Threshold Radiant Exposure Research Articles

Re-Evaluation of Rat Corneal Damage by Short-Wavelength UV Revealed Extremely Less Hazardous Property of Far-UV-C†.

Corneal damage‐induced various wavelength UV (311, 254, 235, 222 and 207 nm) was evaluated in rats. For 207 and 222‐UV‐C, the threshold radiant exposure was between 10 000 and 15 000 mJ cm−2 at 207 nm and between 3500 and 5000 mJ cm−2 at 222 nm. Penetrate depth to the cornea indicated by cyclobutene pyrimidine dimer (CPD) localization immediately after irradiation was dependent on the wavelength. 311 and 254 nm UV penetrate to corneal endothelium, 235 nm UVC to the intermediate part of corneal stroma, 222 and 207 nm UVC only to the most outer layer of corneal epithelium. CPD observed in corneal epithelium irradiated by 222 nm UVC disappeared until 12 h after. The minimum dose to induce corneal damage of short‐wavelength UV‐C was considerably higher than the threshold limit value (TLV®) promulgated by American Conference of Governmental Industrial Hygienists (ACGIH). The property that explains why UV‐C radiation at 207 and 222 nm is extremely less hazardous than longer UV wavelengths is the fact that this radiation only penetrates to the outermost layer of the corneal epithelium. These cells typically peel off within 24 h during the physiological turnover cycle. Hence, short‐wavelength UV‐C might be less hazardous to the cornea than previously considered until today.

Potential of sub-microsecond laser pulse shaping for controlling microcavitation in selective retinal therapies.

Pilot results showing the potential of sub-microsecond laser pulse shaping to optimize thermomechanical confinement in laser-tissue interactions involving microcavitation are presented. Model samples based on aqueous suspensions of retinal melanosomes and eumelanin particles were irradiated at 532 nm with nanosecond laser pulses and picosecond laser pulse trains having differing shapes and durations. The cavitation threshold radiant exposure and the bubble lifetime above the threshold were measured using a pump-probe setup and sub-nanosecond time-resolved imaging. Both quantities were found to strongly depend on the pulse format. These results suggest that sub-microsecond laser pulse shaping could be exploited to optimize precision and control in numerous applications of laser-directed microcavitation, including selective retinal laser treatments.

Towards Automatically Controlled Dosing for Selective Laser Trabeculoplasty.

PurposeSelective laser trabeculoplasty (SLT) is a treatment option for open-angle glaucoma; however, it lacks an instant evidence for successful irradiation. So far ophthalmologists use the visible appearance of permanent champagnelike bubbles (macro bubbles) as an indicator for appropriate pulse energy. We hypothesize that micro bubbles, which start energetically far below the appearance of macro bubbles, already trigger the therapeutic benefit. Here we present two methods to capture the onset of these micro bubbles.MethodsThe trabecular meshwork of freshly enucleated porcine eye globes was irradiated with a series of 15 pulses with a pulse duration of 1.7 μs and with increasing energy at a repetition rate of 100 Hz per each spot of 200 μm in diameter. An optical and an optoacoustic method have been developed and appropriate algorithms investigated towards the real-time detection of the onset of micro bubbles.ResultsBoth observation methods are capable of detecting micro bubble nucleation. Threshold radiant exposures were found at 310 ± 137 mJ/cm2. By combination of both methods a sensitivity and specificity of 0.96 was reached.ConclusionsIn case that the therapeutically demanded pressure reduction is already achieved with these micro bubbles, which needs to be proven clinically, then the methods presented here can be used in an automatic feedback loop controlling the laser irradiation. This will unburden the clinicians from any dosing during SLT.Translational RelevanceAutomatic real-time pulse energy dosing based on the formation of micro bubbles in SLT significantly improves and facilitates the treatment for the physician.

The influence of morphological distribution of melanin on parameter selection in laser thermotherapy for vascular skin diseases.

