Cryptic lineage differentiation among Indo-Pacific bottlenose dolphins (Tursiops aduncus) in the northwest Indian Ocean.

Phylogeography can provide insight into the potential for speciation and identify geographic regions and evolutionary processes associated with species richness and evolutionary endemism. In the marine environment, highly mobile species sometimes show structured patterns of diversity, but the processes isolating populations and promoting differentiation are often unclear. The Delphinidae (oceanic dolphins) are a striking case in point and, in particular, bottlenose dolphins (Tursiops spp.). Understanding the radiation of species in this genus is likely to provide broader inference about the processes that determine patterns of biogeography and speciation, because both fine-scale structure over a range of kilometers and relative panmixia over an oceanic range are known for Tursiops populations. In our study, novel Tursiops spp. sequences from the northwest Indian Ocean (including mitogenomes and two nuDNA loci) are included in a worldwide Tursiops spp. phylogeographic analysis. We discover a new 'aduncus' type lineage in the Arabian Sea (off India, Pakistan and Oman) that diverged from the Australasian lineage ∼261 Ka. Effective management of coastal dolphins in the region will need to consider this new lineage as an evolutionarily significant unit. We propose that the establishment of this lineage could have been in response to climate change during the Pleistocene and show data supporting hypotheses for multiple divergence events, including vicariance across the Indo-Pacific barrier and in the northwest Indian Ocean. These data provide valuable transferable inference on the potential mechanisms for population and species differentiation across this geographic range.

Ancient and modern DNA track temporal and spatial population dynamics in the European fallow deer since the Eemian interglacial.

Anthropogenic factors have impacted the diversity and evolutionary trajectory of various species. This can be through factors such as pressure on population size or range, habitat fragmentation, or extensive manipulation and translocation. Here we use time-calibrated data to better understand the pattern and processes of evolution in the heavily manipulated European fallow deer (Dama dama). During the Pleistocene, fallow deer had a broad distribution across Europe and were found as far north as Britain during the Eemian interglacial. The last glacial period saw fallow deer retreat to southern refugia and they did not disperse north afterwards. Their recolonisation was mediated by people and, from northern Europe and the British Isles, fallow deer were transported around the world. We use ancient and modern mitochondrial DNA (mtDNA) and mitogenomic data from Eemian Britain to assess the pattern of change in distribution and lineage structure across Europe over time. We find founder effects and mixed lineages in the northern populations, and stability over time for populations in southern Europe. The Eemian sample was most similar to a lineage currently in Italy, suggesting an early establishment of the relevant refuge. We consider the implications for the integration of anthropogenic and natural processes towards a better understanding of the evolution of fallow deer in Europe.

Population divergence and gene flow in an endangered and highly mobile seabird.

Seabirds are highly vagile and can disperse up to thousands of kilometers, making it difficult to identify the factors that promote isolation between populations. The endemic Hawaiian petrel (Pterodroma sandwichensis) is one such species. Today it is endangered, and known to breed only on the islands of Hawaii, Maui, Lanai and Kauai. Historical records indicate that a large population formerly bred on Molokai as well, but this population has recently been extirpated. Given the great dispersal potential of these petrels, it remains unclear if populations are genetically distinct and which factors may contribute to isolation between them. We sampled petrels from across their range, including individuals from the presumably extirpated Molokai population. We sequenced 524 bp of mitochondrial DNA, 741 bp from three nuclear introns, and genotyped 18 microsatellite loci in order to examine the patterns of divergence in this species and to investigate the potential underlying mechanisms. Both mitochondrial and nuclear data sets indicated significant genetic differentiation among all modern populations, but no differentiation was found between historic samples from Molokai and modern birds from Lanai. Population-specific nonbreeding distribution and strong natal philopatry may reduce gene flow between populations. However, the lack of population structure between extirpated Molokai birds and modern birds on Lanai indicates that there was substantial gene flow between these populations and that petrels may be able to overcome barriers to dispersal prior to complete extirpation. Hawaiian petrel populations could be considered distinct management units, however, the dwindling population on Hawaii may require translocation to prevent extirpation in the near future.

The effect of temperature on the autofluorescence of scattering and non-scattering tissue.

