Stanley D. Spilman

Photonic detection of bacterial pathogens in living hosts.

The study of pathogenic is often limited to ex vivo assays and cell‐culture correlates. A greater understanding of infectious diseases would be facilitated by in vivo analyses. Therefore, we have developed a method for detecting bacterial pathogens in a living host and used this method to evaluate disease processes for strains of Salmonella typhimurium that differ in their virulence for mice. Three strains of Salmonella were marked with bioluminescence through transformation with a plasmid conferring constitutive expression of bacterial luciferase. Detection of photons transmitted through tissues of animals infected with bioluminescent Salmonella allowed localization of the bacteria to specific tissues. In this manner progressive infections were distinguished from those that were persistent or abortive. We observed patterns of bio‐luminescence that suggested the caecum may play a pivotal role in Salmonella pathogenesis. In vivo efficacy of an antibiotic was monitored using this optical method. This study demonstrates that the real time non‐invasive analyses of pathogenic events and pharmacological monitoring can be performed in vivo.

Visualizing gene expression in living mammals using a bioluminescent reporter

Abstract— Control of gene expression often involves an interwoven set of regulatory processes. As information regarding regulatory pathways may be lost in ex vivo analyses, we used bioluminescence to monitor gene expression in living mammals. Viral promoters fused to firefly luciferase as transgenes in mice allowed external monitoring of gene expression both superficially and in deep tissues. In vivo bioluminescence was detectable using either intensified or cooled charge‐coupled device cameras, and could be detected following both topical and systemic delivery of substrate. In vivo control of the promoter from the human immunodeficiency virus was demonstrated. As a model for DNA‐based therapies and vaccines, in vivo transfection of a luciferase expression vector (SV‐40 promoter and enhancer controlling expression) was detected. We conclude that gene regulation, DNA delivery and expression can now be noninvasively monitored in living mammals using a luciferase reporter. Thus, real‐time, noninvasive study of gene expression in living animal models for human development and disease is possible.

STATIONARY HEADBAND FOR CLINICAL TIME-OF-FLIGHT OPTICAL IMAGING AT THE BEDSIDE

Conventional brain‐imaging modalities may be limited by high cost, difficulty of bedside use, noncontinuous operation, invasiveness or an inability to obtain measurements of tissue function, such as oxygenation during stroke. Our goal was to develop a bedside clinical device able to generate continuous, noninvasive, tomographic images of the brain using low‐power nonionizing optical radiation. We modified an existing stage‐based time‐of‐flight optical tomography system to allow imaging of patients under clinical conditions. First, a stationary headband consisting of thin, flexible optical fibers was constructed. The headband was then calibrated and tested, including an assessment of fiber lengths, the existing system software was modified to collect headband data and to perform simultaneous collection of data and image reconstruction, and the existing hardware was modified to scan optically using this headband. The headband was tested on resin models and allowed for the generation of tomographic images in vitro; the headband was tested on critically ill infants and allowed for optical tomographic images of the neonatal brain to be obtained in vivo.

Non-Recursive Linear Algorithms for Optical Imaging in Diffusive Media

Optical imaging has been used to image phantoms, animals, and humans. It offers the potential for the production of functional images of tissues, such as oxygenation of brain during stroke. Fast algorithms are needed to allow diagnostically useful images to be generated under realistic conditions, including the likelihood that transmission geometries will not be possible. We proposed a linear algorithm, while less than ideal, may allow rapid reconstruction of images and avoid the pitfalls of recursive, nonlinear solutions. Such techniques may also facilitate the use of varied but physiologic imaging geometries. We found that linear backprojection tomography is feasible for clinical use. Conversion of the present mechanically scanning device to a clinical scanner should be possible with retention of the current processing algorithms. Such a clinical scanner should ultimately be able to generate images in less than one minute with centimeter resolution at the center of living human brain.

