Study of urinary tract infection in children in one health district.

OBJECTIVES--To determine the number of children who had urine specimens sent for culture, who had infections or sterile pyuria, and who were investigated further. To relate the laboratory findings to the results of imaging. DESIGN--One year survey of urine specimens submitted to a laboratory; review of previous and subsequent laboratory reports; review of the findings of imaging of the urinary tract. SETTING--Portsmouth and South East Hampshire health district. SUBJECTS--An estimated population of 89,086 children aged 12 years or under. MAIN OUTCOME MEASURES--Urine bacterial count and results of imaging. RESULTS--12,551 urine specimens were submitted from 7450 children, 3138 boys and 4312 girls. 2238 children had infection or sterile pyuria at least once during the study (13.9/1000 boys, 37/1000 girls). 996 (45%) of the children with infection or sterile pyuria underwent some form of imaging. 128 children who had infection or sterile pyuria were already known to have urinary tract abnormalities and 114 children had newly identified abnormalities (1.0/1000 boys, 1.5/1000 girls). 50 (44%) of the children with newly detected abnormalities had no pyuria and 48 (42%) had bacterial counts below 10(8)/l. Eight children who had sterile pyuria on presentation were found to have abnormalities on imaging. CONCLUSIONS--Urinary tract infection is much commoner in children than is widely believed. Low bacterial counts, the absence of pyuria, or a finding of sterile pyuria should not be disregarded.     

A yeast splicing factor is localized in discrete subnuclear domains.

Digital imaging microscopy has been used to visualize the splicing protein PRP6p and three other yeast nuclear proteins. The results show that PRP6p is uniquely localized to discrete subnuclear regions. A combination of cytological and biochemical assays suggests that these sites can be saturated when the protein is overexpressed and likely correspond to the location of U4/U6 snRNPs. The observations indicate that some splicing components are located in discrete subregions of the yeast nucleus, similar to the situation described for the mammalian nucleus.     

Progress in n.m.r. zeugmatography imaging

Applications of nuclear magnetic resonance (n.m.r.) zeugmatographic imaging to medical diagnosis and to medical, physiological, and biological research require the development of appropriate imaging instrumentation and ancillary techniques, as well as an understanding of the biological significance of the imaging results. A whole body imaging system, relying primarily upon reconstruction from projections, is under development in the expectation that the reconstruction approach will be the most practical one for many purposes. In addition, injectable magnetic reagents that can selectively change tissue water relaxation times and image contrast are under development so as to increase the specificity and versatility of the measurements. If very high magnetic fields are employed, 31P n.m.r. zeugmatography may be practical at very low resolution for human diagnostic studies and for experiments on perfused organs and small animals. Preliminary images, showing the spatial distributions of different phosphorus metabolites in the compartments of test objects, have been obtained at 146 MHz by reconstruction techniques.     

N.m.r. imaging of intact biological systems

Internal images of structured objects may be obtained with n.m.r. by labelling component parts with different magnetic field strengths and therefore recognizably different n.m.r. frequencies. A linear field gradient generates a one-dimensional projection of nuclear density and a variety of techniques are employed to manipulate this one-dimensional probe to yield internal images in two and three dimensions. In the past few years, n.m.r. imaging, sometimes also called zeugmatography or spin mapping, has been applied progressively to provide proton images of small phantoms, fruit, vegetables and small animals, and finally to in vivo imaging of the human body; it promises to provide a valuable means of interior investigation of intact biological systems generally. For medical imaging the method is non-invasive, does not use ionizing radiations, appears to be without hazard and penetrates bony cavities without attenuation. Furthermore, other n.m.r. parameters, for example, relaxation times and fluid flow, may also be mapped; there is evidence that the relaxation times from tumours are significantly longer than those from corresponding normal tissue. Effort to date has mostly been concentrated on proton n.m.r., but some work has been done with other nuclei. Three examples are shown of n.m.r. images of intact biological systems: a fruit, an animal and a human system. The discussion includes the quantitative nature of the images, tissue discrimination, the relation between the resolution in the image and image acquisition time, attenuation and phase shift of the r.f. field in the biological tissue, and magnets suitable for n.m.r. imaging. In principle, all conventional n.m.r. techniques can be combined with n.m.r. methods in order to investigate heterogeneous systems. Overhauser imaging is briefly discussed.     

