Michael W. Berns

Epidermal heating during laser-induced photothermolysis of port wine stains: modeling melanosomal heating after dynamic cooling of the skin surface

The clinical objective in treatment of port wine stains is to maximize thermal damage to the abnormal blood vessels without introducing thermal damage to the normal overlying epidermis. The rationale of dynamic cooling is to protect the epidermis from thermal damage by selectively cooling this layer down immediately before delivering the laser pulse. This work discusses the thermal dynamics of epidermal cooling by milliseconds cryogen spurts and melanosomal heating by the laser pulse.

Laser-assisted versus mechanical recanalization of femoral arterial occlusions

A randomized clinical trial was performed to test the hypothesis that a laser-heated probe is superior to standard techniques to reopen occluded femoral arteries. Twenty patients were treated with a standard guidewire and balloon dilation method. In a second group of 20 patients, the laser probe was initially used as a nonheated mechanical device. If the probe was unsuccessful in mechanically reopening the artery, an Argon laser was activated to heat the probe. The mean length of occlusion was 15.9 +/- 10.3 cm. The success rate for the laser probe was 15 of 20 (75%), which was not significantly different from the standard method, 19 of 20 (95%). Most of the success in the laser-probe group was due to the probes mechanical properties. The laser probe was successful as a cold, mechanical device in 13 of 15 (87%) arteries. It was necessary to heat the probe in 5 patients. When heated, the laser probe assisted recanalization in 2 but perforated the artery in 3 cases. The results of this randomized trial do not support the hypotheses behind the use of the thermal laser probe. The laser probe functions primarily as a mechanical device. The thermal activation does not significantly improve the success rate without increasing the risk of perforation. This small additional benefit does not justify the large cost of current thermal laser devices. This controlled study also demonstrates a higher success rate in long occlusions than previous reports of mechanical balloon recanalization. This is due to a combination approach of retrograde and anterograde probing of the occluded segment.

Optical determination of motility forces in human spermatozoa with laser tweezers

Laser tweezers may act as optical force transducers. We report on the determination of intrinsic motility forces of human spermatozoa by employing an 800 nm optical trap. The cellular forces were calculated from calibrated trapping forces. The determination of trapping forces based on a hydrodynamic model for ellipsoidal specimens, the measurement of the minimum laser power required to confine a single cell in the trap, and the calculation of viscus forces during the movement of optically trapped sperm heads through a laminar fluid. A mean motility force of 44 plus or minus 24 pN was calculated for spermatozoa of healthy donors.

Influence of tissue parameters on visual reflectance spectra of port wine stains and normal skin

The visual appearance of port-wine stain lesions is often a red to purple color due to an enlarged blood volume in the upper dermis. The purpose of the treatment is to re-establish normal skin coloration. Visual reflectance spectra should therefore, in principle, contain all relevant information about the lesion. The influence on the spectra from the different tissue parameters, such as melanin, blood content and scattering, is rather composite. However, a simple mathematical model can give a good understanding of the relevance of the different components. This knowledge can be used to optimize the laser treatment of port-wine stain. In vivo reflectance spectra were obtained using an integrating sphere spectrophotometer. A simple mathematical model based on the diffusion approximation was used to simulate port- wine stain and normal skin reflectance spectra. The absorption coefficients of epidermis and dermis are mainly due to melanin and blood. These parameters were measured in separate in vivo experiments and obtained from skin biopsies. The scattering coefficients were based on reported values. Simulated reflectance spectra show good agreement with the measured ones. Even though the diffusion model has limited validity for wavelengths shorter than 600 nm, the simulated spectra from 450 to 600 nm give a qualitative understanding of the influence of the tissue parameters. The results show that dark red to almost dark grey port-wine stains contain enlarged blood fraction in the entire upper dermis. The red port-wine stains appear when the abnormal density of blood is confined to a thin layer. High amount of epidermal melanin results in reduced reflectance throughout the visible spectrum. The characteristic spectrum due to the blood is suppressed. The reflectance spectra are strongly dependent on the dermal and epidermal scattering coefficient; even minor changes as naturally occurring with age, might have a significant impact. A permanently reduced scattering coefficient might result in a reddish coloration even when a normal blood content is restored. Correlation between measured and simulated spectra give a good understanding of the importance of the parameters determining the visual appearance of port-wine stain.

Fluorescence imaging and spectroscopy of motile sperm cells and CHO cells in an optical trap (laser tweezers)

We describe fluorescence spectroscopy and imaging studies of optically trapped single Chinese hamster ovary (CHO) and motile human sperm cells. The NIR trapping beam was provided by a tunable, multimode continuous wave Ti:Sapphire laser. The beam was introduced into an inverted confocal laser scanning microscope. Fluorescence of cells in the single- beam gradient force optical trap was excited with a 488 nm microbeam (laser scanning microscopy) or with 365 nm radiation from a high- pressure mercury lamp. Modifications to NADH-attributed autofluorescence and Rhodamine- and Propidium Iodide-attributed xenofluorescence indicate a significant cell-damaging effect of 760 nm trapping beams. 760 nm effects produce a biological response comparable to UVA-induced oxidative stress and appear to be a consequence to two-photon absorption.