BACKGROUND AND OBJECTIVES: With the increasing use of fluorescence in medical applications, a comprehensive understanding of the effect of temperature on tissue autofluorescence is essential. The purpose of this study is to explore the effect of temperature on the fluorescence of porcine cornea and rat skin and determine the relative contributions of irreversible changes in optical properties and in fluorescence yield. STUDY DESIGN/MATERIALS AND METHODS: Fluorescence, diffuse reflectance, and temperature measurements were acquired from excised porcine cornea and rat skin over a temperature range of 0-80 °C. A dual excitation system was used with a 337 nm pulsed nitrogen laser for the fluorescence and a white light source for the diffuse reflectance measurements. A thermal camera measured tissue temperature. Optical property changes were inferred from diffuse reflectance measurements. The reversibility of the change in fluorescence was examined by acquiring measurements while the tissue sample cooled from the highest induced temperature to room temperature. RESULTS: The fluorescence intensity decreased with increasing tissue temperature. This fluorescence change was reversible when the tissue was heated to a temperature of 45 °C, but irreversible when heated to a temperature of 80 °C. CONCLUSION: Auto-fluorescence intensity dependence on temperature appears to be a combination of temperature-induced optical property changes and reduced fluorescence quantum yield due to changes in collagen structure. Temperature-induced changes in measured fluorescence must be taken into consideration in applications where fluorescence is used to diagnose disease or guide therapy.

Investigations on laser hard tissue ablation under various environments.

The purpose of this study was to investigate the effect of liquid environments upon laser bone ablation. A long-pulsed Er,Cr:YSGG laser was employed to ablate bovine bone tibia at various radiant exposures under dry, wet (using water or perfluorocarbon) and spray environmental conditions. Energy loss by the application of liquid during laser irradiation was evaluated, and ablation performance for all conditions was quantitatively measured by optical coherence tomography (OCT). Microscope images were also used to estimate thermal side effects in tissue after multiple-pulse ablation. Wet using water and spray conditions equally attenuated the 2.79 microm wavelength laser beam. Higher transmission efficiency was obtained utilizing a layer of perfluorocarbon. Dry ablation exhibited severe carbonization due to excessive heat accumulation. Wet condition using water resulted in similar ablation volume to the dry case without carbonization. The perfluorocarbon layer produced the largest ablation volume but some carbonization due to the poor thermal conductivity. Spray induced clean cutting with slightly reduced efficiency. Liquid-assisted ablation provided significant beneficial effects such as augmented material removal and cooling/cleaning effects during laser osteotomy.

Hard tissue ablation with a spray-assisted mid-IR laser.

The objective of this study was to understand the dominant mechanism(s) for dental enamel ablation with the application of water spray. A free-running Er,Cr:YSGG (yttrium, scandium, gallium, garnet) laser was used to ablate human enamel tissue at various radiant exposures. During dental ablation, distilled water was sprayed on the sample surface, and these results were compared to ablation without a spray (dry ablation). In order to identify dominant ablation mechanisms, transient acoustic waves were compared to ablation thresholds and the volume of material removed. The ablation profile and depth were measured using optical coherence tomography (OCT). Irregular surface modification, charring and peripheral cracks were associated with dry ablation, whereas craters for spray samples were relatively clean without thermal damage. In spite of a 60% higher ablation threshold for spray associated irradiations owing to water absorption, acoustic peak pressures were six times higher and ablation volume was up to a factor of 2 larger compared to dry ablation. The enhanced pressure and ablation performance of the spray-assisted process was the result of rapid water vaporization, material ejection with recoil stress, interstitial water explosion and possibly liquid-jet formation. With water cooling and abrasive/disruptive mechanical effects, the spray ablation can be a safe and efficient modality for dental treatment.

Using sandpaper for noninvasive transepidermal optical skin clearing agent delivery.

We present a gentle mechanical method for the noninvasive transepidermal delivery of topically applied optical skin clearing agents. Optical skin clearing reduces light scattering in highly turbid skin with the aid of hyperosmotic chemicals such as glycerol, polyethylene glycol, and solutions of dextrose. Transepidermal delivery of such agents is believed to be most patient compliant and most likely to be used in a clinical environment. Optical skin clearing has the potential to expand the current limited use of laser light in medicine for diagnostic and therapeutic applications. Light scattering limits the penetration depth of collimated light into skin. In order to increase the diffusion of topically applied optical skin clearing agents into skin, we present a gentle mechanical delivery method involving glycerol and dextrose as optical skin clearing agents and fine 220-grit sandpaper to rub the clearing agent into the tissue. Gentle rubbing causes abrasion of the superficial skin layer including the stratum corneum, which otherwise prevents these optical skin clearing agents from freely diffusing into skin. Results indicate very fast optical skin clearing rates. In vivo hamster skin turned transparent within 2 min. The 1e light penetration depth increased by 36+/-3.75% for dextrose and 43+/-8.24% for glycerol. Optical skin clearing was reversed using phosphate buffered saline solution. Skin viability was observed 70 h post-treatment and showed scabbing and erythema on a few percent of the total optically cleared skin surface.

