Gene expression microarray analyses of mixtures of cells approximate a weighted average of the gene expression profiles (GEPs) of each cell type according to its relative abundance in the overall cell sample being analyzed. If the targeted subpopulation of cells is in the minority, or the expected perturbations are marginal, then such changes will be masked by the GEP of the normal/unaffected cells. We show that the GEP of a minor cell subpopulation is often lost when that cell subpopulation is of a frequency less than 30 percent. The GEP is almost always masked by the other cell subpopulations when that frequency drops to 10 percent or less. Several methodologies can be employed to enrich the target cells submitted for microarray analyses. These include magnetic sorting and laser capture microdissection. However, high-throughput flow cytometry/cell sorting overcomes many restrictions of experimental enrichment conditions. This technology can also be used to sort smaller numbers of cells of specific cell subpopulations and subsequently amplify their mRNAs before microarray analyses. When purification techniques are applied to unfixed samples, the potential for changes in gene levels during the process of collection is an additional concern. High-throughput cell separation technologies are needed that can process the necessary number of cells expeditiously in order to avoid such uncontrolled changes in the target cells GEP. In cases where even the use of HTS yields only a small number of cells, the mRNAs (after reverse transcription to cDNA's) must be amplified to yield enough material for conventional microarray analyses. However, the problem of using microamplification PCR methods to expand the amount of cDNAs (from mRNAs) is that it is very difficult to amplify equally all of the mRNAs. Unequal amplification leads to a distorted gene expression profile on the microarray. Linear amplifications is difficult to achieve. Unfortunately, present-day gene-chips need to be about 100 times more sensitive than they are now to be able to do many biologically and biomedically meaningful experiments and clinical tests.

In order to minimize hospitalization and morbidity with optimized therapy for a patient with a tumor of the parotid gland a malignancy must be confirmed or excluded as soon as possible. Up to now, non- and minimal-invasive methods do not yield this information. For fine needle aspirate biopsies (FNABs), analysis by a specialized cytologist yields subjective and qualitative but not objective and quantitative data. LSC is a semi-automated microscope-based technology and offers ideal prerequisites for the analysis of specimens fixed on a slide. We have established an assay for FNABs from parotid gland tumors. Cells are stained for cytokeratin and DNA. The analysis quantitatively determines the ploidy of the cells and the degree of condensation of the DNA; on this basis the percentage of cells undergoing mitosis can be determined. Subsequently the cells are stained by H&E and are re-localized on the slide at their fixed position. Micrographs are taken for objective documentation of the cells' morphology. Using this assay FNABs from parotid gland tumors were analyzed; tumors that were diagnosed as benign by routine histopathology showed no aneuploidy whereas malignant tumors were aneuploid. This preliminary study demonstrates the capacities of LSC for minimal-invasive assays yielding quantitative and objective data.

The CellTracks system is a Compact Disk-based cell analyzer that, similar to flow cytometry, differentiates cells that are aligned while passing through focused laser beams. In CellTracks, only immuno-magnetically labeled cells are aligned and remain in position for further analysis. This feature is important in those cases were the cells are relatively infrequent. Epithelium derived tumor cells in peripheral blood are extremely rare but can be present in the blood of cancer patients. The certainty that an event present in a gate is, indeed, an epithelial cell with the assumed characteristics diminishes with the number of events in the analysis gate. Additional and preferably independent information on the individual events aids in the correct classification of the event as an epithelium derived tumor cell. Epithelial cells are immuno-magnetically selected from 7.5 mL of blood and magnetically aligned in the sample chamber between a series of parallel thin film nickel lines. The CD head scans along all nickel lines and captures the fluorescence signals of the objects between the lines. Objects that immuno-phenotypically classify as epithelial tumor cells are revisited for imaging to determine if the identified objects indeed classify as epithelial tumors cells or as debris derived from epithelial cells.