Port wine stains (PWSs) are congenital vascular malformations that progressively darken and thicken with age. Currently, laser therapy is the most effective way in clinical management of PWS. It is known that skin pigmentation (melanin content) affects the radiant exposure that can be safely applied to treat PWS. However, the effect of melanin distribution in the epidermis on the maximum safe radiant exposure has not been studied previously. In this study, 10 different morphological distributions of melanin were proposed according to the formation and migration characteristics of melanin, and the two-scale heat transfer model was employed to investigate the influence of melanin distribution on the threshold radiant exposure of epidermis and blood vessels. The results show that melanin distributions do have a strong effect on laser parameter selection. When uniform melanin distribution is assumed, the threshold radiant exposure to damage a typical PWS blood vessel (50μm diameter) is 8.62J/cm2 lower than that to injure epidermis. The optimal pulse duration is 1-5ms for a typical PWS blood vessel of 50μm when melanin distribution is taken into consideration. PWS blood vessels covered by non-uniformly distributed melanin are more likely to have poor response to laser treatment.

A novel waveguide design that produces an elongated laser beam output for soft tissue ablation

In this work, we have fabricated and experimentally demonstrated a purpose-built 365 μm optical fibre device that produces an elongated laser beam output for efficient tissue ablation. The proposed fibre device has several advantages and potential for application in surgery. The off-axis firing beam from the fibre lowers the risk of past-pointing injury as compared to forward-firing laser fibres. A pulsed Holmium:YAG (Ho:YAG) laser at a wavelength of 2.1 μm at a repetition rate of 6 Hz was used in this experiment. The fibre was angle-polished at ∼25° to create a deviated beam that resulted in a longer ablation length ∼1 cm; it was five times longer than the forward-firing laser output and had sufficient energy intensity to perform an ablation rate of 7.7 μm per pulse. Fresh squid (Cephalopod Theutida) was used as the biological tissue to determine the ablation length and crater. The threshold radiant exposure, Φth for tissue ablation was estimated to be 1.84 J/cm2 and the effective divergence angle was ∼24° by using the highest pulsed energy of 1.8 J.

Thermal cataract induced by near infrared radiation (IRR)

PurposeTo estimate the threshold radiant exposure for 1090 nm IRR and the time evolution of lens damage; To determine the temperature time evolution in the eye during the previously found threshold exposure, and the associated heat diffusion; To investigate if 1090 nm IRR induces cataract photochemically considering irradiance exposure time reciprocity.MethodsThe 6‐weeks‐old albino rats were anesthetized intraperitoneally, ten min before exposure. The pupils of both eyes were dilated with tropicamide. Five min after pupil dilation, the animals were unilaterally exposed to 1090 nm IRR within the pupil area. Temperature was recorded with thermocouples placed in the selected positions of the eye. At the planned post‐exposure time, the animal was sacrificed and the lenses were extracted for measurements of forward light scattering and macroscopic imaging.ResultsThe in vivo exposure to 197 W/cm2 1090 nm IRR required a minimum 8 s for cataract induction with a delayed onset of approx 16 h. The same radiant exposure was found to cause a temperature increase of 10°C at the limbus and 26°C close to the retina. The in vivo exposure to 96 W/cm2 1090 nm IRR with exposure time up to 1 h resulted in an average temperature elevation of 7°C at the limbus with the cornea humidified and no significant light scattering was induced one week after exposure.ConclusionsAn in vivo exposure to 197 W/cm2 IRR at 1090 nm within the pupil for 8 s induces cataract with a time delay. This threshold exposure causes a temperature rise of 10°C at the anterior segment of the rat eye. IRR at 1090 nm produces thermal cataract, probably by indirect heat conduction from absorption in tissues surrounding the lens. There is no cataract development given that the limbal temperature increase is below 8ºC.

