The use of exogenous probes to gain a deeper understanding of physiological and molecular processes in vivo through the acquisition of optical signals, particularly via enhanced contrast using molecular probes ~physiologically transported, site-directed, or via reporter genes! has emerged with tremendous vigor in the past few years. One such area of expanded activity is in the area of early cancer detection, in great part because it is so critical to the clinical outcome in the treatment.1–3 As an example, in colon cancer, which accounts for 15% of all U.S. cancer-related deaths, only 37% are found early enough for moderate treatment1 and once these types of cancer reach metastatic activity the survival rate is only 7%.

Genomic DNA contains many higher-order structural deviations from the Watson–Crick global average. The massive expansion and hypermethylation of the duplex triplet repeat (CCG)n(CGG)n has characteristic higher-order structures that are associated with the fragile X syndrome. We have used luminescent mineral nanoparticles of protein-sized cadmium sulfide in optical assays to detect anomalous DNA structures. The photoluminescence of these particles is sensitive to the presence and nature of adsorbates. We previously found that our nanoparticles bind the fragile X repeat well but do not bind to normal double-helical DNA. In this study, we have determined that these particles are also able to detect the hypermethylated forms of these triplet repeats. Therefore, these nanoparticles may form the basis for future optical assays of higher-order DNA structures, especially those associated with human disease.

Oxygen plays a very important role in living cells. The intracellular level of oxygen is under tight control, as even a small deviation from normal oxygen level affects major cellular metabolic processes and is likely to result in cellular damage or cell death. This paper describes the use of the oxygen sensitive fluorescent dye tris (1,10-phenanthroline) ruthenium chloride [Ru(phen)3] as an intracellular oxygen probe. Ru(phen)3 exhibits high photostability, a relatively high excitation coefficient at 450 nm (18 000 M?1 cm?1), high emission quantum yield (~0.5), and a large Stoke shift (peak emission at 604 nm). It is effectively quenched by molecular oxygen due to its long excited state lifetime of around 1µs. The luminescence of Ru(phen)3 decreases with increasing oxygen concentrations and the oxygen levels are determined using the Stern–Volmer equation. In our studies, J774 Murine Macrophages are loaded with Ru(phen)3, which passively permeates into the cells. Fluorescence spectroscopy and digital fluorescence imaging microscopy are used to observe the cells and monitor their response to changing oxygen levels. The luminescence intensity of the cells decreases when exposed to hypoxia and recovers once normal oxygen conditions are restored. The analytical properties of the probe and its application in monitoring the cellular response to hypoxia are described.

We have designed, synthesized, and evaluated the efficacy of novel dye-peptide conjugates that are receptor specific. Contrary to the traditional approach of conjugating dyes to large proteins and antibodies, we used small peptide-dye conjugates that target overexpressed receptors on tumors. Despite the fact that the peptide and the dye probe have similar molecular mass, our results demonstrate that the affinity of the peptide for its receptor and the dye fluorescence properties are both retained. The use of small peptides has several advantages over large biomolecules, including ease of synthesis of a variety of compounds for potential combinatorial screening of new targets, reproducibility of high purity compounds, diffusiveness to solid tumors, and the ability to incorporate a variety of functional groups that modify the pharmacokinetics of the peptide-dye conjugates. The efficacy of these new fluorescent optical contrast agents was evaluated in vivo in well-characterized rat tumor lines expressing somatostatin (sst2) and bombesin receptors. A simple continuous wave optical imaging system was employed. The resulting optical images clearly show that successful specific tumor targeting was achieved. Thus, we have demonstrated that small peptide-dye conjugates are effective as contrast agents for optical imaging of tumors.