Optical microcavities can be used to enhance the detection sensitivity of evanescent-wave fluorescence biosensors to the binding of a labeled analyte to a biospecific monolayer. The enhancement results form the buildup of intensity within the microcavity on resonance, which thereby increases fluorescence output from species specifically bound on the surface of the microcavity. Target studies are directed at nucleic acid hybridization, and initial results using high-Q dielectric microspheres have been obtained.

The possibility of using a pressure monitoring system based on differential pressure sensors to detect contaminant effects on cellular cultures metabolic activity is discussed using Saccharomyces cerevisiae cell cultures: differential pressure curves' shape, starting slope and maximum are affected both by physical and chemical contamination. Aim of the present study is the investigation of the effects generated by a 72h exposition of Saccharomyces cerevisiae, human lymphocytes and AHH1 cellular line cultures to 50Hz, 60(mu) T electromagnetic field. No significant differences have been recorded between irradiated and control yeast samples. On other hand irradiated lymphocytes samples, cultures in a PHA medium, grow less than control ones, but exhibit a greater metabolic activity: changes in the exposure system configuration influence neither sample growth differences nor metabolic response variations between control and irradiated samples. Control and irradiated lymphocyte samples, without PHA in culture medium, show the same behavior both during irradiation and metabolic test. AHH1 control and irradiated samples show no difference both in growth percentage during irradiation and in metabolic test. Different cell cultures respond to the same stimulus in different manners.

Our work is devoted to the study of Saccharomyces cerevisiae and human lymphocytes cellular metabolism in order to develop a reference model to assess biological systems responses to chemical or physical agents exposure. CO₂ variations inside test-tubes are measured by differential pressure sensors; pressure values are subsequently converted in voltage. The system allows to test up to 16 samples at the same time. Sampling manages up to 100 acquisitions per second. Values are recorded by a data acquisition card connected to a computer. This procedure leads to a standard curve (pressure variation versus time), typical of the cellular line, that describe cellular metabolism. The longest time lapse used is of 170 h. Different phases appear in this curve: an initial growth up to a maximum, followed by a decrement that leads to a typical depression (pressure value inside the test-tubes is lower than the initial one) after about 35 h from the beginning of yeast cells. The curve is reproducible within an experimental error of 4%. The analysis of many samples and the low cost of the devices allow a good statistical significance of the data. In particular as a test we will compare two sterilizing agents effects: UV radiation and amuchina.

A hydroxyl radical nanoprobe has been developed by covalently attaching the dye, coumarin-3-carboxylic acid (CCA), to amine-functionalized polyacrylic acid (AFPAA) nanoparticles. These nanoparticles are referred to as PEBBLEs (Probes Encapsulated By Biologically Localized Embedding). This probe has been shown to be highly specific and sensitive. The formation of hydroxyl radical is monitored by an increase in fluorescence over time.

The current trend in diagnostic assay development is toward the use of smaller and smaller biological samples. The assay will then be required to identify a trace amount of a particular protein, nucleic acid or chemical species within a complex mixture of molecules. Due to the inherently low amount of analyte in these samples, a very sensitive detection device is required to make the measurement. In addition, many assays are becoming multi-parametric in order to reduce cost and lower the turnaround time of analysis. The increase in sample numbers can be dealt with by highly parallel analysis of samples either in an array format or in adjacent micro-channels. Another factor pushing the development toward highly parallel analysis is the desire to use high throughput methods to do measurements on multiple source samples in parallel. The highly parallel analysis can be readily achieved with imaging methods in contrast to the point measurements usually employed in current instrumentation. This paper will examine the recent trends in scientific grade CCD based imaging systems that are being driven by new development in CCD sensors, intensifiers and camera designs.

