reactor, a temperature controller, a versatile gas handling system, and a data acquisition and analysis system. The performance of the system has been tested under standard experimental conditions for CO oxidation over well-characterized Ce1-x-y(La/Y)(y)O2-delta catalysts. Testing of 3-way catalysis with CO, NO and C2H2 to convert to CO2, N-2 and H2O is done with this catalyst which shows complete removal of pollutants below 325 degrees C. Fixed oxide-ion defects in Pt substituted Ce1-y(La/Y)(y)O2-y/2 show higher catalytic activity than Pt ion-substituted CeO2

Diagnosis of dominant forcing factors for large-scale vertical velocities during active and break phases of the monsoon

An attempt to diagnose the dominant forcings which drive the large-scale vertical velocities over the monsoon region has been made by computing the forcings like diabatic heating fields,etc. and the large-scale vertical velocities driven by these forcings for the contrasting periods of active and break monsoon situations; in order to understand the rainfall variability associated with them. Computation of diabatic heating fields show us that among different components of diabatic heating it is the convective heating that dominates at mid-tropospheric levels during an active monsoon period; whereas it is the sensible heating at the surface that is important during a break period. From vertical velocity calculations we infer that the prime differences in the large-scale vertical velocities seen throughout the depth of the atmosphere are due to the differences in the orders of convective heating; the maximum rate of latent heating being more than 10 degrees Kelvin per day during an active monsoon period; whereas during a break monsoon period it is of the order of 2 degrees Kelvin per day at mid-tropospheric levels. At low levels of the atmosphere, computations show that there is large-scale ascent occurring over a large spatial region, driven only by the dynamic forcing associated with vorticity and temperature advection during an active monsoon period. However, during a break monsoon period such large-scale spatial organization in rising motion is not seen. It is speculated that these differences in the low-level large-scale ascent might be causing differences in convective heating because the weaker the low level ascent, the lesser the convective instability which produces deep cumulus clouds and hence lesser the associated latent heat release. The forcings due to other components of diabatic heating, namely, the sensible heating and long wave radiative cooling do not influence the large-scale vertical velocities significantly.