Optical mammography with near-infrared (NIR) light using time-domain, frequency-domain, or continuous-wave techniques is a novel imaging modality to locate human breast tumors. By investigating excised specimens of normal and diseased mamma tissue we were able to demonstrate that differences in their scattering properties are a poor predictive parameter for normal and diseased mamma tissue. This paper describes the application of a NIR dye to improve the differentiation between breast tumors and normal tissue in a rat model. The NIR dye furnished a high tumor-to-tissue contrast ratio (6:1) in fluorescence images. Furthermore, this dye was used to develop liquid scattering phantoms with absorbing and fluorescent inhomogeneities. Using frequency-domain and time-domain instrumentation these inhomogeneities were localized at sufficient contrast by their increased absorption and fluorescence. Contrast between inhomogeneities and surrounding medium could be improved by combining fluorescence and transmittance images.

Ultrasound contrast agents are small microbubbles that can be readily destroyed with sufficient acoustic pressure, typically, at a frequency in the low megaHertz range. Microvascular flow rate may be estimated by destroying the contrast agent in a vascular bed, and estimating the rate of flow of contrast agents back into the vascular bed. Characterization of contrast agent destruction provides important information for the design of this technique. In this paper, high-speed optical observation of an ultrasound contrast agent during acoustic insonation is performed. The resting diameter is shown to be a significant parameter in the prediction of microbubble destruction, with smaller diameters typically correlated with destruction. Pressure, center frequency, and transmission phase are each shown to have a significant effect on the fragmentation threshold. A linear prediction for the fragmentation threshold as a function of pressure, when normalized by the resting diameter, has a rate of change of 300 kPa/mm for the range of pressures from 310 to 1200 kPa, and a two-cycle excitation pulse with a center frequency of 2.25 MHz. A linear prediction for the fragmentation threshold as a function of frequency, when normalized by the resting diameter, has a rate of change of ?1.2 MHz/µm for a transmission pressure of 800 kPa, and a two-cycle excitation pulse with a range of frequencies from 1 to 5 MHz.

A common method to induce enhanced short-term endogenous porphyrin synthesis and accumulation in cell is the topical, systemic application of 5-aminolevulinic acid or one of its derivatives. This circumvents the intravenous administration of photosensitizers normally used for photodynamic therapy (PDT) of fluorescence photodetection. However, in the majority of potential medical indications, optimal conditions with respect to the porphyrin precursor or its pharmaceutical formulation have not yet been found. Due to ethical restrictions and animal right directives, the number of available test objects is limited. Hence, definition and use of nonanimal test methods are needed. Tissue and organ cultures are a promising approach in replacing cost intensive animal models in early stages of drug development. In this paper, we present a tissue culture, which can among others be used routinely to answer specific questions emerging in the field of photodynamic therapy and fluorescence photodetection. This technique uses mucosae excised from sheep paranasal sinuses or pig bladder, which is cultured under controlled conditions. It allows quasiquantitative testing of different protoporphyrin IX precursors with respect to dose-response curves and pharmacokinetics, as well as the evaluation of different incubation conditions and/or different drug formulations. Furthermore, this approach, when combined with the use of electron microscopy and fluorescence-based methods, can be used to quantitatively determine the therapeutic outcome following protoporphyrin IX-mediated PDT.

We report the development of novel luminescent nanoparticles composed of inorganic luminescent dye, Tris(2,2'-bipyridyl) dichlororuthenium (II) hexahydrate, doped inside a silica network. These dye doped silica (DDS) nanoparticles have been synthesized using a water-in-oil microemulsion technique in which controlled hydrolysis of the tetraethyl orthosilicate leads to the formation of monodispersed nanoparticles. They are prepared with a variety of sizes: small (561 nm), medium (63±4 nm), and large (400±10 nm), which shows the efficiency of the microemulsion technique for the synthesis of uniform nanoparticles. All these nanoparticles are suitable for biomarker application since they are much smaller than cellular dimension. These nanoparticles are highly photostable in comparison to most commonly used organic dyes. These nanoparticles have been characterized by various microscopic and spectroscopic techniques. The amount of dye content in these nanoparticles has been optimized to eliminate self-quenching. It has been observed that maximum luminescence intensity is achieved when the dye content is around 20 wt%. Silica surface of DDS nanoparticles is available for surface modification and bioconjunction. For demonstration as a biomarker, the DDS nanoparticle’s surface has been biochemically modified to attach membrane-anchoring groups and applied successfully to stain human leukemia cells.