An array of ~50,000 individual polymeric micropost sensors was patterned across a glass coverslip by a photoimprint lithographic technique. Individual micropost sensors were ~3-micrometers tall and ~8-micrometers wide. The O₂-sensitive micropost array sensors (MPASs) comprised a ruthenium complex encapsulated in a gas permeable photopolymerizable siloxane. The pH-sensitive MPASs comprised a fluorescein conjugate encapsulated in a photocrosslinkable poly(vinyl alcohol)-based polymer. PO₂ and pH were quantitated by acquiring MPAS luminescence images with an epifluorescence microscope/charge coupled device imaging system. O₂-sensitive MPASs displayed linear Stern-Volmer quenching behavior with a maximum I_o/I of ~8.6. pH-sensitive MPASs displayed sigmoidal calibration curves with a pK_a of ~5.8. The adhesion of undifferentiated rat pheochromocytoma (PC12) cells across these two polymeric surface types was investigated. The greatest PC12 cell proliferation and adhesion occurred across the poly(vinyl alcohol)-based micropost arrays relative to planar poly(vinyl alcohol)-based surfaces and both patterned and planar siloxane surfaces. An additional advantage of the patterned MPAS layers relative to planar sensing layers was the ability to direct the growth of biological cells. Preliminary data is presented whereby nerve growth factor-differentiated PC12 cells grew neurite-like processes that extended along paths defined by the micropost architecture.

Molecular cytometry refers to ultrasensitive analysis tools that are used to separate and identify entire classes of molecules in single cells. Recently, we described two molecular cytometry methods to analyze proteins at the single cell level. The first one was based on capillary gel electrophoresis with sheath-flow cuvette laser-induced fluorescence (LIF). A vacuum pulse was employed to introduce a single HT29 human colon cancer cell into the capillary. Once the cell was lysed, proteins were denatured with SDS, labeled with 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ), and then separated according to their size by using pullulan as the sieving matrix. The second one was based on submicellar capillary electrophoresis with sheath-flow cuvette LIF. Once a single cell was introduced and lysed, the cellular proteins were labeled with FQ and then separated in a submicellar buffer. This method has been applied to analysis of proteins in a single HT29 human cancer cell as well as single-cell stage Caenorhabditis elegans embryo.

The MexAB-OprM efflux pump of Pseudomonas aeruginosa consists of two inner and one outer membrane proteins and exports xenobiotics rendering the cells resistant to structurally diverse antimicrobial agents. We used fluorescent probes to monitor the pump function. Fluorescence intensity of hydrophobic cation, 2-(4-dimethylaminostyryl)-1-ethylpyridinium, was 7 to 4 times higher in the mutant lacking the pump subunit than the wild type strain. Water-soluble fluorescent probe, ethidium bromide, accumulated in the Mex mutants at the rate 5 times faster than in the wild-type cells. The results demonstrated that the fluorescent probes are powerful tools in real-time monitoring the function of the efffux pump. Topological of the MexB subunit showed that the protein span the membrane 12 times and had two huge periplasmic domains. MexA is an inner membrane anchoring lipoprotein and exposing entire polypeptide domain to the periplasmic space. OprM anchored the outer membrane via fatty acids and exposed the protein portion to the periplasmic space. These results suggested that the MexAB-OprM pump exports xenobiotics as that MexB drafts the substrates at the cytoplasmic membrane and export to periplasmic space and MexA and OprM convey the substrate bypassing the periplasmic space to the outer membrane.

Cytokines have a strong potential for triggering anticancer immunity if released in the tumor microenvironment. Successful vaccines have been engineered using tumor cells genetically modified to secrete the cytokines. Unfortunately, this approach remains difficult and hazardous to perform in the clinic. We describe a new way of combining cytokines with tumor cells to prepare anticancer vaccines. This consists in anchoring recombinant cytokines to the membrane of killed tumor cells. Attachment is mediated by a fragment of diphtheria toxin (T) genetically connected to the cytokine. It is triggered by an acid pH pulse. The method was applied to IL-2, a potent anti-tumor cytokine. IL-2 anchored to the surface of tumor cells by the T anchor retained its IL-2 activity and remained exposed several days. Interestingly, vaccination of mice with these modified tumor cells induced a protective anti-tumor immunity mediated by tumor-specific cytotoxic T lymphocytes. This procedure presents several advantages as compared to the conventional approaches based on the transfection of tumor cells with cytokine genes. It does not require the culture of tumor cells from the patients and eliminates the safety problems connected with viral vectors while allowing the control of the amount of cytokines delivered with the vaccine.