Porcine skin damage thresholds for 0.6 to 9.5 cm beam diameters from 1070-nm continuous-wave infrared laser radiation

There is an increasing use of high-power fiber lasers in manufacturing and telecommunications industries operating in the infrared spectrum between 1000 and 2000 nm, which are advertised to provide as much as 10 kW continuous output power at 1070 nm. Safety standards have traditionally been based on experimental and modeling investigations with scant data available for these wavelengths. A series of studies using 1070-nm infrared lasers to determine the minimum visible lesion damage thresholds in skin using the Yucatan miniature pig (Sus scrofa domestica) for a range of beam diameters (0.6, 1.1, 1.9, 2.4, 4.7, and 9.5 cm) and a range of exposure durations (10 ms to 10 s) is presented. Experimental peak temperatures associated with each damage threshold were measured using thermal imaging. Peak temperatures at damage threshold for the 10-s exposures were ∼10°C lower than those at shorter exposures. The lowest and highest experimental minimum visible lesion damage thresholds were found to have peak radiant exposures of 19 and 432 J/cm2 for the beam diameter-exposure duration pairs of 2.4 cm, 25 ms and 0.6 cm, 10 s, respectively. Thresholds for beam diameters >2.5 cm had a weak to no effect on threshold radiant exposure levels for exposure times ≤0.25 s, but may have a larger effect on thresholds for exposures ≥10 s.

Infrared Neural Stimulation of Thalamocortical Brain Slices

Infrared neural stimulation (INS) has been well characterized in the peripheral nervous system, and has been shown to enable stimulation with high spatial precision and without causing the typical electrical stimulation artifact on the recording electrode. The next step in the development of INS is to demonstrate feasibility to stimulate neurons located in the central nervous system (CNS). Thalamocortical brain slices were used to establish feasibility of INS in the CNS and to optimize laser parameters. Infrared light was used to evoke action potentials in the brain slice with no electrical stimulation artifact. This response was blocked by the application of tetrodotoxin demonstrating neurological origin of the recorded signal. Threshold radiant exposure decreased as the absorption coefficient of the wavelength of light increased. Higher repetition rates lead to a decrease in threshold radiant exposure, and threshold radiant exposure was found to decrease as the spot size diameter increased. Additionally, neuronal responses to INS were intracellularly recorded demonstrating artifact free electrical recordings. The results from this paper lay the foundation for future in vivo studies to develop INS for CNS stimulation.

飞秒激光诱导水光学击穿阈值

For studying the relationship between breakdown thresholds and laser parameters, numerical simulation for ellipsoidal time and space model of femtosecond laser-induced optical breakdown in water was done at different pulse duration (40 fs to 540 fs), wavelength (400 nm to 1 200 nm) and spot size (0 to 200 μm) with a Runge-Kutta method. The figures of threshold intensity as a function of pulse duration, wavelength or spot size were given via variable-controlled method. Some conclusions were presented about the relationship between threshold intensity and laser parameters. At last, the figures of threshold power, energy, radiant exposure or electric field strength as a function of pulse duration, wavelength or spot size were presented, which gives a further theoretical support for the study

Selective retina therapy (SRT) in patients with geographic atrophy due to age-related macular degeneration

For geographic atrophy (GA) due to age-related macular degeneration (AMD) there is so far no approved treatment option. Usually, increased autofluorescence (AF) levels of different patterns adjacent to the atrophic area indicate lipofuscin-laden retinal pigment epithelium (RPE) cells at a high risk for apoptosis. Herein, SRT was used to selectively treat these cells to stimulate RPE proliferation, in order to reduce or ideally stop further growth of the atrophic area. Six eyes of six patients with bilateral equally pronounced GA were treated by SRT, while the fellow eye served as control. Irradiation was performed using a prototype SRT laser (Medical Laser Center Lübeck, Nd:YLF laser; 527 nm; 200 ns/1.7 micros pulse duration; 30 repetitive pulses at 100 Hz). Test lesions with increasing energies were applied at the lower vessel arcade to determine the individual angiographic and ophthalmoscopic threshold radiant exposures. Treatment was then performed in the area of increased AF adjacent to the GA using energies between both thresholds. The GA progression rates of treated and fellow eyes were evaluated. After a 1-year follow-up, a progression of the atrophic area was observed in the treated eyes (0.7-8.0 mm(2)/yr, mean 3.0 mm(2)/yr; 46%/yr) whereas the progression rates of the fellow eyes were insignificantly lower (0.46-4.04 mm(2)/yr, mean 1.9 mm(2)/yr; 30%/yr; p = 0.134). The progression rate in the treated eyes of two patients increased significantly, while in the other four patients, the progression rates were nearly the same between both eyes. Moreover, one of these two eyes showed an unexpected RPE reaction after treatment, since all laser lesions led to RPE atrophy and thus an accelerated enlargement of the GA occurred. SRT in the hyperautofluorescent areas of GA was not able to stop or slow down the progression of GA. However, modified treatment strategies might be more promising, e.g. placing the spots outside the hyperautofluorescent areas where RPE apoptosis is postulated. Moreover, SRT studies on GA might be more successfully performed on specific subgroups of GA, based on autofluorescence and other findings.

