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.

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.

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.

The difference between initial arteriole and venule damage could be explained by the threefold higher absorption coefficient at 595 nm in (oxygen-poor!) arterioles. In human patients, PWS consist of ectatic venules (characterized by higher absorption), so that these considerations favor the use of 595-nm irradiation for laser photothermolysis. For optimal treatment of PWS it is proposed that t(p) be between 0.1 and 1.5 milliseconds. This is based on a modified relaxation time tau'(d), defined as the time required for heat conduction into the full thickness of the vessel wall, which is assumed to have a thickness DeltaD approximately 0.1D. The corresponding tau'(d) will be a factor of about six smaller than given in the literature. For vessels with D between 30 and 300 mum, tau'(d) ranges from 0.1 to 1.5 milliseconds.

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The antibacterial photodynamic effects of uncharged (o-tetrahydroxyphenyl porphine [THPP], m-THPP and p-THPP), cationic (5,10,15,20-tetra[4-N-methylpyridyl]porphine [TMPyP]) and anionic (5,10,15,20-tetra[4-sulfonatophenyl porphine] [TPPS4]) porphines on Staphylococcus aureus and Escherichia coli bacteria inactivation were examined. The results show that uncharged porphines provoked antibacterial photodynamic activity on S. aureus, and also on E. coli in the presence of the membrane-disorganizing peptide polymixin B nonapeptide (PMNP). The TMPyP compound was highly photoactive toward gram-positive bacteria but only marginally effective on gram-negative cells, whereas TPPS4 showed no activity on either gram-positive or gram-negative bacteria. The photoactivity of TMPyP is due to the electrostatic attraction between the positively charged sensitizer molecule and the negatively charged membrane of the gram-positive target cells. For TPPS4, the inactivity toward gram-positive bacteria is due to electrostatic repulsion between the charged sensitizer molecule and the cell membrane. For gram-negative bacteria, the inactivity is conceivably due to preferential (electrostatic) binding to the positively charged PMNP, which is an adjuvant for membrane disorganization, but has no effect on cell viability. For hydrophobic sensitizers, the photoactivity depends on the state of aggregation. The extent of deaggregation of the different THPP isomers was determined by fluorescence measurements of bound sensitizers and could be positively correlated with their photoinactivation capacity. We conclude that the structure-activity relationships of these porphines are affected by their net charge and by aggregation.

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.

Abstract-The production of singlet oxygen was measured indirectly for three classes of photosensitizers: porphyrins (Photofrin 11, TPPS,), 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: TPPS, > MACE > PI1 > 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 PI1 > MACE i = CASPc. Expressed in terms of oxygen depletion per cell the order was CASPc = PI1 > MACE. When performing cell clonogenicity studies the order of efficiencies, expressed as percentage cell kill per unit weight of sensitizer, was CASPc > PI1 > 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), intraccllular localization (for PII), and contributions from a Type I mechanism (for CASPc).