Porcine skin visible lesion thresholds for near-infrared lasers including modeling at two pulse durations and spot sizes.

With the advent of such systems as the airborne laser and advanced tactical laser, high-energy lasers that use 1315-nm wavelengths in the near-infrared band will soon present a new laser safety challenge to armed forces and civilian populations. Experiments in nonhuman primates using this wavelength have demonstrated a range of ocular injuries, including corneal, lenticular, and retinal lesions as a function of pulse duration. American National Standards Institute (ANSI) laser safety standards have traditionally been based on experimental data, and there is scant data for this wavelength. We are reporting minimum visible lesion (MVL) threshold measurements using a porcine skin model for two different pulse durations and spot sizes for this wavelength. We also compare our measurements to results from our model based on the heat transfer equation and rate process equation, together with actual temperature measurements on the skin surface using a high-speed infrared camera. Our MVL-ED50 thresholds for long pulses (350 micros) at 24-h postexposure are measured to be 99 and 83 J cm(-2) for spot sizes of 0.7 and 1.3 mm diam, respectively. Q-switched laser pulses of 50 ns have a lower threshold of 11 J cm(-2) for a 5-mm-diam top-hat laser pulse.

Dehydration mechanism of optical clearing in tissue.

Previous studies identified various mechanisms of light scattering reduction in tissue induced by chemical agents. Our results suggest that dehydration is an important mechanism of optical clearing in collagenous and cellular tissue. Photographic and optical coherence tomography images indicate that air-immersed skin and tendon specimens become similarly transparent to glycerol-immersed specimens. Transmission electron microscopy images reveal that dehydration causes individual scattering particles such as collagen fibrils and organelles to become more densely packed, but does not significantly alter size. A heuristic particle-interaction model predicts that the scattering particle volume fraction increase can contribute substantially to optical clearing in collagenous and cellular tissue.

Spotsize effects on guinea pig skin following pulsed irradiation.

Laser irradiation parameters such as wavelength, irradiance (W/cm2), and pulse duration have been clearly shown to influence the extent to which tissue is damaged. The careful choice of these parameters can result in confining laser injury to specific targets in tissue. Spotsize, a parameter not commonly appreciated in the application of lasers to medicine and surgery, has been shown, in this study, to contribute to the ultimate outcome of laser effects in tissue. A series of histological events occurring in the skin are demonstrated to be directly related to the effects of spotsize on tissue at a fixed exposure time and wavelength. Many of these changes could contribute to unwanted adverse effects, such as scarring, which occur following certain laser therapies.

Reflectance feedback control of photocoagulation in vivo.

OBJECTIVE: The objective of this research was to build a real-time feedback system that controlled lesion size. Two-dimensional reflectance images were acquired with a charge-coupled device camera during irradiation, and argon laser exposure was ended when parameters of the image reached prespecified values. METHODS: The real-time feedback control system was tested by creating lesions at different power levels in pigmented rabbits. Laser exposure time was controlled by monitoring the central reflectance. RESULTS: Results of feedback-controlled lesions formed in vivo in pigmented rabbits are presented. An ability to produce uniform lesions despite variation in tissue absorption or changes in laser power is demonstrated. CONCLUSIONS: Reflectance control of photocoagulation is possible; incorporation of feedback during photocoagulation has numerous therapeutic and safety benefits over current ballistic delivery.

Laser-tissue interaction during transmyocardial laser revascularization.

BACKGROUND: The clinical procedure known as transmyocardial revascularization has recently seen its renaissance. Despite the promising preliminary clinical results, the associated mechanisms are subject to much discussion. This study is an attempt to unravel the basics of the interaction between 800-W CO2 laser radiation and biological tissue. METHODS: Time-resolved flash photography was used to visualize the laser-induced channel formation in water and in vitro porcine myocardium. In addition, laser-induced pressures were measured. Light microscopy and birefringence microscopy were used to assess the histologic characteristics of laser-induced thermal damage. RESULTS: The channel depth increased logarithmically with time (ie, with pulse duration) in water and porcine myocardium. Pressure measurements showed the occurrence of numerous small transients during the laser pulse, which corresponded with channel formation, as well as local and partial channel collapse during the laser pulse. Twenty millimeters of myocardium was perforated in 25 ms. Increasing the pulse duration had a small effect on the maximum transversable thickness, but histologic analysis showed that thermal damage around the crater increased with increasing pulse duration. CONCLUSIONS: Several basic aspects of the interaction of high-power CO2 laser radiation with myocardial tissue and tissue phantoms were studied in vitro. Although the goal of this study was not to unravel the mechanisms responsible for the beneficial effects of transmyocardial revascularization, it provided important information on the process of channel formation and collapse and tissue damage.