Optimal pulse durations for the treatment of leg telangiectasias with an alexandrite laser

Determine optimal settings using a long pulse 755 nm alexandrite laser in the treatment of superficial leg veins. STUDY DESIGN\ Fifteen patients with Fitzpatrick skin types I-III with telangiectasia ranging from 0.2 to 1.0 mm were enrolled. Spot size varied from 3 to 6 mm. Pulse durations ranged from 3 to 100 milliseconds. For each pulse duration, test sites were performed to determine threshold radiant exposures using persistent bluing and/or immediate stenosis (closure) as the clinical endpoint. Test sites were re-evaluated 21 days later. Optimal settings, those that resulted in the greatest clearance with minimal side effects (pain, purpura, epidermal damage, pigment changes), were used to treat a larger area of like-sized vessels. Follow-up evaluations were conducted 12 weeks after a single treatment using the optimal setting. Polarized digital photographs were obtained at each visit. Improvement was determined by blinded evaluation of pre/post-treatment photographs. Fourteen patients completed the study. Radiant exposure thresholds for immediate vessel changes depended on vessel diameter, with larger radiant exposures required for smaller spot sizes and smaller vessels. The average threshold radiant exposure for closure was 89 J/cm(2). The optimal pulse duration was 60 milliseconds for most of the patients. With this pulse width, clearance approached 65% 12 weeks after a single treatment. Transient hyperpigmentation occurred in four patients. Increasing the pulse duration improved epidermal tolerance and decreased the likelihood of purpura. By lengthening the pulsewidth beyond 3 milliseconds, a long pulse alexandrite laser achieves satisfactory clearance with an improved side effect profile.

Combined optical and electrical stimulation of neural tissue in vivo

Low-intensity, pulsed infrared light provides a novel nerve stimulation modality that avoids the limitations of traditional electrical methods such as necessity of contact, presence of a stimulation artifact, and relatively poor spatial precision. Infrared neural stimulation (INS) is, however, limited by a 2:1 ratio of threshold radiant exposures for damage to that for stimulation. We have shown that this ratio is increased to nearly 6:1 by combining the infrared pulse with a subthreshold electrical stimulus. Our results indicate a nonlinear relationship between the subthreshold depolarizing electrical stimulus and additional optical energy required to reach stimulation threshold. The change in optical threshold decreases linearly as the delay between the electrical and optical pulses is increased. We have shown that the high spatial precision of INS is maintained for this combined stimulation modality. Results of this study will facilitate the development of applications for infrared neural stimulation, as well as target the efforts to uncover the mechanism by which infrared light activates neural tissue.

