Sol Kimel

Wavelength dependence of cell cloning efficiency after optical trapping.

A study on clonal growth in Chinese hamster ovary (CHO) cells was conducted after exposure to optical trapping wavelengths using Nd:YAG (1064 nm) and tunable titanium-sapphire (700-990 nm) laser microbeam optical traps. The nuclei of cells were exposed to optical trapping forces at various wavelengths, power densities, and durations of exposure. Clonal growth generally decreased as the power density and the duration of laser exposure increased. A wavelength dependence of clonal growth was observed, with maximum clonability at 950-990 nm and least clonability at 740-760 nm and 900 nm. Moreover, the most commonly used trapping wavelength, 1064 nm from the Nd:YAG laser, strongly reduced clonability, depending upon the power density and exposure time. The present study demonstrates that a variety of optical parameters must be considered when applying optical traps to the study of biological problems, especially when survival and viability are important factors. The ability of the optical trap to alter either the structure or biochemistry of the process being probed with the trapping beam must be seriously considered when interpreting experimental results.

Selective cooling of biological tissues: application for thermally mediated therapeutic procedures

The ability to control the degree and spatial distribution of cooling in biological tissues during a thermally mediated therapeutic procedure would be useful for several biomedical applications of lasers. We present a theory based on the solution of the heat conduction equation that demonstrates the feasibility of selectively cooling biological tissues. Model predictions are compared with infrared thermal measurements of in vivo human skin in response to cooling by a cryogen spurt. The presence of a boundary layer, undergoing a liquid-vapour phase transition, is associated with a relatively large thermal convection coefficient (approximately 40 kW m-2 K-1), which gives rise to the observed surface temperature reductions (30-40 degrees C). The degree and the spatial-temporal distribution of cooling are shown to be directly related to the cryogen spurt duration.

In vivo TUMOR OXYGEN TENSION MEASUREMENTS FOR THE EVALUATION OF THE EFFICIENCY OF PHOTODYNAMIC THERAPY

Among the sequence of events which occur during photodynamic therapy (PDT) are depletion of oxygen and disruption of tumor blood flow. In order to more clearly understand these phenomena we have utilized transcutaneous oxygen electrodes to monitor tissue oxygen disappearance. These results provide, for the first time, non‐invasive real‐time information regarding the influence of light dose on tissue oxygenation during irradiation. Measurements were conducted on transplanted VX‐2 skin carcinomas grown in the ears of New Zealand white rabbits. Rabbits were treated with Photofrin II and tumors were irradiated with up to 200 kj/m2 (500 W/m2) of 630‐nm light. Substantial reductions in tumor oxygen tension were observed upon administration of as little as 20 kJ/m2. For a series of brief irradiations, oxygen tension was modulated by the appearance of laser light. Tissue oxygen reversibility appeared to be dependent upon PDT dose. Long‐term, irreversible tissue hypoxia was recorded in tumors for large (200 kJ/m2) fluences. These results suggest that transcutaneous oxygen tension may be useful as a general indicator of the effectiveness of PDT and as an in situ predictor of the energy required to elicit tumor damage.

A theoretical study of the thermal response of skin to cryogen spray cooling and pulsed laser irradiation: implications for treatment of port wine stain birthmarks.

The successful treatment of port wine stain (PWS) patients undergoing laser therapy is based on selective thermal coagulation of blood vessels without damaging the normal overlying epidermis. Cryogen spray cooling of skin may offer an effective method for minimizing epidermal thermal injury. Inasmuch as the density of melanosomes and depth of PWS blood vessels can vary considerably, an optimum cooling strategy is required on an individual patient basis. We present a theoretical study of the thermal response of various pigmented PWS lesions to spray cooling in conjunction with flashlamp-pumped pulsed dye laser irradiation (585 nm). Results of our model indicate that precooling of skin using tetrafluoroethane as the cryogen spray is sufficient to eliminate epidermal thermal injury when using incident fluences less than 10 J cm-2 and 8 J cm-2 on patients with intermediate and high epidermal melanin content, respectively. Cryogens that have lower boiling points than tetrafluoroethane may allow successful treatment when using fluences equal to or greater than those indicated.

SINGLET OXYGEN GENERATION OF PORPHYRINS, CHLORINS, AND PHTHALOCYANINES

The production of singlet oxygen was measured indirectly for three classes of photosensitizers: porphyrins (Photofrin II, TPPS4), chlorins (MACE, DACE), and a phthalocyanine (CASPc). Buffered solutions of sensitizers and singlet oxygen acceptors were irradiated with a CW dye laser and the oxygen depletion was monitored electrochemically with a Clark‐type microelectrode. A comparison of oxygen‐depletion rate constants and quantum efficiencies yields the order of efficiency of the sensitizers: TPPS4 > MACE > PII > DACE > CASPc. For singlet oxygen acceptors the order was: furfuryl alcohol > imidazole > tryptophan. CHO cell suspensions were also used as acceptors. Here the order of efficiency (per absorbed photon) was PII > MACE = CASPc. Expressed in terms of oxygen depletion per cell the order was CASPc = PII > MACE. When performing cell clonogenicity studies the order of efficiencies, expressed as percentage cell kill per unit weight of sensitizer, was CASPc > PII > MACE = DACE. The discrepancy between the efficiencies of sensitizers to generate singlet oxygen and their cytotoxicity was explained in terms of photodegradation (for the chlorins), intracellular localization (for PII), and contributions from a Type I mechanism (for CASPc).

