The development of optical pulses is crucial to the elucidation of complex biological and chemical problems. Existing fluorescent labels tend to photobleach, have low luminescence, and area sensitive to the environment. These problems can be overcome by the use of semiconductor quantum dots. These ZnS-capped CdSe quantum dots are synthesized in an organic media. To use them for biological labeling, the quantum dots are made water-soluble by adsorbing bifunctional molecules onto the surface. This step is crucial for the successful conjugation of biomolecules onto the surface. In this report, a new method for the direct adsorption of biomolecules onto the surface of quantum dots is demonstrated. Biomolecules such as glutathione, mercaptusuccic acid, and histidine are directly conjugated to luminescent quantum dots by S-Zn or N-Zn bonding.

The generic global structure of the DNA double helix is well known, but at the base pair level there are a multitude of deviations from the `ideal'. These deviations can result in sequence-directed curvature of DNA over a few base pairs. We have used luminescent mineral nanoparticles of CdS in optical assays to detect these curved DNAs. The nanoparticles, originally developed by the materials science community as `quantum dots,' are approximately 20 - 100 angstroms in diameter, similar to proteins, and their photoluminescence is sensitive to the presence and nature of adsorbates. In this paper, we have investigated higher-order structures of nucleic acids that are correlated with human disease: triplet repeats of the single-stranded oligonucleotides 5'-(CCG)n-3' and 5'-(CGG)n-3'. These sequences fold into structures that have not been determined, but give characteristic spectra in circular dichroism spectroscopy. Under salt conditions where 5'- (CCG)7-3' and 5'-(CGG)7-3' fold into these higher-order structures, our nanoparticles bind them well but do not bind to normal double-helical DNA. This result may form the basis for future assays of higher-order DNA structures.

The local structure of biological membranes is critically important to membrane function. Regions of very high positive and negative curvature are found in the membranes of many cells, and rapid changes in membrane curvature are integral parts of many cell activities (e.g. endo/exocytosis, cell crawling, cell division). Our goal is to understand the effects of changes in local membrane structure on membrane properties. Optical tweezers are used to control the local structure of the lipid bilayer by controlling the curvature of the membrane. We use giant (`cell-sized'), thin-walled vesicles as our membrane models. Optically trapped latex microspheres are used to deform the liposome bilayer, forming large areas of altered membrane curvature. In contrast to literature reports in which 514.5 nm light was used in optical trapping, we have not observed adhesion of uncoated latex microspheres to liposome vesicles, nor have we observed signs of rapidly increased osmotic pressure within irradiated vesicles. This indicates that the longer wavelength used in our studies (647.1 nm) is less damaging to biological membranes. Furthermore, optical trapping of vesicles with coexisting gel and fluid phase lipids did not lead to gross changes in domain structure, which would be expected upon laser-induced heating of the membrane.

Fluorescein dye covalently attached to anionic and cationic carriers of molecular weight 0.5 to 30 kDa were prepared and evaluated as potential fluorescent tracer agents for monitoring real-time dynamic renal function. These fluorescein bioconjugates were generally prepared by the reaction of fluorescein isothiocyanate with the desired polypeptides. The mode of clearance from the blood, and the clearance rate, is substantially altered in these conjugated compounds with respect to the dye alone. For example, the blood clearance of fluorescein is known to be through both the hepatic and renal systems, while several of the newly synthesized anionic fluorescein bioconjugates exhibit renal clearance only. Increasing the number of anionic residues in such compounds yields an increase in the renal clearance rate until a lower limit (saturation value) is reached. Thus, an optimum fluorescent renal tracer agent with respect to clearance rate and chain length can be identified. More lipophilic anionic conjugates clear at slower rates, and are not exclusively renal. In contrast, fluorescein bioconjugates composed of cationic carriers do not clear from the blood within the time frame of measurement.

We describe the design and properties of cyanine dye-peptide conjugates synthesized on solid supports for screening assays. With this approach, we demonstrate the feasibility of including a diagnostic molecule into a spot synthesis of membrane-bound peptide libraries in order to permit screening of complete dye-peptide conjugates for their potential as fluorescent contrast agents in biomedical optical imaging. A cellulose support, which is modified with a linker permitting cleavage of the dye-peptide conjugates from the support, was prepared. The attachment of dyes to the peptides is exemplified with carboxy-substituted indotricarbocyanines, which can be covalently linked to the N-terminal amino group or a lysin in the course of the synthesis. Several dye-peptide conjugates were obtained by automated peptide synthesis on resins. Furthermore, model sequences consisting of up to 11 amino acids were synthesized on cellulose in sufficient amounts and purity, thus permitting direct testing of these compounds in cell- based assays and fluorescence microscopy. The dyes show only negligible alterations in their absorption and fluorescence properties when attached to low molecular weight peptides. In conclusion, membrane-bound peptide libraries provide a powerful tool to generate large diversities of dye-labeled peptides and to make these compounds available for biological screening assays.