Tomographic Time-of-Flight Optical Imaging Device

Time-resolved optical imaging has been used to image phantoms, animals, and humans, and offers the potential for the production of functional images of human tissues, such as the oxygenation of brain during stroke. We had previously reported a transmission scanner, and now give an early report on conversion to a rotational tomographic scanner with a non-parallel ray geometry similar to early CAT scanners. Initial scans show that 1) spatial imaging in turbid media using time-of-flight measurements, non-recursive algorithms, and standard tomographic geometry is possible, 2) separation of absorbance and scattering as an image is attainable, a key step in performing spatially-resolved chemometric analysis, 3) imaging of multiple objects buried within scattering material is feasible, demonstrating that equations derived for homogeneous media can be applied in at least some cases to inhomogeneous media such as tissue-like phantoms, and 4) imaging of brain pathology produces recognizable images with sufficient resolution for diagnostic decisions. We conclude that optical tomography is feasible for clinical use and that conversion of the present mechanically scanning device to a clinical scanner should be possible with retention of the current processing algorithms. Such a clinical scanner should ultimately be able to generate images in a few minutes with centimeter resolution at the center of living human brain.

Laser imaging for clinical applications

Medical optical imaging (MOI) uses light emitted into opaque tissues in order to determine the interior structure and chemical content. These optical techniques have been developed in an attempt to prospectively identify impending brain injuries before they become irreversible, thus allowing injury to be avoided or minimized. Optical imaging and spectroscopy center around the simple idea that light passes through the body in small amounts, and emerges bearing clues about tissues through which it passed. Images can be reconstructed from such data, and this is the basis of optical tomography. Over the past few years, techniques have been developed to allow construction of images from such optical data at the bedside. We have used a time-of-flight system reported earlier to monitor oxygenation and image hemorrhage in neonatal brain. This article summarizes the problems that we believe can be addressed by such techniques, and reports on some of our early results.

Noninvasive Monitoring of Acute Susceptibility and Host Response to Salmonella Infection in Living Neonatal Mice • 886

Noninvasive Monitoring of Acute Susceptibility and Host Response to Salmonella Infection in Living Neonatal Mice • 886

MONITORING GENE EXPRESSION IN LIVING ANIMALS USING BIOLUMINESCENT REPORTERS. • 844

The introduction of nucleic acid sequences into the cells of living animals is the basis of generating transgenic and chimeric animals, which themselves serve as basic tools in genetics and as a foundation for emerging gene therapies, gene vaccines, and antisense oligonucleotide-based therapeutics. The detection of gene-modifying events now requires that large numbers of cells be altered, selected, grown, and analyzed in a time-consuming and labor-intensive sequence of steps. A method for rapid, in situ assessment of the uptake and expression of nucleic acids would thus facilitate evaluation of gene delivery systems and DNA-based therapies. We report on development of a real-time bioluminescent reporter that noninvasively indicates the level of promoter activity in living animals. Such a system differs from existing green-fluorescent-protein-(GFP)-based systems, as our reporters spontaneously emit light without a need for outside light sources. A gene fusion product consisting of HIV-1 LTR (promoter) and firefly luciferase gene complex (reporter) was studied in a transgenic mouse. Photons from thein vivo luciferase reaction were detected by a CCD camera, after transmission through the animals tissues, and used as an indication of the level of gene expression. This allowed us to estimate, in real time, the extent of promoter activity. We were able to assess activity, both on the surface of the animal as well as in deep tissues. In the skin of living transgenic mice, we assessed the levels of induction of HIV-LTR in response to various stimuli (in murine and human tissues, the HIV-1 LTR promoter can be activated by chemical, photo, and thermal signals). Reduction of photon emission from luciferase expressed in the skin was observed during ATP depletion, suggesting use of bioluminescence as an in situ ATP sensor. Photons emitted from internal organs were also detected externally, indicating that a wide variety of promoters expressed at various tissues sites may be studied using this approach. Use of such a reporter system may facilitate an in vivo assessment of new therapies targeting regulation of viral and host gene expression. Our approach has advantages over dye-producing methods (e.g., GFP), as a low background signal makes near single-event detection possible, the signal is real-time rather than integrated, and no cytotoxic photosensitizing dyes are produced. These studies open a window through which biological processes can be viewed in vivo, illuminating the temporal and spatial distribution of gene expression in animals and humans. Supported by NIH N43-NS-4-2315 & RR-00081, Universitywide AIDS Prog., Packard Foundation (Stanford), and ONR N-00014-94-1-1024.