Photodynamic parameters in the chick chorioallantoic membrane (CAM) bioassay for topically applied photosensitizers

The relative efficacy of Photofrin-based photodynamic therapy (PDT) has been compared with that of the second-generation photosensitizers 5-aminolevulinic acid (ALA), sulfonated chloro-aluminum phthalocyanine (AlPcSn), benzoporphyrin derivative monoacid ring A (BPD-MA), and lutetium texaphyrin (Lutex). PDT-induced vascular damage in the chick chorioallantoic membrane (CAM) is measured following topical application of the photosensitizers. In order to make meaningful comparisons, care is taken to keep treatment variables the same. These include light dose (5 and 10 J/cm2), power density (33 and 100 mW/cm2), and drug uptake time (30 and 90 min). The drug dose ranges from 0.1 microgram/cm2 for BPD to 5000 micrograms/cm2 for ALA. Results are also analyzed statistically according to CAM vessel type (arterioles versus venules), vessel diameter, and vessel development (embryonic age). For each photosensitizer, the order of importance for the various PDT parameters is found to be unique. The differences between the sensitizers are most likely due to variation in biophysical and biochemical characteristics, biodistribution, and uptake kinetics.

An, Acute Light and Electron Microscopic Study of Ultraviolet 193-nm Excimer Laser Corneal Incisions

The 193-nm ultraviolet beam from an argon fluoride excimer laser was focused on the corneas of rabbits to produce incisions of the type necessary for radial keratotomy. The energy densities used were in two ranges, 1.0 to 2.1 J/cm2 per pulse and 200 to 700 mJ/cm2 per pulse. The eyes were enucleated and fixed for histologic and electron microscopic examination immediately after exposure. Structural analysis of the higher energy density exposures showed ridging on the surface of the cornea, micro-pitting on the stromal surface inside the cut, and denudation of the endothelium under the ablation zone. The lower energy density incisions did not exhibit significant surface ridging or endothelial cell loss but did exhibit significant stromal swelling during the laser exposure thus making it difficult to produce incisions of a precisely controlled depth. Beam profile measurements and infrared thermal measurements of the cornea surface during laser exposure were made.

Systemic application of photosensitizers in the chick chorioallantoic membrane (CAM) model: Photodynamic response of CAM vessels and 5-aminolevulinic acid uptake kinetics by transplantable tumors

The aim of this study is to modify the chick chorioallantoic membrane (CAM) model into a whole-animal tumor model for photodynamic therapy (PDT). By using intraperitoneal (i.p.) photosensitizer injection of the chick embryo, use of the CAM for PDT has been extended to include systemic delivery as well as topical application of photosensitizers. The model has been tested for its capability to mimic an animal tumor model and to serve for PDT studies by measuring drug fluorescence and PDT-induced effects. Three second-generation photosensitizers have been tested for their ability to produce photodynamic response in the chick embryo/CAM system when delivered by i.p. injection: 5-aminolevulinic acid (ALA), benzoporphyrin derivative monoacid ring A (BPD-MA), and Lutetium-texaphyrin (Lu-Tex). Exposure of the CAM vasculature to the appropriate laser light results in light-dose-dependent vascular damage with all three compounds. Localization of ALA following i.p. injections in embryos, whose CAMs have been implanted with rat ovarian cancer cells to produce nodules, is determined in real time by fluorescence of the photoactive metabolite protoporphyrin IX (PpIX). Dose-dependent fluorescence in the normal CAM vasculature and the tumor implants confirms the uptake of ALA from the peritoneum, systemic circulation of the drug, and its conversion to PpIX.

Tumor Oxygen Tension During Photodynamic Therapy

Journal ofPhotochemistry and Photobiology, B: Biology, 5 (1990) News and Views Tumor Oxygen Tension During Photodynamic Therapy B. J. TROMBERG, S. KIMEL, A. ORENSTEIN, S. J. BARKER, J. S. NELSON, W. G. ROBERTS and M. W. BERNS Beckman Laser Institute and Medical 92715 (U.S.A.) Clinic, University J. HYATT, of California at Irvine, Iruine, CA 1. Introduction The administration of a sufficient dose of radiation at the appropriate wavelength to photosensitizer-containing neoplasms is essential for photo- dynamic therapy (PDT). Accordingly, monitoring the effects of light delivery in tissue during PDT has great clinical significance. In order to understand PDT on the molecular level, one or more “participants” (e.g. reaction products or intermediates) in the photodynamic reactions have to be measured. We will concentrate on the role of oxygen as a participant since PDT has been shown to depend critically, although in a rather complicated way, on the presence of oxygen [l]. Three consecutive processes can be distinguished. In the first phase, PDT involves the photogeneration of cytotoxic oxygen intermediates, primarily singlet molecular oxygen IO,, at the expense of ground state molecular oxygen O,, which by photochemical processes cause damage to essential cellular components. In this “oxidative” phase, the presence of oxygen is crucial for the initiation of PDT. The photo-oxidation reactions cause a dynamic, reversible depletion of ambient oxygen which is propor- tional to the fluence rate. In the second phase, oxygen-induced pathophysiological alterations lead to occlusion of blood vessels and hypoxia [2 - 41. During this stage, measurements of oxygen reflect a depletion of tissue oxygen because of the decreased supply. For low light doses the “hypoxic” phase is reversible, since in the absence of light the blood flow is restored. In the third phase of in uiuo PDT, regional breakdown of oxygen and nutrient delivery occurs due to vascular collapse which causes tumor necrosis [4]. In this “ischemic” stage the depletion of oxygen levels is, of course, irreversible. Even if the exact role of oxygen in each phase is still being debated, it is clear that the progress of PDT can be followed by monitoring tissue oxygen depletion. Most in viuo studies have dealt in a descriptive fashion with photo- dynamic reactions in the third phase. These efforts have used histological techniques [ 5 - 71. observation chambers [4] or blood flow measurements loll-1344/90/

Effects of mass flow rate and droplet velocity on surface heat flux during cryogen spray cooling

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