Comparison of threshold irradiances and online dosimetry for selective retina treatment (SRT) in patients treated with 200 nanoseconds and 1.7 microseconds laser pulses

Selective retina therapy (SRT) solely affecting the RPE while sparing of the photoreceptors is usually performed with a train of repetitive laser pulses of 1.7 microseconds in duration. It was our purpose to evaluate the principle feasibility of SRT with shorter 200 nanoseconds laser pulses in patients. Nineteen patients with macular disorders [diabetic maculopathy (DMP), geographic atrophy (GA), drusen maculopathy and central serous chorioretinopathy (CSR)] were treated with a prototype of a SRT laser (Nd:YLF laser; 527 nm; 1.7 microseconds and 200 nanoseconds pulse duration; 30 pulses at 100 Hz; spot size: 200 microm). Test lesions (n = 175) with increasing energy were applied at the lower arcade to determine the individual angiographic and ophthalmoscopic threshold radiant exposures (therapeutic window) before applying the central treatment lesions within these ranges additionally guided by online optoacoustic measurements. Postoperatively RPE damage was visualized and confirmed by fluorescein angiographic leakage and correlated with optoacoustic results. Additionally ED(50) damage thresholds were calculated by probit analysis. None of the short repetitive 200 nanoseconds laser pulses led to retinal hemorrhages or retinal ruptures. Nearly all of the test- and treatment lesions could be visualized by angiography indicating desired RPE damage but were ophthalmoscopically invisible suggesting intact neurosensory retinal structures. ED(50) cell damage threshold energies were significantly lower using 200 nanoseconds (99.6 microJ; n = 122) instead of 1.7 microseconds (196.3 microJ; n = 53) laser pulses. Optoacoustic and angiographic visibility correlated in 83.7% (200 nanoseconds) and 87.5% (1.7 microseconds). Selective RPE effects can safely be achieved using shorter 200 nanoseconds laser pulses in patients without adverse effects to the neurosensory retina. The required pulse energy compared to the standard 1.7 microseconds regime was reduced by about a factor of 2 suggesting a reduced heat generation and flow into adjacent tissues during the shorter laser impact and thus possibly enhancing selectivity. Optoacoustics also seem to be a viable alternative in 200 nanoseconds treatment for a non-invasive online dosimetry control system.

Corneal minimal visible lesion thresholds for 20 μm laser radiation

To support refinement of the ANSI Maximum Permissible Exposure safety limits, a series of experiments were conducted in vivo on Dutch Belted rabbit corneas to determine corneal minimum visible lesion thresholds for 2.0 microm continuous-wave laser irradiation. Single pulse radiant exposures were made at specified pulse durations of 0.1, 0.25, 0.5, 1.0, 2.0, and 4.0 s for spot 1/e(2) diameters of 1.17 mm and 4.02 mm. Threshold lesions were defined as the presence of a superficial surface whitening one hour after irradiation. Temperature measurements indicated that threshold peak temperatures were dependent on spot size and exposure duration. The exposure duration dependence of threshold average radiant exposure was described by an empirical power law equation: threshold radiant exposure[J/cm(2)]=a x exposure duration[s](b).

Nucleation dynamics around single microabsorbers in water heated by nanosecond laser irradiation

Suspensions containing micro- and nanoabsorbers, which are irradiated by short laser pulses, are used for a manifold of procedures in medicine, biotechnology, and other fields. Detailed knowledge of the bubble nucleation and dynamics, which is induced by the heat transfer from the absorber to the surrounding transparent water, is essential for understanding the underlying processes occurring on a microscopic scale. We investigated the rapid phase change phenomena including temperature, heating rates, pressure generation, bubble nucleation, and initial bubble growth around absorbing micron-sized melanin particles (retinal pigment epithelial melanosomes) during irradiation with 12ns (full width at half maximum) laser pulses at a wavelength of 532nm. The melanosomes were heated at rates in the order of 1010K∕s. A mean bubble nucleation temperature of 136°C was found. The initial bubble expansion was observed by time-resolved microscopy. The expansion velocities range from 10m∕s at 1.5-fold to 85m∕s at 8.5-fold threshold radiant exposure for bubble formation, respectively. The expansion velocity increases in the investigated range almost linearly with the applied radiant exposure.