Imaging and force-distance analysis of human fibroblasts in vitro by atomic force microscopy.

The structure of human fibroblasts have been characterised in vitro by atomic force microscopy (AFM) operated in the imaging or in the force versus distance (F-d) modes. The choice of cell substrate is important to ensure good adhesion. Of greater significance in the context of AFM analysis, is the observation that the substrate affects the imaging conditions for in vitro analysis of live cells. For instance, very rarely will glass coverslips lead to acceptable outcomes (i.e., resolved cytoskeletal structure). Activated tissue culture dishes, on the other hand, promote conditions that routinely result in good quality images. Those conditions are then unaffected by adoption of relatively high force loadings (more than 10 nN), large fields of view (100 x 100 microm2) and high scan speeds (up to ca. 200 microm/sec), all of which exceed values recommended in the literature. Plasma membranes are fragile in the context of AFM analysis (F-d analysis gives an equivalent Young's Modulus of ca. 5 kPa). However, the present work suggests that fragility per se need not be a problem, rather it is the adhesive interactions with the tip, which under some circumstances may exceed 20 nN, that are the source of poor imaging conditions. The present results, being supported by a qualitative model, suggest that the activated substrate acts as a preferential scavenger of cellular debris thus preventing the tip from biofouling, and will therefore promote low adhesion between tip and membrane. Good imaging conditions provide non-destructive in vitro information about cytoskeletal structure and dynamics, as shown in two examples concerned with cytochalasin treatment and with the MTT assay.     

Hemodynamic responses in cortex investigated with optical imaging methods. Implications for functional brain mapping.

During the last 20 years, optical imaging methods - either alone or in combination with other recording techniques - has proven a fruitful approach to explore both the physiological and the functional aspects of activity-evoked hemodynamic responses in cortex. One of the main advantages of optical imaging consists in its high spatio-temporal resolution (in the order of few microns and milliseconds), allowing not only to unambiguously distinguish between activity patterns relating to the underlying functional architecture and those originating from the activation of medium/large blood vessels, but also to investigate the various activity-evoked hemodynamic processes at very fine detail. Here, we briefly review the principal findings obtained by optical imaging about the spatio-temporal properties of the various hemodynamic responses in cortex, i.e., changes in blood-oxygenation, blood-volume, and, to some extent, blood-flow. We also discuss the implications of those findings for non-invasive high-resolution functional brain imaging, in particular for fMRI. Finally, we underscore the importance of novel approaches for high-resolution blood-flow imaging, in the context of the need to gather information at fine spatial detail about the blood-flow response, necessary to constrain the multiple free parameters of hemodynamic response models.     

In vivo cytometry: a spectrum of possibilities.

BACKGROUND: We investigate whether optical imaging can reliably detect abnormalities in tissue, in a range of specimens (live cells in vitro; fixed, fresh ex-vivo and in vivo tissue), without the use of added contrast agents, and review our promising spectral methods for achieving quantitative, real-time, high resolution intrasurgical optical diagnostics. METHODS: We use reflectance, fluorescence, two-photon, and Mie scattering imaging, performed with instrumentation we developed or modified, to detect intrinsic tissue signatures. Emphasis is on spectral/hyperspectral imaging approaches allowing the equivalent of in vivo pathology. RESULTS: With experimental focus on unstained specimens, we demonstrate the ability to segment tissue images for cancer detection. Spectral reflectance imaging, coupled with advanced analysis, typically yields 90% specificity and sensitivity. Autofluorescence is also shown to be diagnostically useful, with lymph nodes results highlighted here. Elastic scattering hyperspectral imaging endoscopy, using a new instrument we designed and built, shows promise in bronchoscopic detection of dysplasia and early cancer in patients. CONCLUSIONS: The results demonstrate that advanced optical imaging can detect and localize cellular signatures of cancer in real-time, in vivo, without the use of contrast agents, in animals and humans. This is an important step towards tight spatio-temporal coupling between such detection and clinical intervention.     

Real-time functional magnetic resonance imaging.