In this paper we present the absorption coefficient µa and the isotropic scattering coefficient µ's for 22 human skin samples measured using a double integrating sphere apparatus in the wavelength range of 1000–2200 nm. These in vitro results show that values for µa follow 70% of the absorption coefficient of water and values for µ's range from 3 to 16 cm?1. From the measured optical properties, it was found that a 2% Intralipid solution provides a suitable skin tissue phantom.

Visible-near infrared spectroscopy was successfully used for the determination of total hemoglobin concentration in whole blood. Absorption spectra of whole blood samples, whose hemoglobin concentrations ranged between 6.6 and 17.2 g/dL, were measured from 500 to 800 nm. Two different types of transmission were measured: conventional transmission spectroscopy which collected primarily collimated radiation transmitted through the sample, and total transmission spectroscopy which used an integrating sphere to collect all scattered light as well. Different preprocessing techniques in conjunction with a partial least squares regression calibration model to predict hemoglobin concentrations were applied to the above two types of transmission. Depending on different preprocessing methods, the standard error of predictions ranged from 0.37 to 2.67 g/dL. Mean centering gave the most accurate prediction in our particular data set. Preprocessing methods designed for compensation of the scattering effect produced the worst results contrary to expectations. For univariate analysis, better prediction was achieved by total transmission measurement than by conventional transmission measurement. No significant difference was observed for multivariate analysis on the other hand. Careful selection of the data preprocessing methods and of the multivariate statistical model is required for reagentless determination of hemoglobin concentration in whole blood.

It is well known that the reconstruction problem in optical tomography is ill-posed. In other words, many different spatial distributions of optical properties inside the medium can lead to the same detector readings on the surface of the medium under consideration. Therefore, the choice of an appropriate method to overcome this problem is of crucial importance for any successful optical tomographic image reconstruction algorithm. In this work we approach the problem within a gradient-based iterative image reconstruction scheme. The image reconstruction is considered to be a minimization of an appropriately defined objective function. The objective function can be separated into a least-square-error term, which compares predicted and actual detector readings, and additional penalty terms that may contain a priori information about the system. For the efficient minimization of this objective function the gradient with respect to the spatial distribution of optical properties is calculated. Besides presenting the underlying concepts in our approach to overcome illposedness in optical tomography, we will show numerical results that demonstrate how prior knowledge, represented as penalty terms, can improve the reconstruction results.

When carrying out medical imaging based on detection of isotopic radiation levels of internal organs such a lungs or heart, distortions, and blur arise as a result of the organ motion during breathing and blood supply. Consequently, image quality declines, despite the use of expensive high resolution devices and, such devices are not exploited fully. A method with which to overcome the problem is image restoration. Previously, we suggested and developed a method for calculating numerically the optical transfer function (OTF) for any type of image motion. The purpose of this research is restoration of original isotope images (of the lungs) by restoration methods that depend on the OTF of the real time relative motion between the object and the imaging system. This research uses different algorithms for the restoration of an image, according to the OTF of the lung motion, which is in several directions simultaneously. One way of handling the three-dimensional movement is to decompose the image into several portions, to restore each portion according to its motion characteristics, and then to combine all the image portions back into a single image. An additional complication is that the image was recorded at different angles. The application of this research is in medical systems requiring high resolution imaging. The main advantage of this approach is its low cost versus conventional approaches.

