Dear Colleagues, This is an exciting time for biomedical optics. We are in the midst of an extraordinary knowledge expansion in biology which, in turn, demands technologies that provide better, faster, more quantitative measurements on ever more challenging systems.

Photodynamic therapy (PDT) was used to treat endometrial G1 cancer tissue derived from patients who had undergone a total hysterectomy and bilateral salpingo-oophorectomy. After surgical treatment the cancerous tissue was kept in a medium containing Dulbecco solution, fetal calf serum, and antibiotics. The tissue was then exposed to hematoporphyrin derivative (0.1 mg/l ) and 24 h later exposed to light (total light dose—18 J/sq cm). Necrosis depth was evaluated 24 h later using a light microscope. In order to assess the possible role of the basal membrane component laminin, as well as epidermal growth factor receptor susceptibility to PDT, immunohistochemical studies were carried out. Additionally, nucleolar organizer regions evaluation was performed. Our experiment confirmed that PDT results in the necrosis in the treated endometrial cancer, while not affecting the laminin in the cancerous tissue. In contrast, PDT strongly affects the epidermal growth factor receptor and nucleolar organizer regions in cancer cells. We suggest that laminin may contribute to the prevention of cancer dissemination in the cases where PDT has to be repeated, and that after PDT the cells become less susceptible to a mitogen, like, e.g., epidermal growth factor.

In the present paper information about the synthesis and results on the pharmacokinetic and experimental photodynamic therapy (PDT) of naphthalocyanines are given. The photodynamic activity of differently substituted zinc(II)- and silicon(IV)-naphthalocyanines using liposomes or Cremophor EL as drug-delivery systems is shown on different tumor models. For the evaluation of the phototherapeutic effect different assessment criteria were used, including light and electron microscopy observations. The main conclusions which can be arrived at on the basis of our findings are the following: silicon(IV)-naphthalocyanine seems to be not a very effective tumor sensitizer, especially in the treatment of pigmented melanoma, while zinc(II)- naphthalocyanines appear to be very promising for PDT of tumors. Their selective targeting and slow clearance from tumor tissue, fast clearance from skin and pronounced phototherapeutic effect on different tumor models and especially at melanotic tumors, even after application of low drug doses, make this group of photosensitizers very attractive for successful PDT of cancer.

5-aminolevulinic acid (5-ALA) is an interesting photosensitizing substance for photodynamic therapy (PDT), successfully applied topically for urological malignancy. In gastroenetrology it has proven efficacy for treatment of some GI neoplasms after systemic administration. This study was aimed at investigating the possibility of topical 5-ALA administration also for the PDT of gut cancer in a mice model. 5-ALA solution at different concentrations (5%, 1.5%, and 0.5%) was instilled in the colon of mice, which was later removed and examined by fluorescence microscopy. The results of fluorescence studies were compared with those obtained in a control group treated with 5-ALA given systemically. Satisfactory epithelial fluorescence levels and good selectivity between gut layers was obtained after intracolonic 5-ALA instillation. However, mean fluorescence intensity was higher after systemic drug application. Our results suggest that 5-ALA may probably be used topically for the PDT of some gut neoplasms.

Virus inactivation in red blood cell concentrates (RBCC) is being studied in order to increase the safety of the blood supply. For this purpose we have been studying the silicon phthalocyanine (Pc 4), a photosensitizer activated with red light. Two approaches were used to achieve enhanced selectivity of Pc 4 for virus inactivation. One was formulation of Pc 4 in liposomes that reduce its binding to red cells. The other was the use of a light emitting diode (LED) array emitting at 700 nm. Vesicular stomatitis virus (VSV) infectivity served as an endpoint for virus kill in treated RBCC. Red cell hemolysis and circulatory survival in rabbits served as measures for red cell damage. Treatment of small aliquots of human RBCC with 2 µM Pc 4 in liposomes and 10 J/cm2 of 700 nm LED light in the presence of the quenchers of reactive oxygen species glutathione and trolox resulted in 6 log10 inactivation of VSV. Under these conditions hemolysis of treated red cells stored at 4 °C for 21 days was only slightly above that of control cells. Rabbit RBCC similarly treated circulated with a half life of 7.5 days compared with 10.5 days of control. It is concluded that Pc 4 used as described here may be useful for viral decontamination of RBCC, pending toxicological and clinical studies.