Temperature rise required for production of minimal lesions in the Macaca mulatta retina.

Fundus temperatures of the rhesus monkey were measured with argon laser (488 nm) irradiations that produced minimal, ophthalmoscopically visible lesions five minutes after exposure. Measurements were made with 10- to 20-mu tip diameter, copper-nickel thermocouples. Preliminary data included measurements at eight paramacular sites and seven macular sites. The threshold temperature rise was 17 to 26 degrees C for a ten-second exposure at these sites. Decreasing the exposure duration to 20 msec increased the threshold temperature rise range to the interval between 30 and 40 degrees C. The temperature measurements in the eye were compared to a computer solution of the heat conduction equation.

Surgical adhesives for laser-assisted wound closure.

Solid protein solder-doped polymer membranes were developed for laser-assisted tissue repair. Biodegradable polymer membranes of controlled porosity were fabricated with poly(L-lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) (PEG), and salt particles, using a solvent-casting and particulate-leaching technique. The membranes provided a porous scaffold that readily absorbed the traditional protein solder composed of serum albumin, indocyanine green dye, and de-ionized water. In vitro investigations were conducted to assess the influence of various processing parameters on the strength of tissue repairs formed using the new membranes. These parameters included PLGA copolymer and PLGA/PEG blend ratios, membrane pore size, initial albumin weight fraction, and laser irradiance used to denature the solder. Altering the PLGA copolymer ratio had little effect on repair strength, however such variations are known to influence the degradation rate of the membranes. The repair strength increased with increased membrane pore size and bovine serum albumin concentration. The addition of PEG during the membrane casting stage increased the flexibility of the membranes but not necessarily the repair strength. Typically, the repair strength increased with increasing irradiance from 12 to 18 W/cm(2). The new solder-doped polymer membranes provided all of the benefits associated with solid protein solders including high repair strength and improved edge coaptation. In addition, the flexible, moldable nature of the new membranes offers the capability of tailoring the membranes to a wide range of clinically relevant geometries.

Laser assisted soldering: effects of hydration on solder-tissue adhesion.

Wound stabilization is critical in early wound healing. Other than superficial skin wounds, most tissue repair is exposed to a hydrated environment postoperatively. To simulate the stability of laser-soldered tissue in a wet environment, we studied the effects of hydration on laser soldered rat dermis and baboon articular cartilage. In this in vitro study, we used a solder composed of human serum albumin, sodium hyaluronate, and Indocyanine Green. A 2 µL solder droplet was deposited on each tissue specimen and then the solder was irradiated with a scanning laser beam (808 nm and 27 W/cm2). After photocoagulation, each tissue specimen was cut into two halves dividing the solder. One half was reserved as control while the other half was soaked in saline for a designated period before fixation (1 h, 1, 2, and 7 days). All tissue specimens were prepared for scanning electron microscopy (SEM). SEM examinations revealed nonuniform coagulation across the solder thickness for most of the specimens, likely a result of the temperature gradient generated by laser heating. Closer to the laser beam, the uppermost region of the solder formed a dense coagulum. The solder aggregated into small globules in the region anterior to the solder-tissue interface. All cartilage specimens soaked in saline suffered coagulum detachment from tissue surface. We noted a high concentration of the protein globules in the detached coagulum. These globules were likely responsible for solder detachment from the cartilage surface. Solder adhered better to the dermis than to cartilage. The dermal layer of the skin, composed of collagen matrix, provided a better entrapment of the solder than the smooth surface of articular cartilage. Insufficient laser heating of solder formed protein globules. Unstable solder-tissue fusion was likely a result of these globules being detached from tissue substrate when the specimen was submerged in a hydrated environment. The solder-tissue bonding was compromised as a result of this phenomenon. © 1998 Society of Photo-Optical Instrumentation Engineers.

Initial in vivo results of a hybrid retinal photocoagulation system.

We describe initial in vivo experimental results of a new hybrid digital and analog design for retinal tracking and laser beam control. An overview of the design is given. The results show in vivo tracking rates which exceed the equivalent of 38 degrees/s in the eye. A robotically assisted lesion pattern is created for laser surgery to treat conditions such as diabetic retinopathy and retinal breaks.