Many gastroenteropancreatic tumors express receptors for somatostatin (SST) and/or vasoactive intestinal peptide (VIP). These receptors can be used as molecular targets for the delivery of contrast agents for tumor diagnostics. We have synthesized conjugates consisting of a cyanine dye and an SST analogue or VIP for use as contrast agents in optical imaging. Receptor binding and internalization of these compounds were examined with optical methods in transfected RIN38 tumor cells expressing the SST2 receptor or a GFP- labeled VIP (VPAC1) receptor. Furthermore, biodistribution of the conjugates was examined by laser-induced fluorescence imaging in nude mice bearing SST2 or VPAC1 receptor- expressing tumors. After incubation of RIN38 SSTR2 cells in the presence of 100 nM indotricarbocyanine-SST analogue, cell-associated fluorescence increased, whereas no increase was observed when receptor-medicated endocytosis was inhibited. Indodicarbocyanine-VIP accumulated in RIN38 VPAC1 cells and co-localization with the GFP-labeled VPAC1 receptor was observed. After injection of indotricarbocyanine-SST analogue into tumor-bearing nude mice, SST2 receptor-positive tumors could be visualized for a time period from 10 min to at least 48 h. After application of indodicarbocyanine-VIP, a fluorescence signal in VIP1 receptor-expressing tumors was only detected during the first hour. We conclude that cyanine dye-labeled VIP and SST analogue are novel, targeted contrast agents for the optical imaging of tumors expressing the relevant receptor.

Tuberculosis (TB) remains the leading cause of death in the world from a single infectious disease and the threat is becoming more critical with the emergence and spread of multi-drug resistant tuberculosis (MDR-TB). Existing methods for detection of various strains of mycobacterium tuberculosis are complex, time consuming and expensive, and therefore, not suitable for use in developing countries where the spread of the disease is most rampant. Currently, a digital detection system based on advanced digital imaging technology, including CMOS and image intensification technology, is being developed by InterScience, Inc. for use with the luciferase reporter mycobacteriophages technique as developed at the Albert Einstein College of Medicine. This compact, low cost and high sensitivity system for rapid diagnosis and drug susceptibility testing for TB will have an immediate impact for both research and clinical applications. It is envisioned that the instrument will be suitable for use as a portable tool for rapid screening of MDR-TB in both developed and developing countries. The development of the system, recent results and a comparison to competing technologies will be presented.

Cytosolic Ca²⁺ ([Ca²⁺]^cyt) regulates several cellular functions, e.g. cell growth, contraction, secretion, etc. In many cell types, ion homeostasis appears to be coupled with glucose metabolism. In certain cell types, a strict coupling between glycolysis and the activity of Sarcoplasmic/Endoplasmic Reticulum Ca²⁺-ATPases (SERCA) has been suggested. Glucose metabolism is altered in diabetes. We hypothesize that: (1) Ca²⁺ homeostasis is altered in microvascular endothelial cells from diabetic animals due to the dysfunction of glycolysis coupling the activity of SERCA; (2) endosomal/lysosomal compartments expressing SERCA are involved in the dysfunction associated with diabetes.

We have previously shown the relationship between metastatic potential and plasmalemmal V-H⁺-ATPase (pmV-ATPase) expression in tumor cells. This led us to hypothesize that pmV-ATPase activity is involved in invasion. Angiogenesis involves invasion of adjacent tissues by microvascular endothelial cells, thus we hypothesized that pmV-ATPases contribute to pHⁱⁿ regulation and invasion in microvascular endothelial cells.

Development of fluorescent probes which sense cellular constituents (metabolites, ions) has revolutionized studies of cell physiology. To take full advantage of these probes for physiological studies, techniques which simultaneously monitor signal from multiple probes with high temporal resolution are required. In addition,the response of unique cells within a population may be heterogeneous making spatial sampling important for specific applications.

Contrary to conventionally used serial discrete dilutions of a ligand to construct concentration-response curves with a few experimental points, we describe here an automated system, HT-PS^TM 100, that produces continuous ligand concentration gradients for interrogating cells in flow, and concurrently measures the ligand-induced functional response.

Fluorescent in situ hybridization (FISH) is an excellent method for detection of gene copy number alterations in cancer and other diseases. A limitation of the technology is the tedious, inaccurate and often highly subjective spot counting. For a number of reasons, automation of FISH spot, counting has not been accomplished. FISH signals are often at different focal planes, resulting in interfering out-of- focus light. This paper describes current progress towards automated FISH spot counting, with particular reference to the previous technical limitations.

Digital imaging spectroscopy combines image processing and optical spectroscopy such that each picture element (pixel) or group of pixels in a 2D scene also includes additional dimension(s) of spectral and/or temporal information. Imaging spectrophotometers developed at KAIROS are literally equivalent to hundreds of thousands of conventional spectrophotometers running in parallel, i.e., each pixel is functionally equivalent to a single `instrument'. Because of the amount of data acquired by this massively parallel technique--often in the gigabyte range for a single experiment--it was necessary to develop a new generation of graphical tools for data analysis and display.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to study, among many of its important activities, the exonuclease activity of exonuclease III (EC 3.1.11.2), a metal-dependent enzyme. The technique is capable of analyzing the mixture of DNA substrates in the reaction. Time dependent measurement of the reaction mixture provided kinetics data of the enzyme. It was found that among divalent metals studied, Mn, which increased the rate of reactions by two orders of magnitude, is the best cofactor for exonuclease III.