The synthesis and characterization of amphiphilic glycoconjugated porphyrins, benzochlorin, and azaporphyrins were reported. Among these molecules, several were found to be efficient photosensitizers in an in vitro assay using the human tumoral cell line HT29. Moreover, glycosylated benzochlorin and azaporphyrins, whose absorption bands in the red region of the visible spectrum are substantially increased as compared to porphyrins, display a good photocytotoxicity on tumor cells after irradiation with wavelength above 590 nm.

The porphyrins seem to be the most effective photosensitizers in photodynamic therapy of cancer. Monomers and dimers of sulfonated and nonsulfonated porphyrins [5,10,15,20-tetra-phenyl-porphyrin, 5,10,15,20-tetranaphtyl- porphyrin (TNP), 5,10,15,20-tetra-p-sulfonato-phenyl-porphyrin (TS4PP) and 5,10,15,20-tetra-psulfonato- naphtyl-porphyrin], are studied in this paper by means of different spectral methods (UV-vis, fluorescence and polarization fluorescence). The porphyrins as TNP seem to be the most effective photosensitizer especially in a DMSO:water binary mixture of solvent. Monomer–dimer and J-aggregation equilibria and the temperature dependence of TS4PP spectra are also studied in this paper. The incorporation of the forms of these porphyrins into blood cells is studied by means of the cytofluorimetric method.

Photothermal tissue welding has been investigated as an alternative surgical tool to improve bonding of a variety of severed tissues. Yet, after almost two decades of research, inconsistencies in interpretation of experimental reports and, consequently, mechanism of this photothermal process as well as control of dosimetry remain an enigma. Widespread clinical use may greatly depend on full automation of light dosimetry to perform durable and reproducible welds with minimal thermal damage to surrounding and/or underlying tissues. Recognizing photothermal damage as a rate process, radiometrically measured tissue surface temperature has been studied as an indirect marker of tissue status during laser irradiation. Dosimetry control systems and surgical devices were developed to perform controlled temperature tissue welding using surface temperature feedback from the site of laser impact. Nevertheless, end points that mark the completion of a durable and stable weld have not been precisely identified, and subsequently, not incorporated into dosimetry control algorithms. This manuscript reviews thermal dosimetry control systems of the 1990s in an attempt to systematically indicate the difficulties encountered so far and to elaborate on major issues for photothermal tissue welding to become a clinical reality in the new millennium.

Laser damage threshold energies produced from ultrashort (i.e., ?1 ns) laser pulses are investigated as a function of both pulse width and spot size for an artificial retina. A piece of film acts as the absorbing layer and is positioned at the focus of a variant on the Cain artificial eye [C. Cain, G. D. Noojin, D. X. Hammer, R. J. Thomas, and B. A. Rockwell, ‘‘Artificial eye for in vitro experiments of laser light interaction with aqueous media,’’ J. Biomed. Opt. 2, 88–94 (1997)]. Experiments were performed at the focal point and at two and ten Rayleigh ranges (RR) in front of the focus with the damage end point being the presence of a bubble imaged at the film plane. Pulse energy thresholds were determined for wavelengths of 1064, 580, and 532 nm with pulse durations ranging from the nanosecond (ns) to the femtosecond (fs) regime. For the at-focus data in the visible regime, the threshold dropped from 0.25 µJ for a 532 nm, 5 ns pulse to 0.11 µJ for a 580 nm, 100 fs pulse. The near-infrared (NIR) threshold changed from 5.5 µJ for a 5 ns pulse to 0.9 µJ for a 130 fs pulse at a distance two RR in front of the focus. The experiment was repeated using the same pulse widths and wavelengths, except the water path was removed to determine the impact of nonlinear self-focusing in water. A vertical microscope imaging system was employed in order to observe the threshold event. The NIR fluence threshold of 0.5 J/cm2 remained constant within an experimental uncertainty for all pulse widths, which corresponds to values in the literature [C. P. Lin and M. W. Kelly, ‘‘Ultrafast time-resolved imaging of stress transient and cavitation from short pulsed laser irradiated melanin particles,’’ SPIE Laser-Tissue Interactions VI, Proc. SPIE 2391, 294–299 (1995)]. The visible data also demonstrated a nearly constant fluence of 0.07 J/cm2. The disparity in thresholds between the two techniques arises from nonlinear optical phenomena related to propagation differences in the ocular fluid.