Higher-order optical errors of the human eye are often responsible for reduced visual acuity in spite of an optimal spherical or cylindrical refraction. These optical aberrations are of natural origin or can result from operations in the eye that involve optical structures. The ocular aberrometer presented is based on Tscherning’s aberroscope. A collimated laser beam (532 nm, 10 mW) illuminates a mask with a regular matrix of holes which forms a bundle of thin parallel rays of 0.3 mm diameter. These rays are focused by a lens in front of the eye so that their intraocular focus point is located a certain distance in front of the retina, generating a corresponding pattern of light spots on it. According to the existing ocular optical errors, this spot pattern is more or less distorted in comparison to the mask matrix. For a 6 mm pupil diameter 68 retinal spots are plottable for assessment of the optical aberrations. The retinal spot pattern is imaged onto the sensor of a low-light charge coupled device video camera by indirect ophthalmoscopy. Deviations of all spots from their ideal regular positions are measured by means of a PC, and from these values the intraocular wave front aberration is computed in the form of the sum of Zernike polynomials up to sixth order.

Diode lasers [? = 1480 nm] are used with in vitro fertilization to dissect the zona pellucida (shell) of pre-embryos. A focused laser beam is applied in vitro to form a channel or trench in the zona pellucida. The procedure is used to facilitate biopsy or as a promoter of embryo hatching. We present examples and measurements of zona pellucida ablation using animal models. In using the laser it is vital not to damage pre-embryo cells, e.g., by overheating. In order to define safe regimes we have derived some thermal side effects of zona pellucida removal. The temperature profile in the beam and vicinity is predicted as function of laser pulse duration and power. In a crossedbeam experiment a HeNe laser probe is used to detect the temperature-induced change in the refractive index of an aqueous solution, and estimate local thermal gradient. We find that the diode laser beam produces superheated water approaching 200°C on the beam axis. Thermal histories during and following the laser pulse are given for regions in the neighborhood of the beam. We conclude that an optimum regime exists with pulse duration ?5 ms and laser power ~100 mW.

Pulsed photothermal radiometry (PPTR) is known to be suitable for in vivo investigations of tissue optical properties. As a noncontact, nondestructive method it is a very attractive candidate for on-line dosimetry of laser treatments that rely on thermal laser–tissue interaction. In this article, we extend the one-dimensional (1D) analytical formalism that has widely been used to describe PPTR signals to a two-dimensional treatment of a simplified model of a blood vessel. This approach leads to quantitative description of a PPTR signal that, unlike in an 1D treatment, not only shows changes in time, but also varies in space. Using this approach, we are able to gain instructive understanding on how target characteristics of a blood vessel-like structure influence such a spatiotemporal PPTR signal. Likewise, the ability of extracting target features from those measurements is evaluated. Subsequently, we present experimental realization of the idealized model of a blood vessel as used in our theory. Comparison of actual PPTR measurements with theoretical predictions allow vessel localization laterally and in depth. Using our setup, we furthermore demonstrate the influence of flow inside the vessel on the measured signal.

The purpose of this study was to investigate the behavior of the tooth–bone interface on the nature of stress distribution in the tooth and its supporting alveolar bone for various occlusal loads using an advanced digital photoelastic technique. A digital image processing system coupled with a circular polariscope was used for the stress analysis. The phase shift technique and a phase unwrapping algorithm was utilized for fringe processing. This aids in obtaining qualitative and quantitative information on the nature of stress distribution within the dento-osseous structures. The experiments revealed bending stresses within dento-osseous structures. However, the compressive stress magnitude was larger than the tensile stress. Zero stress regions were also identified within the dento-osseous structures. The results suggest that the geometry of the dento-osseous structures and the structural gradients at the tooth–bone interface play a significant role in the distribution of stresses without stress concentrations. Further, the application of an advanced image-processing system with the circular polariscope showed notable advantages and could be applied in other biomechanical investigations.