Magnetic resonance imaging (MRI) has been shown to be useful in the detection of brain activity via the relatively indirect coupling of neural activity to cerebral blood flow and subsequently to magnetic resonance signal intensity. Recent technical advances have made possible the continuous collection of successive images at a rate rapid compared with such signal changes and in the statistical processing of these image time series to produce tomographic maps of brain activity in real time, with updates of 10 frames/s or better. We describe here our preferred method of real-time functional MRI and some of the early results we have obtained with its use.     

Multispectral imaging in biology and medicine: slices of life.

Multispectral imaging (MSI) is currently in a period of transition from its role as an exotic technique to its being offered in one form or another by all the major microscopy manufacturers. This is because it provides solutions to some of the major challenges in fluorescence-based imaging, namely ameliorating the consequences of the presence of autofluorescence and the need to easily accommodate relatively high levels of signal multiplexing. MSI, which spectrally characterizes and computationally eliminates autofluorescence, enhances the signal-to-background dramatically, revealing otherwise obscured targets. While this article concentrates on examples derived from liquid-crystal tunable filter-based technology, the intent is to showcase the advantages of multispectral imaging in general. Some technologies used to generate multispectral images are compatible with only particular optical configurations, such as point-scanning laser confocal microscopy. Band-sequential approaches, such as those afforded by liquid-crystal tunable filters (LCTFs), can be conveniently coupled with a variety of imaging modalities, which, in addition to fluorescence microscopy, include brightfield (nonfluorescent) microscopy as well as small-animal, noninvasive in-vivo imaging. Brightfield microscopy is the chosen format for histopathology, which relies on immunohistochemistry to provide molecularly resolved clinical information. However, in contrast to fluorescent labels, multiple chromogens, if they spatially overlap, are much harder to separate and quantitate, unless MSI approaches are used. In-vivo imaging is a rapidly growing field with applications in basic biology, drug discovery, and clinical medicine. The sensitivity of fluorescence-based in-vivo imaging, as with fluorescence microscopy, can be limited by the presence of significant autofluorescence, a limitation which can be overcome through the utilization of MSI.     

Oesophageal carcinoma demonstrated by whole-body nuclear magnetic resonance imaging.

The quality of the images produced by nuclear magnetic resonance (NMR) imaging has steadily improved over the past five years. Images of the head, thorax, and abdomen have clearly shown the normal anatomy. A clinical trial of NMR imaging has therefore been started in Aberdeen to assess its diagnostic accuracy and compare it with conventional radiography and other imaging technique. The first patient examined by whole-body NMR imaging had carcinoma of the oesophagus diagnosed on barium meal examination. A technetium-99m-sulphur colloid liver scan also showed hepatic metastases. NMR imaging showed a large tumour in the lower third of the oesophagus, and areas of increased proton spin-lattice relaxation time (T1) on a section through the liver corresponded with the metastases shown on the radionuclide scan. Increased areas of T1 were present in some vertebrae, and a technetium-99m bone scan confirmed the presence of bone metastases. The NMR images in this patient compared well with the images from other techniques. The continuing clinical trial may show that NMR is an accurate diagnostic aid which will complement existing techniques for diagnosing intrathoracic and intra-abdominal conditions.     

Photoprotective implications of leaf variegation in E. dens-canis L. and P. officinalis L

Variegated leaves occur rarely in nature, but there are some species, primarily in the forest understory, that possess this characteristic. We recently studied two variegated plants: Erytronium dens-canis L., which is characterised by a pattern of red patches and Pulmonaria officinalis L., with light green spots. These non-green areas could attenuate light reaching mesophyll cells with respect to green sections. The aim of the study was to verify whether such red and light green parts are more photoprotected than green ones and if this trait could be of adaptive value. Red patches in E. dens-canis were due to a single layer of red cells in the upper parenchyma, which accumulated anthocyanins. Light green spots in P. officinalis were caused by the presence of loosely arranged cells instead of a well-established layer of packed cells in the palisade parenchyma. Chlorophyll fluorescence imaging was performed under light treatment, showing a greater decrease of photochemical efficiency in red and light green patches than in green sections. Differences in the extent of photochemical efficiency among patches were not attributable to different activation of the xanthophyll cycle. These observations failed to confirm our initial hypothesis, but they questioned the physiological reason for this higher sensitivity in red and light green patches of photosynthetic tissues. Chlorophyll fluorescence imaging was therefore performed in the field. The same pattern of photochemical efficiency was maintained only in E. dens-canis. The current results demonstrate that in both species the benefits of variegation, if any, are different from enhanced photosynthetic performance.     