Laser induced thermal damage to the retina is investigated. The one step Arrhenius type thermal damage integral of Henriques is analyzed for its strengths and weaknesses. The zero-order activated rate process is shown to well represent the data for pulse durations greater than 10 µs. A zero-order biochemical damage mechanism involving free radical formation and thermal disruption of the melanosome’s protein coat is proposed as the initial molecular process that leads to cellular damage which appears after a delay. Data are presented that show the photoactivation of melanin granule oxidative reactivity. This in vitro data is evidence for an important step in our hypothesized damage pathway.

Side-firing fibers are used to provide coagulative therapy to the urologic tract. These fibers use different optical technologies to deflect the beam transverse to the fibers’ optical axis. This produces emitted beams which differ in both beam direction and divergence angles. The relative optical performance of 13 fibers was studied in an optical phantom suspension. The fluence rate distribution created by each side-firing fiber was determined. The fluence rate distribution accounts for both the direct and spurious beams emitted from side-firing fibers as well as the light scattering produced by the target tissue. Based upon limited clinical dosimetry studies, the relative fluence rate distribution appears to indicate general exposure conditions for the evaluated fibers.

The effects of spectral shape on two photon fluorescence excitation are investigated experimentally using an acousto-optic pulse shaper to modify femtosecond pulses from a Ti:sapphire laser. By using different spectral window shapes, we find that the measured two photon efficiency can vary by a factor of 2 for differently shaped spectra with the same full width at half maximum. We find that these effects are described well by a simple model assuming transform-limited pulses. The fact that even small changes in the spectral wings can significantly affect the efficiency of nonlinear processes has implications for biological multiphoton imaging, where it may be desirable to minimize sample exposure to radiation and maximize fluorescence or harmonic efficiency.

The holographic interferometry (HI) technique commonly used for nondestructive testing of laminate materials was applied to create fringe contour distortion near the site of indwelling breast lesions. For this medical imaging application, the HI technique was successful in demonstrating abnormal mechanical properties of living tissue. Adequate density and contrast of fringes, crucial factors necessary for analysis of surface deformation of an object, can be made only with an appropriate stressing method. We have applied vibration and mild pressure to the surface of female breasts for the purpose of detecting localized densities and mass alterations of the tissue, which may be indicative of an abnormality of that tissue. Even though each stressing method had both positive and negative aspects, pneumatic pressure was adopted for the present study because it was more suitable for a noninvasive and noncontact breast examination. We also developed a computer based holographic imaging system to precisely control the stressing phase for the pressure and laser triggering so the resultant holograms had manageable fringe density and repeatability.

A wavelet tomographic algorithm is presented for statistical reconstruction of a lymphocyte nucleus. The investigation is concerned with lymphocyte nuclei of peripheral blood from two groups of patients: those who live in the area affected by the Chernobyl accident and the control group. The purpose of reconstruction is to find the probability density function of radial distribution of condensed chromatin. The difference between the two test groups is seen from considering the results of reconstruction and the wavelet energy spectra. The Radon transform is treated here as a singular wavelet transform, which allows us to reconstruct the essential scales from projection and to denoise them simultaneously. The algorithm of reconstruction involves back projecting and continuous wavelet synthesis with denoising. The use of a special local filter insures the stability of reconstruction.