Differential cell photosensitivity in photodynamic therapy of the rat endometrium

The purpose of this study was to determine the optical dose needed for both lasting endometrial destruction and prevention of implantation by photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) as a photosensitizer. Three hours after topical drug administration 74 female Sprague-Dawley received varying optical doses of 630 nm light delivered by an intrauterine cylindrical light diffusing fiber. Histologic evaluation of the endometrium 1 and 21 days after PDT as well as the number of implantation sacs after mating were assessed. Irreversible endometrial destruction was determined measuring the thickness of the endometrial layer 3 weeks after treatment. An in situ dose of 64 J/cm2 was required to eradicate endometrial glands and prevent regeneration. In contrast, a 43 J/cm2 in situ dose visibly damaged the endometrial stroma and myometrium but the endometrial glands survived and the endometrium regenerated to its full thickness within 21 days. However, implantation potential was significantly reduced at these low light levels. Due to differential cell photosensitivity, the optical threshold for lasting endometrial destruction is higher than for functional damage. For lasting endometrial destruction the endometrial glands must be destroyed, whereas for reproductive impairment, damage to the endometrial stroma seems to be sufficient.

Laser trapping microscopy as a diagnostic technique for the study of cellular resopnse and laser-cell interactions

Optical laser trapping microscopy has emerged as a powerful tool not only for the optical manipulation of cells and macromolecules, but also for the study of cellular physiological responses via force transduction and fluorescence imaging. We describe here the most recent results from our laboratory in the use and application of laser trapping microscopy to a variety of studies at the cellular and molecular levels. Fluorescence spectroscopy and imaging have been successfully combined with optical micromanipulation. A single near-infrared laser beam is used for two-photon fluorescence excitation and micromanipulation of trapped biological specimens. Cell viability is observed and monitored with a Nd:YAG laser ((lambda) equals 1064 nm) and an Al:GaAs diode laser ((lambda) equals 809 nm). Traps and conventional fluorescence imaging are also used simultaneously to examine T-cell activation dynamics.

Micromanipulation and physiological monitoring of cells using two-photon excited fluorescence in cw laser tweezers

We report the observation of two-photon fluorescence excitation and cell confinement, simultaneously, in a continuous-wave (cw) single-beam gradient force optical trap, and demonstrate its use as an in-situ probe to study the physiological state of an optically confined cell sample. At the wavelength of 1064 nm, a single focused gaussian laser beam is used to simultaneously confine, and excite visible fluorescence from, a human sperm cell that has been tagged with propidium iodide, a exogenous fluorescent dye that functions as a viability assay of cellular physiological state. The intensity at the dye peak emission wavelength of 620 nm exhibits a near-square-law dependence on incident trapping beam photon laser power, a behavior consistent with a two-photon absorption process. In addition, for a sperm cell held stationary in the optical tweezers for a period of several minutes at a constant trapping power, red fluorescence emission was observed to increase the time, indicating that the cell has gradually transitioned between a live and dead state. Two-photon excited fluorescence was also observed in Chinese hamster ovary cells that were confined by cw laser tweezers and stained with either propidium iodide or Snarf, a pH-sensitive dye probe. These results suggest that, for samples suitably tagged with fluorescent probes and vital stains, optical tweezers can be used to generate their own in-situ diagnostic optical probes of cellular viability or induced photodamage, via two-photon processes.

How safe is gamete micromanipulation by laser tweezers

Laser tweezers, used as novel sterile micromanipulation tools of living cells, are employed in laser-assisted in vitro fertilization (IVF). For example, controlled spermatozoa transport with 1064 nm tweezers to human egg cells has been performed in European clinics in cases of male infertility. The interaction of approximately 100 mW near infrared (NIR) trapping beams at MW/cm2 intensity with human gametes results in low mean less than 2 K temperature increases and less than 100 pN trapping forces. Therefore, photothermal or photomechanical induced destructive effects appear unlikely. However, the high photon flux densities may induce simultaneous absorption of two NIR photons resulting in nonlinear interactions. These nonlinear interactions imply non-resonant two-photon excitation of endogenous cellular chromophores. In the case of less than 800 nm tweezers, UV- like damage effects may occur. The destructive effect is amplified when multimode cw lasers are used as tweezer sources due to longitudinal mode-beating effects and partial mode- locking. Spermatozoa damage within seconds using 760 nm traps due to formation of unstable ps pulses in a cw Ti:Sa ring laser is demonstrated. We recommend the use of greater than or equal to 800 nm traps for optical gamete micromanipulation. To our opinion, further basic studies on the influence of nonlinear effects of laser tweezers on human gamete are necessary.

Effect of photodynamic therapy in intimal hyperplasia by phthalocyanine conjugated to the scavenger-receptor ligand, maleylated bovine serum albumin

Initial hyperplasia is major cause of restenosis after vascular interventions for arterial occlusive disease. We reported that a fluorescent probe, Texas Red, conjugated to the scavenger-receptor ligand, maleylated bovine serum albumin accumulated almost exclusively in the injured, hyperplastic sites. The purpose of this study is to test the feasibility of enhanced drug delivery to the hyperplastic lesion by targeting the scavenger-receptors.