Circular dichroism and fluorescence polarization anisotropy are important tools for characterizing biomolecular systems. Both are used extensively in kinetic experiments involving stopped - or continuous flow systems as well as titrations and steady-state spectroscopy. This paper presents the theory for determining circular dichroism and fluorescence polarization anisotropy simultaneously, thus insuring the two parameters are recorded under exactly the same conditions and at exactly the same time in kinetic experiments. The approach to measuring circular dichroism is that used in almost all conventional dichrographs. Two arrangements for measuring fluorescence polarization anisotropy are described. One uses a single fluorescence detector and signal processing with a lock-in amplifier that is similar to the measurement of circular dichroism. The second approach uses classic T format detection optics, and thus can be used with conventional photon-counting detection electronics. Simple extensions permit the simultaneous measurement of the absorption and excitation intensity corrected fluorescence intensity.

A continuing issue in chemical oceanography and environmental monitoring is the need for frequent and continuous monitoring of analytes in complex matrices such as sea water and ground waters. Particularly for analytes at trace levels such as Cu(II) in sea water, sampling and analysis of discrete specimens is costly, slow, labor intensive, employs ship time inefficiently, and risks error by contamination. We have developed a fluorescence lifetime- based fiber optic biosensor which demonstrates real time determination of free Cu(II) in coastal waters, in situ, with a subpicomolar detection limit.

HPTLC (High Performance Thin Layer Chromatography) is a well known and versatile separation method which shows a lot of advantages and options in comparison to other separation techniques. The method is fast and inexpensive and does not need time-consuming pretreatments. Using fiber-optic elements for controlled light-guiding, the TLC-method was significantly improved: the new HPTLC-system is able to measure simultaneously at different wavelengths without destroying the plate surface or the analytes on the surface. For registration of the sample distribution on a HPTLC-plate we developed a new and sturdy diode-array HPTLC- scanner which allows registration of spectra on the TLC- plates in the range of 198 nm to 610 nm with a spectral resolution better than 1.2 nm. The spatial resolution on plate is better than 160 micrometers . In the spectral mode, the new HPTLC-scanner delivers much more information than the commonly used TLC-scanner. The measurement of 450 spectra of one separation track does not need more than three minutes. However, in the fixed wavelength mode the contour plot can be measured within 15 seconds. In this case, the signal will be summarized and averaged over a spectral range having FWHM from 10 nm to 25 nm depending on the substance under test. The new diode-array HPTLC-scanner makes various chemometric applications possible. The new method can be used easily in clinical diagnostic systems easily, e.g. for blood and uring investigations. In addition, new applications are possible. For example, the rich structured PAHs were studied. Although the separation is incomplete the 16 compounds can be quantified using suitable wavelengths.

We demonstrate the application of Terahertz Pulse Imaging (TPI) in reflection geometry for the study of skin tissue and related cancers. The terahertz frequency regime of 0.1-100THz excites the vibrational modes of molecules, allowing for spectroscopic investigation. The sensitivity of terahertz to polar molecules, such as water, makes TPI suitable for studying the hydration levels in the stratum corneum and the determination of the lateral spread of skin cancer pre-operatively. By studying the terahertz pulse shape in the time domain we have been able to differentiate between diseased and normal tissue for the study of basal cell carcinoma (BCC). Measurements on scar tissue, which is known to contain less water than the surrounding skin, and on regions of inflammation, show a clear contrast in the THz image compared to normal skin. We discuss the time domain analysis techniques used to classify the different tissue types. Basal cell carcinoma shows a positive terahertz contrast, and inflammation and scar tissue shows a negative terahertz contrast compared to normal tissue. This demonstrates for the first time the potential of TPI both in the study of skin cancer and inflammatory related disorders.