Spectral imaging of multi-color chromogenic dyes in pathological specimens.

We have investigated the use of spectral imaging for multi-color analysis of permanent cytochemical dyes and enzyme precipitates on cytopathological specimens. Spectral imaging is based on Fourier-transform spectroscopy and digital imaging. A pixel-by-pixel spectrum-based color classification is presented of single-, double-, and triple-color in situ hybridization for centromeric probes in T24 bladder cancer cells, and immunocytochemical staining of nuclear antigens Ki-67 and TP53 in paraffin-embedded cervical brush material (AgarCyto). The results demonstrate that spectral imaging unambiguously identifies three chromogenic dyes in a single bright-field microscopic specimen. Serial microscopic fields from the same specimen can be analyzed using a spectral reference library. We conclude that spectral imaging of multi-color chromogenic dyes is a reliable and robust method for pixel color recognition and classification. Our data further indicate that the use of spectral imaging (a) may increase the number of parameters studied simultaneously in pathological diagnosis, (b) may provide quantitative data (such as positive labeling indices) more accurately, and (c) may solve segmentation problems currently faced in automated screening of cell- and tissue specimens.     

Radiologic investigation of low back pain.

Low back pain is one of the commonest disorders, yet is the most confusing. The cost in work-time lost and in the search for and treatment of its many causes amounts to billions of dollars annually. The traditional techniques for anatomic visualization have been plain-film radiography and myelography, but they have limitations. The development of computed tomography and magnetic resonance imaging have substantially improved anatomic imaging. However, invasive procedures, such as discography, percutaneous nerve-root blocking and percutaneous facet injection, may be helpful in patients with disabling pain in whom noninvasive methods give negative findings, show abnormalities that do not correlate with the symptoms or identify multiple sites of disease. The invasive procedures are believed by some to be associated with too many complications. We have attempted to clarify the strengths and weaknesses of the currently available methods of investigating low back pain and the indications for their use.     

Poor sensitivity of multiple-gated blood-pool imaging in diagnosing left ventricular aneurysms.

A study was carried out to determine the accuracy of multiple-gated blood-pool imaging in diagnosing left ventricular aneurysm. Fifteen patients with an aneurysm and 17 with left ventricular hypokinesia were studied by contrast ventriculography and multiple-gated blood-pool imaging. The results of blood-pool imaging were examined blind by five independent observers, the results of contrast ventriculography being used as the standard. The mean sensitivity of the procedure was 56%, the specificity 61%, and diagnostic accuracy 59%. These results indicate that contrast ventriculography remains the best method for diagnosing left ventricular aneurysms. Moreover, ventriculography provides additional information-for example, on wall thickness-not provided by multiple-gated blood-pool imaging.     

Radiologic assessment in the pediatric intensive care unit.

The severely ill infant or child who requires admission to a pediatric intensive care unit (PICU) often presents with a complex set of problems necessitating multiple and frequent management decisions. Diagnostic imaging plays an important role, not only in the initial assessment of the patient''s condition and establishing a diagnosis, but also in monitoring the patient''s progress and the effects of interventional therapeutic measures. Bedside studies obtained using portable equipment are often limited but can provide much useful information when a careful and detailed approach is utilized in producing the radiograph and interpreting the examination. This article reviews some of the basic principles of radiographic interpretation and details some of the diagnostic points which, when promptly recognized, can lead to a better understanding of the patient''s condition and thus to improved patient care and management. While chest radiography is stressed, studies of other regions including the upper airway, abdomen, skull, and extremities are discussed. A brief consideration of the expanding role of new modality imaging (i.e., ultrasound, CT) is also included. Multiple illustrative examples of common and uncommon problems are shown.     

