Design of a simple radio frequency circuit for implementing the open-ended coaxial probe method for permittivity measurement.

The development of electromagnetic (EM)-based therapeutic and diagnostic tools, as well as safety assessment of EM interactions with the human body, requires adequate measurement of the complex permittivity of different biological tissues. Such measurement techniques must be low-cost, readily available, and easy to implement. In this study, a simple circuit with basic radio frequency electronics was used to implement the open-ended coaxial probe method for permittivity measurement, as opposed to the widely used vector network analyzers. The non-ideal behavior of the circuit due to spurious reflections and ohmic losses was accounted for by a scattering matrix (SM) that relates the measured reflection coefficient to the true reflection coefficient at the probe tip. Parameters of SM were obtained using three calibration standards, and the circuit was used to measure the complex permittivity of a standard, tissue-equivalent, American Society of Testing Materials (ASTM) polymer gel. A more intuitive approach to circuit analysis is also introduced. For both methods, the dielectric constant and electrical conductivity of the gel were found to agree with the recommended uncertainties of the ASTM standard and validate the utility of the circuit at the test frequency.

Photo- and bio-physical characterization of novel violet and near-infrared lipophilic fluorophores for neuronal tracing.

Lipophilic fluorescent dyes have been used to trace neuronal connections because of their ability to diffuse laterally within nerve cell membranes. Given the hundreds to thousands of connections that a typical neuron makes with its neighbours, a diffusion-matched set of spectrally distinct dyes is desirable. To extend a set of these dyes to obtain six independent labels, we have characterized the properties of novel violet and near-infrared candidates. By combining two-photon and confocal microscopy all of these candidates can be imaged using a single Titanium Sapphire laser. Here we present measurements of the two-photon action cross-sections and diffusion properties of the dyes, using either the relative diffusion distance or fluorescence recovery after photobleaching techniques, and demonstrate six-colour neuronal tracing within the spinal cord and brain tissue.

Near-infrared laser illumination transforms the fluorescence absorbing X-Gal reaction product BCI into a transparent, yet brightly fluorescent substance.

The beta-galactosidase protein generated by the bacterial LacZ gene is widely used to map gene expression patterns. The ease of its use is only rivaled by green fluorescent protein, which can be used in combination with various other procedures such as immunocytochemistry, flow cytometry, or tract tracing. The beta-galactosidase enzymatic reaction potentially provides a more sensitive assay of gene expression than green fluorescent protein. However, the virtual impermeability and tendency to absorb light over a wide range limit the use of the most frequently used beta-galactosidase substrate, X-Gal, in combination with other fluorescent labeling procedures. Here, we provide details on a simple photoactivation procedure that transforms the light-absorbing X-Gal product, 5-bromo-4-chloro-3-indolyl (BCI) precipitate, into an intensely fluorescent product excited by 488 and 633 nm light. Photoactivation is achieved through exposure to 730 nm near-infrared light emitted from a femtosecond titanium-doped Sapphire laser. Photoactivation of BCI occurs in tissue sections suspended in buffered saline, glycerol, or even embedded in epoxy resin. A protocol for the use of BCI photoactivation is here provided. Importantly, the BCI photoactivated product is photoswitchable, displaying bistable photochromism. This permits the use of the fluorescent product in a variety of co-localization studies in conjunction with other imaging modalities. As with other bistable and photoswitchable products, the BCI reaction product shows concentration quenching at high density and can be degraded by continuous exposure to intense 730 nm illumination. Therefore, care must be taken in developing imaging strategies. Our findings have implications for the use of X-Gal in gene and protein detection and provide a novel substrate for high density digital information storage.

Effect of molecular structure on the performance of triarylmethane dyes as therapeutic agents for photochemical purging of autologous bone marrow grafts from residual tumor cells.

Extensively conjugated cationic molecules with appropriate structural features naturally accumulate into the mitochondria of living cells, a phenomenon typically more prominent in tumor than in normal cells. Because a variety of tumor cells also retain pertinent cationic structures for longer periods of time compared with normal cells, mitochondrial targeting has been proposed as a selective therapeutic strategy of relevance for both chemotherapy and photochemotherapy of neoplastic diseases. Here we report that the triarylmethane dye crystal violet stains cell mitochondria with efficiency and selectivity, and is a promising candidate for photochemotherapy applications. Crystal violet exhibits pronounced phototoxicity toward L1210 leukemia cells but comparatively small toxic effects toward normal hematopoietic cells (murine granulocyte-macrophage progenitors, CFU-GM). On the basis of a comparative examination of chemical, photochemical, and phototoxic properties of crystal violet and other triarylmethane dyes, we have identified interdependencies between molecular structure, and selective phototoxicity toward tumor cells. These structure-activity relationships represent useful guidelines for the development of novel purging protocols to promote selective elimination of residual tumor cells from autologous bone marrow grafts with minimum toxicity to normal hematopoietic stem cells.

Quantitative broadband near-infrared spectroscopy of tissue-simulating phantoms containing erythrocytes.

