Evaluating performance in three-dimensional fluorescence microscopy

In biological fluorescence microscopy, image contrast is often degraded by a high background arising from out of focus regions of the specimen. This background can be greatly reduced or eliminated by several modes of thick specimen microscopy, including techniques such as 3-D deconvolution and confocal. There has been a great deal of interest and some confusion about which of these methods is ‘better’, in principle or in practice. The motivation for the experiments reported here is to establish some rough guidelines for choosing the most appropriate method of microscopy for a given biological specimen. The approach is to compare the efficiency of photon collection, the image contrast and the signal-to-noise ratio achieved by the different methods at equivalent illumination, using a specimen in which the amount of out of focus background is adjustable over the range encountered with biological samples. We compared spot scanning confocal, spinning disk confocal and wide-field/deconvolution (WFD) microscopes and find that the ratio of out of focus background to in-focus signal can be used to predict which method of microscopy will provide the most useful image. We also find that the precision of measurements of net fluorescence yield is very much lower than expected for all modes of microscopy. Our analysis enabled a clear, quantitative delineation of the appropriate use of different imaging modes relative to the ratio of out-of-focus background to in-focus signal, and defines an upper limit to the useful range of the three most common modes of imaging

NASA Contributions to Development of Special-Purpose Thermocouples. A Survey

The thermocouple has been used for measuring temperatures for more than a century, but new materials, probe designs, and techniques are continually being developed. Numerous contributions have been made by the National Aeronautics and Space Administration and its contractors in the aerospace program. These contributions have been collected by Midwest Research Institute and reported in this publication to enable American industrial engineers to study them and adapt them to their own problem areas. Potential applications are suggested to stimulate ideas on how these contributions can be used

Clinical application of high throughput molecular screening techniques for pharmacogenomics.

Genetic analysis is one of the fastest-growing areas of clinical diagnostics. Fortunately, as our knowledge of clinically relevant genetic variants rapidly expands, so does our ability to detect these variants in patient samples. Increasing demand for genetic information may necessitate the use of high throughput diagnostic methods as part of clinically validated testing. Here we provide a general overview of our current and near-future abilities to perform large-scale genetic testing in the clinical laboratory. First we review in detail molecular methods used for high throughput mutation detection, including techniques able to monitor thousands of genetic variants for a single patient or to genotype a single genetic variant for thousands of patients simultaneously. These methods are analyzed in the context of pharmacogenomic testing in the clinical laboratories, with a focus on tests that are currently validated as well as those that hold strong promise for widespread clinical application in the near future. We further discuss the unique economic and clinical challenges posed by pharmacogenomic markers. Our ability to detect genetic variants frequently outstrips our ability to accurately interpret them in a clinical context, carrying implications both for test development and introduction into patient management algorithms. These complexities must be taken into account prior to the introduction of any pharmacogenomic biomarker into routine clinical testing

A high temperature apparatus for measurement of the Seebeck coefficient

A high temperature Seebeck coefficient measurement apparatus with various features to minimize typical sources of error is designed and built. Common sources of temperature and voltage measurement error are described and principles to overcome these are proposed. With these guiding principles, a high temperature Seebeck measurement apparatus with a uniaxial 4-point contact geometry is designed to operate from room temperature to over 1200 K. This instrument design is simple to operate, and suitable for bulk samples with a broad range of physical types and shapes

Determining Microscopic Viscoelasticity in Flexible and Semiflexible Polymer Networks from Thermal Fluctuations

We have developed a new technique to measure viscoelasticity in soft materials such as polymer solutions, by monitoring thermal fluctuations of embedded probe particles using laser interferometry in a microscope. Interferometry allows us to obtain power spectra of fluctuating beads from 0.1 Hz to 20 kHz, and with sub-nanometer spatial resolution. Using linear response theory, we determined the frequency-dependent loss and storage shear moduli up to frequencies on the order of a kHz. Our technique measures local values of the viscoelastic response, without actively straining the system, and is especially suited to soft biopolymer networks. We studied semiflexible F-actin solutions and, as a control, flexible polyacrylamide (PAAm) gels, the latter close to their gelation threshold. With small particles, we could probe the transition from macroscopic viscoelasticity to more complex microscopic dynamics. In the macroscopic limit we find shear moduli at 0.1 Hz of G'=0.11 +/- 0.03 Pa and 0.17 +/- 0.07 Pa for 1 and 2 mg/ml actin solutions, close to the onset of the elastic plateau, and scaling behavior consistent with G(omega) as omega^(3/4) at higher frequencies. For polyacrylamide we measured plateau moduli of 2.0, 24, 100 and 280 Pa for crosslinked gels of 2, 2.5, 3 and 5% concentration (weight/volume) respectively, in agreement to within a factor of two with values obtained from conventional rheology. We also found evidence for scaling of G(omega) as \omega^(1/2), consistent with the predictions of the Rouse model for flexible polymers.Comment: 16 pages, with 15 PostScript figures (to be published in Macromolecules

Molecular biology techniques as a tool for detection and characterisation of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of paratuberculosis, also known as Johne’s disease, a chronic intestinal infection in cattle and other ruminants. Paratuberculosis is characterised by diarrhea and weight loss that occurs after a period of a few months up to several years without any clinical signs. The considerable economic losses to dairy and beef cattle producers are caused by reduced milk production and poor reproduction performance in subclinically infected animals. Early diagnosis of infected cattle is essential to prevent the spread of the disease. Efforts have been made to eradicate paratuberculosis by using a detection and cull strategy, but eradication is hampered by the lack of suitable and sensitive diagnostic methods. This thesis, based on five scientific investigations, describes the development of different DNA amplification strategies for detection and characterisation of M. paratuberculosis. Various ways to pre-treat bacterial cultures, tissue specimens and fecal samples prior to PCR analysis were investigated. Internal positive PCR control molecules were developed and used in PCR analyses to improve the reliability and to facilitate the interpretation of the results. The sensitivity of the ultimate methods was found to be approximate that of culture and allowed detection of low numbers of M. paratuberculosis expected to be found in subclinically infected animals. Genomic DNA of a Swedish mycobacterial isolate, incorrectly identified by PCR as M. paratuberculosis was characterised. The isolate was closely related to M. cookii and harboured one copy of a DNA segment with 94% similarity to IS900, the target sequence used in diagnostic PCR for detection of M. paratuberculosis. This finding highlighted the urgency of developing or evaluating PCR systems based on genes other than IS900. A PCR-based fingerprinting method using primers targeting the enterobacterial intergenic consensus sequence (ERIC) and the IS900 sequence was developed and successfully used to distinguish M. paratuberculosis from closely related mycobacteria, including the above mentioned mycobacterial isolate. In conclusion, the molecular biology techniques developed in these studies have proved useful for accelerating the diagnostic detection and characterisation of M. paratuberculosis