Live-cell imaging in the era of too many microscopes

At the time of this writing, searching Google Scholar for ‘light-sheet microscopy’ returns almost 8500 results; over three-quarters of which were published in the last 5 years alone. Searching for other advanced imaging methods in the last 5 years yields similar results: ‘super-resolution microscopy’ (>16 000), ‘single-molecule imaging’ (almost 10 000), SPIM (Single Plane Illumination Microscopy, 5000), and ‘lattice light-sheet’ (1300). The explosion of new imaging methods has also produced a dizzying menagerie of acronyms, with over 100 different species of ‘light-sheet’ alone, from SPIM to UM (Ultra microscopy) to SiMView (Simultaneous MultiView) to iSPIM (inclined SPIM, not to be confused with iSPIM, inverted SPIM). How then is the average biologist, without an advanced degree in physics, optics, or computer science supposed to make heads or tails of which method is best suited for their needs? Let us also not forget the plight of the optical physicist, who at best might need help with obtaining healthy samples and keeping them that way, or at worst may not realize the impact their newest technique could have for biologists. This review will not attempt to solve all these problems, but instead highlight some of the most recent, successful mergers between biology and advanced imaging technologies, as well as hopefully provide some guidance for anyone interested in journeying into the world of live-cell imaging.     

Exploring large-scale brain networks in functional MRI.

Increasing emphasis has been recently put on large-scale network processing of brain functions. To explore these networks, many approaches have been proposed in functional magnetic resonance imaging (fMRI). Their objective is to answer the following two questions: (1) what brain regions are involved in the functional process under investigation? and (2) how do these regions interact? We review some of the key concepts and corresponding methods to cope with both issues.     

Forensic visualization of foreign matter in human tissue by near-infrared spectral imaging: methodology and data mining strategies.

BACKGROUND: Rapidity of data acquisition, high image fidelity and large field of view are of tremendous value when looking for chemical contaminants or for the proverbial "needle in the haystack" - in this case foreign inclusions in histologic sections of biopsy or autopsy tissues. Near infrared chemical imaging is one of three chemical imaging techniques (NIR, MIR and Raman) based on vibrational spectroscopy, and provides distinct technical advantages for this application. METHODS: We have chosen to utilize and evaluate near infrared (NIR) imaging for studies of foreign materials in tissue because the experimental configuration is relatively simple, data collection is rapid, and large sample areas can be screened with high image fidelity and spatial resolution. RESULTS: We have shown that NIR imaging can readily find and identify silicone gel inclusions in biological tissue samples. Additionally, preliminary results indicate that spectral signatures in the data set are also potentially sensitive to structural changes in the surrounding tissue that may be induced by the foreign body. CONCLUSIONS: NIR chemical imaging is a powerful, non-destructive tool for localization and identifying foreign contaminants in biological tissue. Preliminary results indicate that NIR imaging is also sensitive enough to differentiate tissue types (perhaps based on collagen structural differences), and provide data on the spatial localization of these components.     

Gout. Imaging of gout: findings and utility

Imaging is a helpful tool for clinicians to evaluate diseases that induce chronic joint inflammation. Chronic gout is associated with changes in joint structures that may be evaluated with diverse imaging techniques. Plain radiographs show typical changes only in advanced chronic gout. Computed tomography may best evaluate bone changes, whereas magnetic resonance imaging is suitable to evaluate soft tissues, synovial membrane thickness, and inflammatory changes. Ultrasonography is a tool that may be used in the clinical setting, allowing evaluation of cartilage, soft tissues, urate crystal deposition, and synovial membrane inflammation. Also ultrasound-guided puncture may be useful for obtaining samples for crystal observation. Any of these techniques deserve some consideration for feasibility and implementation both in clinical practice and as outcome measures for clinical trials. In clinical practice they may be considered mainly for evaluating the presence and extent of crystal deposition, and structural changes that may impair function or functional outcomes, and also to monitor the response to urate-lowering therapy.     

A method for anchoring round shaped cells for atomic force microscope imaging.

More and more researchers are interested in imaging living (Henderson, 1994) or fixed cells in their natural environment using the atomic force microscope (AFM). However, the AFM tip interacts strongly with the sample, and its z range freedom is limited to a few micrometers. This means that the cells to be imaged have to be strongly attached to the substrate, and imaging is restricted to cells having a flattened shape. Here we propose a simple and inexpensive solution to overcome these limitations. The method we propose is trapping living round shaped cells in a Millipore filter with a pore size comparable to the dimensions of the cell. The highest part of some of the blocked cells protrude through the holes of the filter and can this way be easily observed using the AFM without detachment.