We report the use of steady-state diffuse reflectance spectroscopy (SSDRS) to measure the near-infrared absorption spectrum of liquid phantoms containing human erythrocytes in aqueous suspensions of polystyrene spheres which simulate the scattering properties of tissue. The absorption spectra obtained from these SSDRS measurements of intact red cells under oxygenated and deoxygenated conditions are compared with several published spectra of 'stripped' haemoglobin prepared from lysed cells. Two fitting algorithms (nonlinear least squares and singular value decomposition) which exploit the broad spectral range provided by these measurements (170 data points spanning 164 nm in a single acquisition) are used to determine haemoglobin oxygen saturation (SO2) from SSDR spectra collected over a wide range of measured oxygen partial pressures. The validity of these algorithms is assessed by comparing literature values of p50 (the oxygen tension at which haemoglobin is 50% saturated) and the Hill coefficient to values of these parameters determined from the SO2 estimates. The singular value decomposition algorithm can also be used to reconstruct the non-haemoglobin background absorption spectrum without a priori assumptions regarding its constituent chromophores or their concentrations. Using this technique, the absorption spectrum of a small amount of India ink (maximum absorption coefficient (mu(a max)) approximately 0.0006 mm(-1)) added to a phantom containing red cells (mu(a max) approximately 0.026 mm(-1)) was reconstructed over a full range of oxygen saturations. The implications of these measurements for detection of weakly absorbing chromophores (such as cytochrome aa3) in the presence of haemoglobin are discussed.

Localization of Luminescent Inhomogeneities in Turbid Media with Spatially Resolved Measurements of cw Diffuse Luminescence Emittance.

We present a steady-state method for localizing a source ofluminescence (i.e., fluorescence or phosphorescence) buried in asemi-infinite turbid medium with unknown optical properties. Adiffusion theory expression describing the emittance of an isotropicpoint source is fit to spatially resolved surface measurements of thediffuse emittance from the luminescent source. The techniquereports the location of the center of a 6.0-mm-diameter, fluorophore-containing spherical bulb embedded in a liquid phantom withan accuracy of 1.0 mm or better for source depths as great as 40.0mm. Monte Carlo data are analyzed to investigate the range and thepossible sources of error in the reconstructed source depth.

Design and testing of a white-light, steady-state diffuse reflectance spectrometer for determination of optical properties of highly scattering systems.

We present a steady-state radially resolved diffuse reflectance spectrometer capable of measuring the absorption and transport scattering spectra of tissue-simulating phantoms over an adjustable 170-nm wavelength interval in the visible and near infrared. Measurements in a variety of phantoms are demonstrated over the relevant range of tissue optical properties, and the accuracy of the instrument is found to be approximately 10% in both scattering and absorption. Monte Carlo simulations designed to test the accuracy of the instrument are presented that support the experimental findings.

The mechanism of Photofrin photobleaching and its consequences for photodynamic dosimetry.

We report experimental results that support a theory of self-sensitized singlet oxygen-mediated bleaching of the porphyrin photosensitizer Photofrin. Microelectrode measurements of photodynamic oxygen consumption were made near the surface of individual, Photofrin-sensitized EMT6 spheroids during laser irradiation. The progressive decrease in photochemical oxygen consumption with sustained irradiation is consistent with a theory in which bleaching occurs via self-sensitized singlet oxygen reaction with the photosensitizer ground state. A bleaching model based solely on absorbed optical energy density is inconsistent with the data. Photobleaching has a significant effect on calculated photodynamic dose distributions in 500 microns diameter spheroids. Dose distributions corrected for the effects of bleaching produce a new estimate (12.1 +/- 1.2 mM) for the threshold dose of reacting singlet oxygen in this system.

Oxygen diffusion and reaction kinetics in the photodynamic therapy of multicell tumour spheroids.

Effects of oxygen diffusion and reaction kinetics in photodynamic therapy are considered in the context of a multicell tumour spheroid model. Steady-state measurements of oxygen made with a Clark-style microelectrode (4 microm diameter tip) enable us to determine the rate of metabolic oxygen consumption and the oxygen diffusion coefficient in 500 microm diameter EMT6/Ro spheroids. These values are 5.77 micromol 1(-1) s(-1) and 1460 microm2 s(-1), respectively. Time-dependent electrode measurements of oxygen concentration during laser irradiation of individual Photofrin-sensitized spheroids are fitted to numerical solutions of a pair of diffusion-with-reaction equations. The analysis yields the rate of photodynamic oxygen consumption and a parameter that governs the oxygen sensitivity of photodynamic therapy. These experimentally derived quantities are used to calculate the temporal and spatial distributions of oxygen and the rate of oxygen consumption in a spheroid during irradiation at several fluence rates. The spatial distribution of photodynamic oxygen consumption is strongly fluence rate dependent. Using the experimental and theoretical results developed in this report, previously published survival data are analysed. The analysis indicates that the threshold dose of reacting singlet oxygen in the EMT6/Ro spheroid is 323 +/- 38 micromol 1(-1) (mean +/- SEM).

Fluence rate effects in photodynamic therapy of multicell tumor spheroids.

EMT6/Ro spheroids approximately 500 microns in diameter were subjected to photodynamic therapy administered at various incident radiation fluence rates. Following 24 h incubation with 10 micrograms/ml Photofrin, groups of spheroids were irradiated at 630 nm with an identical fluence of 60 J/cm2, delivered at fluence rates ranging from 25 to 200 mW/cm2. After treatment, spheroids were dissociated, cell yields were determined, and surviving cells were assayed for their colony-forming ability. A surviving fraction was calculated for each treatment group by computing the product of the fractional cell yield and the plating efficiency. The results exhibit a strong dependence on the fluence rate, with surviving fractions decreasing from approximately 0.5 to 0.07 as the incident fluence rate was lowered from 200 to 25 mW/cm2. These data were analyzed using a mathematical model of photochemical oxygen consumption in spheroids undergoing photodynamic therapy. Calculations showed that therapy-induced oxygen consumption creates hypoxic volumes within which cells would be protected from singlet oxygen-mediated damage and that the magnitude of these hypoxic volumes depends on the radiation fluence rate. The fluence rate dependence of the spheroid cell survival was consistent with such an interpretation.