Pulsed CO2 lasers have been shown to be effective for both removal and modification of dental hard tissue for the treatment of dental caries. In this study, sealed transverse excited atmospheric pressure (TEA) laser systems optimally tuned to the highly absorbed 9.6 µm wavelength were investigated for application on dental hard tissue. Conventional TEA lasers produce an initial high energy spike at the beginning of the laser pulse of submicrosecond duration followed by a long tail of about 1–4 µs. The pulse duration is well matched to the 1–2 µs thermal relaxation time of the deposited laser energy at 9.6 mm and effectively heats the enamel to the temperatures required for surface modification at absorbed fluences of less than 0.5 J/cm2. Thus, the heat deposition in the tooth and the corresponding risk of pulpal necrosis from excessive heat accumulation is minimized. At higher fluences, the high peak power of the laser pulse rapidly initiates a plasma that markedly reduces the ablation rate and efficiency, severely limiting applicability for hard tissue ablation. By lengthening the laser pulse to reduce the energy distributed in the initial high energy spike, the plasma threshold can be raised sufficiently to increase the ablation rate by an order of magnitude. This results in a practical and efficient CO2 laser system for caries ablation and surface modification

This study was performed with the objective of evaluating osseointegration of titanium alloy Ti6Al4V dental implants coated with hydroxylapatite (HA) deposited by a KrF laser. For this a KrF excimer laser and stainless-steel deposition chamber were used. The thickness of the HA films was approximately 1 µm. In this investigation experimental animals minipigs were used; the implants were placed vertically into the lower jaw. After 14 weeks of unloaded osseointegration, metal-ceramic crowns were inserted and, at the same time, fluorescent solution was injected into the experimental animals. Six months after insertion of crowns the animals were sacrificed. The vertical position of the implants was checked by a radiograph. Microscopic sections were cut and ground, and the sections were examined under polarized and fluorescent light using a microscope with a charge coupled device camera. The six month long osseointegration in the lower jaw has confirmed the presence of newly formed bone around all the implants. In the experimental group, which had a laserdeposited coating, the layer of fibrous connective tissue was seen only randomly. In the control group (titanium implant without a cover) the fibrous connective tissue between the implant and the newly formed bone was observed more frequently, but this difference was not significant.

A longer operating time and steeper learning curve in mastering the techniques for transurethral laser resection of the prostate are the main problems faced by surgeons in addition to the existing ones in standard transurethral resection of the prostate (TURP). However, these disadvantages can be alleviated with the introduction of a treatment procedure designed and developed based on an integrated system of computer, robotics and laser technology. In vitro experiments were carried out to determine variables affecting the vaporization and coagulation lesions, in order to study the effectiveness and feasibility of robotics for this procedure. Human cadaveric prostates and fresh tauted chicken breast tissues were irradiated with different parameters using the LaserTrode lightguide in contact with the tissue. The effects of irrigant flow rate, fiber/tissue angle of inclination, number of passes, direction, speed and power of lase on the volume of tissue vaporized and coagulated, were assessed. The final phase of the experiments includes executing the robotic motion plan for the laser resection procedure on the human cadaveric prostate tissue embedded in an anatomically alike prostate phantom. It was concluded from our study that power and speed of lase are the most significant parameters influencing the volume of the vaporized and coagulated lesion. Comparison of removal rate using the new treatment procedure of robotic laser resection of the prostate with TURP and HoLRP evinced equivalent results.

Plasmid DNA is efficiently photocleaved by sodium pheophorbides (Na–Phdes) a and b in the absence of oxygen as well as in the presence of oxygen. Fluorescence microscopic observation shows a rapid incorporation of Na–Phde a into nuclei, mitochondria, and lysosome of human oral mucosa cells. In contrast Na–Phde b is incorporated only into the plasma membrane. The photodynamic activity of these pigments in living tissues is probably determined by the monomeric pigment molecules formed in hydrophobic cellular structures and involves two types of reactions: (i) direct electron transfer between DNA bases (especially guanine) and pheophorbide singlet excited state, and (ii) indirect reactions mediated by reactive oxygen species, including singlet oxygen whose production from molecular oxygen is sensitized by the Na–Phdes triplet state. A preliminary report has appeared in ‘‘Photodynamic Therapy of Cancer II,’’ Proc. SPIE 2325, 416–424 (1994).