Comparison of 1540-nm laser-induced injuries in ex vivo and in vitro rabbit corneal models

Despite the increasing use of infrared lasers in medical, industrial, and military settings, data on threshold radiant exposures and median effective dose (ED(50)) as they relate to laser-tissue interaction are limited. Our goals were to determine the ED(50) for single-pulse, 1540-nm laser exposures in ex vivo and in vitro rabbit corneal models and to characterize the histopathological changes associated with the laser-tissue interaction. An erbium-glass laser was used to deliver single, 1540-nm wavelength pulses to 27 ex vivo and 24 in vitro rabbit corneal models. The ex vivo model was exposed to single pulses of 0.8-ms duration and radiant energies ranging from 17.61 J/cm(2) to 42.26 J/cm(2). The in vitro corneal models were exposed to single pulses of 0.8 ms duration and had radiant exposures ranging from 14.87 to 29.72 J/cm(2). Tissue exposure sites were observed for presence of a lesion immediately post-exposure and 24 h after exposure. Histopathological evaluations of tissue exposure sites were conducted 24 h after exposure. The ED(50) was determined to be 21.24 J/cm(2) for the in vitro rabbit corneal models and 30.86 J/cm(2) for the ex vivo corneal models. Both the in vitro and ex vivo models displayed similar histopathological responses of tissue necrosis and epithelial cell proliferation.

Optical detection of intracellular cavitation during selective laser targeting of the retinal pigment epithelium: dependence of cell death mechanism on pulse duration

Selective laser targeting of the retinal pigment epithelium (RPE) is an attractive method for treating RPE-associated disorders. We are developing a method for optically detecting intracellular microcavitation that can potentially serve as an immediate feedback of the treatment outcome. Thermal denaturation or intracellular cavitation can kill RPE cells during selective targeting. We examined the cell damage mechanism for laser pulse durations from 1 to 40 micros ex vivo. Intracellular cavitation was detected as a transient increase in the backscattered treatment beam. Cavitation and cell death were correlated for individual cells after single-pulse irradiation. The threshold radiant exposures for cell death (ED(50,d)) and cavitation (ED(50,c)) increased with pulse duration and were approximately equal for pulses of up to 10 micros. For 20 micros, the ED(50,d) was about 10% lower than the ED(50,c); the difference increased with 40-micros pulses. Cells were killed predominantly by cavitation (up to 10-micros pulses); probability of thermally induced cell death without cavitation gradually increases with pulse duration. Threshold measurements are discussed by modeling the temperature distribution around laser-heated melanosomes and the scattering function from the resulting cavitation. Detection of intracellular cavitation is a highly sensitive method that can potentially provide real-time assessment of RPE damage during selective laser targeting.

Hexyl‐aminolevulinate‐mediated photodynamic therapy: How to spare normal urothelium. An in vitro approach

Photodynamic therapy (PDT) of superficial bladder cancer may cause damages to the normal surrounding bladder wall. Prevention of these is important for bladder healing. We studied the influence of photosensitizer concentration, irradiation parameters, and production of reactive oxygen species (ROS) on the photodynamically induced damage in the porcine urothelium invitro. The aim was to determine the threshold conditions for the cell survival. Living porcine bladder mucosae were incubated with solution of hexylester of 5-aminolevulinic acid (HAL). The mucosae were irradiated with increasing doses and cell alterations were evaluated by scanning electron microscopy and by Sytox green fluorescence. The urothelial survival score was correlated with Protoporphyrin IX (PpIX) photobleaching and intracellular fluorescence of Rhodamine 123 reflecting the ROS production. The mortality ratio was dependent on PpIX concentration. After 3 hours of incubation, the threshold radiant exposures for blue light were 0.15 and 0.75 J/cm(2) (irradiance 30 and 75 mW/cm(2), respectively) and for white light 0.55 J/cm(2) (irradiance 30 mW/cm(2)). Photobleaching rate increased with decreasing irradiance. Interestingly, the DHR123/R123 reporter system correlated well with the threshold exposures under all conditions used. We have determined radiant exposures sparing half of normal urothelial cells. We propose that the use of low irradiance combined with systems reporting the ROS production in the irradiated tissue could improve the in vivo dosimetry and optimize the PDT.