Improved vascular tissue fusion using new light-activated surgical adhesive on a canine model.

Newly developed light-activated surgical adhesives have been investigated as a substitute to traditional protein solders for vascular tissue fusion without the need for sutures. Canine femoral arteries (n = 14), femoral veins (n = 14), and carotid arteries (n = 10) were exposed, and a 0.3-0.6 cm longitudinal incision was made in the vessel walls. The surgical adhesive, composed of a poly(L-lactic-co-glycolic acid) scaffold doped with the traditional protein solder mix of bovine serum albumin and indocyanine green dye, was used to close the incisions in conjunction with an 805 nm diode laser. Blood flow was restored to the vessels immediately after the procedure and the incision sites were checked for patency. The new adhesives were flexible enough to be wrapped around the vessels while their solid nature avoided the problems associated with "runaway" of the less viscous liquid protein solders widely used by researchers. Assessment parameters included measurement of the ex vivo intraluminal bursting pressure 1-2 h after surgery, as well as histology. The acute intraluminal bursting pressures were significantly higher in the laser-solder group (>300 mmHg) compared to the suture control group (<150 mmHg) where four evenly spaced sutures were used to repair the vessel (n = 4). Histological analysis showed negligible evidence of collateral thermal damage to the underlying tissue in the laser-solder repair group. These initial results indicated that laser-assisted vascular repair using the new adhesives is safe, easy to perform, and contrary to conventional suturing, provides an immediate leak-free closure. In addition, the flexible and moldable nature of the new adhesives should allow them to be tailored to a wide range of tissue geometries, thus greatly improving the clinical applicability of laser-assisted tissue repair.

Laser balloon angioplasty.

Unlike conventional transluminal percutaneous angioplasty (PTCA), which applies only intraluminal pressure, laser balloon angioplasty (LBA) employs simultaneous heat and pressure to reopen heavily occluded arterial lumens. The circumferential irradiation of Nd:YAG (1.06 microns) laser light is directly absorbed by approximately 1 to 2 mm of arterial tissue immediately adjacent to the inflated balloon. Such heating by LBA is able to seal disrupted luminal flaps, thermally remodel the luminal surface topology, reduce arterial recoil, selectively (partially) dehydrate thrombus, and possibly even reduce thrombogenicity at atherosclerotic sites. Criteria for successful LBA are defined based on earlier fundamental in vitro experiments to determine effective welding temperature, laser power doses, and exposure period; in addition, the derivation and validity of a three-part optical-thermal model and its application in parametric dosimetry analysis are presented. Though the lumen remodeled by LBA is acutely satisfactory, recurrence of the lesion is problematic chronically. Because of this, LBA is currently most useful as an adjunctive procedure whenever PTCA fails to produce optimal results or causes acute vessel closure. Perhaps, another potential application of the LBA system is to aid localized delivery of pharmacologic agents and their thermal adhesion to superficial tissue at angioplastied sites.

Laser fluence for permanent damage of cutaneous blood vessels.

Treatment of vascular disorders may be improved by a more thorough understanding of laser-blood vessel interaction. In this study, the probability of permanent damage to a given type and size of blood vessel was determined as a function of fluence at the top (superficial edge) of the vessel lumen. A 532 nm wavelength, 10 ms pulse duration, 3 mm spot size laser was used to perform approximately 250 irradiations of subdermal blood vessels in the hamster dorsal skin flap preparation. The radiant exposure required for a 50% probability of permanent damage was calculated using a probit analysis of experimental results. Threshold radiant exposure increased with larger blood vessel diameters and was greater for arterioles than venules. Monte Carlo modeling of a typical blood vessel geometry revealed that fluence at the top of the blood vessel lumen was amplified by a factor of approximately 2.4 over tissue surface radiant exposure, due to light scattering in the tissue and internal reflection at the skin-air interfaces.

Laser thermal ablation.

Continuous wave and pulsed laser ablation of tissue is described as an explosive event. A subsurface temperature maximum and superheated tissue produce high pressures that eject fragments from the tissue. Decreased water content due to dehydration and vaporization decreases thermal conductivity which reduces heat conduction. Also, a decrease in water content dramatically alters the local rate of heat generation of laser radiation above 1.3 microns since water is the primary absorber. In contrast, at UV wavelengths protein and DNA are the primary absorbers so destruction of tissue bonds is due to direct absorption of the laser light rather than heat transfer from water.