The existence of macular pigment (MP) in human eyes has been found to be of importance in the prevention of age-related macular degeneration (ARMD). A non-invasive technique of two-wavelength (460 nm and 560 nm) imaging fundus reflectometry was developed to measure the density distribution of this yellow pigment in retina in vivo. A total of 54 healthy human subjects were recruited and divided into three groups, which had average ages and sample sizes of 24.8+/- 2.6 (N=24), 40.2+/- 8.3 (N=13) and 67.5+/- 7.1 years (N=17) respectively. The MP distributions only in the horizontal meridian, which are fitted by Gaussian equation are presented in this report. The results show that the average peak MP densities are 0.48+/- 0.17, 0.46+/- 0.21 and 0.52+/- 0.13 in density unit (DU) from young to old groups respectively. No significant difference is shown in the statistical t-test between the groups of the MP density. The half MP distribution width (covers 95% of Gaussian area) are 5.4 degree(s)+/- 1.6 degree(s), 5.7 degree(s)+/- 2.3 degree(s) and 7.9+/- +/- 2.1 degree(s) from young to old groups respectively. It shows that the area of MP is increased approximately by a rate of 0.06 degree(s)/year. However, the cause of the MP extension in the retina with respect to age is unknown.

In this communication, we propose a non-invasive imaging setup which does not require any lateral and longitudinal scanning over the sample. The idea is to image a line over the sample surface with a laser and to collect the photons backscattered by the sample surface and the internal structures. As a result, a single shot 2D image of the sample can be obtained, one axis representing a lateral coordinate and the other one the longitudinal coordinate, e.g. the depth of the sample. To collect the backscattered light from the sample, we use a femtosecond laser source and the potential of a classical single-shot autocorrelator to make a time-to-space conversion by use of non-collinear sum frequency generation in a nonlinear crystal. With this technique, we performed single shot longitudinal imaging of an ex vivo mouse ear and in vivo human skin with a 35 micrometers lateral and 15 micrometers depth resolution in tissues. Inside the mouse ear, the epidermis, dermis and cartilage have been observed. In the in vivo human skin, the stratum corneum, the epidermis and the dermis have been observed. Especially, the epidermal-dermal junction has been clearly revealed and the thickness of the epidermis has been measured.

Optical Coherence Tomography (OCT) is a relatively new type of imaging system for medical diagnosis. Because most current OCT systems use a sharply focused beam in tissues, they have a short depth of field (high image resolution is near the focus only). In this paper, limited diffraction beams of different orders are used to increase depth of field and to reduce sidelobes in OCT. Results show that the proposed OCT system has a lateral resolution of about 4.4 wavelengths (the central wavelength of the source is about 940 nm with a bandwidth of about 70 nm) and lower than -60 dB sidelobes over an entire depth of field of 4.5 mm with the diameter of the objective lens of 1 mm.

Advanced sensor device for shape analysis of the tissue- reflected mean single period photoplethysmography (SPPPG) signals has been designed and clinically tested. The SPPPG signal shape reveals individual features of the patient's cardio-vascular state. Clinical studies of several patient groups (e.g. diabetes mellitus, atherosclerosis obliterans, Raynaud's syndrome) made possible to specify components of the SPPPG signal that are sensitive to the corresponding organic or functional pathologies. Comparison of the right and left arm finger SPPPG signal shapes, for instance, appears to be efficient tool for early screening of unilateral atherosclerosis obliterans.

We have developed a new series of glucose sensitive fluorophores that display shifts in emission wavelengths upon the binding of glucose. Complexation of glucose with the boronic acid moiety changes both its orbital hybridization and its ability to accept and donate electrons. This change results in distinct emission spectra for the fluorophores when free in solution or complexed with glucose.