Analytical modeling of laser pulse heating of embedded biological targets: An application to cutaneous vascular lesions

Detailed understanding of the thermal processes in biological targets undergoing laser irradiation continues to be a challenging problem. For example, the contemporary pulsed dye laser (PDL) delivers a complex pulse format which presents specific challenges for theoretical understanding and further development. Numerical methods allow for adequate description of the thermal processes, but are lacking for clarifying the effects of the laser parameters. The purpose of this work is to derive a simplified analytical model that can guide the development of future laser designs. A mathematical model of heating and cooling processes in tissue is developed. Exact analytical solutions of the model are found when applied to specific temporal and spatial profiles of heat sources. Solutions are reduced to simple algebraic expressions. An algorithm is presented for approximating realistic cases of laser heating of skin structures by heat sources of the type found to have exact solutions. The simple algebraic expressions are used to provide insight into realistic laser irradiation cases. The model is compared with experiments on purpura threshold radiant exposure for PDL. These include data from four independent groups over a period of 20 years. Two of the data sets are taken from previously published articles. Two more data sets were collected from two groups of patients that were treated with two PDLs (585 and 595nm) on normal buttocks skin. Laser pulse durations were varied between 0.5 and 40ms; radiant exposures were varied between 3 and 20J∕cm2. Treatment sites were evaluated 0.5, 1, and 24 hours later to determine purpuric threshold. The analytical model is in excellent agreement with a wide range of experimental data for purpura threshold radiant exposure. The data collected by independent research groups over the last 20 years with PDLs with wavelengths ranged from 577 to 595nm were described accurately by this model. The simple analytical model provides an accurate description of a wide range of experimental data. The model can be used to guide the development of future laser designs and help refine laser parameters.

Comparative study of cryogen spray cooling with R-134a and R-404a: implications for laser treatment of dark human skin

Cutaneous laser treatment in dark skin patients is challenging due to significant light absorption by the melanin at the basal layer of epidermis, which can result in irreversible nonspecific thermal injury to the epidermis. Cryogen spray cooling (CSC) with R-134a (boiling point approximately -26.2 degrees C at 1 atm), which is currently used during cutaneous laser treatment, has shown poor efficacy in protecting dark human skin. We investigated the potential of CSC with R-404a (boiling point approximately -46.5 degrees C at 1 atm), which has a lower boiling point than R-134a, for improved therapeutic outcome in dark human skin at three levels: in vitro (epoxy resin skin phantom), ex vivo (normal dark human skin sample), and in vivo (skin of the rabbit external ear). The skin phantom was used to acquire the surface and internal temperature profiles in response to CSC with R-134a or R-404a at various spurt durations, based upon which CSC-induced heat removal from the skin phantom was estimated using an algorithm that solved a one-dimensional inverse heat conduction problem. CSC with R-404a increased the temperature reductions within the phantom and subsequently the amount of heat removal from the phantom in comparison to that with R-134a. Normal ex vivo Fitzpatrick types V-VI human skin samples were used to investigate the thermal response of dark human skin epidermis to CSC (R-134a or R-404a) at various spurt durations in conjunction with 595-nm pulsed dye laser irradiation at various radiant exposures. Cryogen R-404a increased the threshold radiant exposures for irreversible thermal injury to the epidermis in dark pigmentation skin. No obvious CSC-induced morphological changes to human skin was observed when sprayed with R404-a spurts using durations up to 300 ms. In vivo rabbit ear vasculature was used as a model of cutaneous anomalies to assess the influences of CSC (with R-134a or R-404a) on the photothermolysis of dermal blood vessels. CSC (R-134a or R-404a) with the spurt durations of 100 to 300 ms increased the most superficial depth of thermally damaged dermal blood vessel compared with the sites without CSC, implying possible nonspecific cooling of superficial dermal blood vessels by the cryogen spurts with the settings applied.