Hyperspectral imaging of atherosclerotic plaques in vitro.

Vulnerable plaques constitute a risk for serious heart problems, and are difficult to identify using existing methods. Hyperspectral imaging combines spectral- and spatial information, providing new possibilities for precise optical characterization of atherosclerotic lesions. Hyperspectral data were collected from excised aorta samples (n = 11) using both white-light and ultraviolet illumination. Single lesions (n = 42) were chosen for further investigation, and classified according to histological findings. The corresponding hyperspectral images were characterized using statistical image analysis tools (minimum noise fraction, K-means clustering, principal component analysis) and evaluation of reflectance/fluorescence spectra. Image analysis combined with histology revealed the complexity and heterogeneity of aortic plaques. Plaque features such as lipids and calcifications could be identified from the hyperspectral images. Most of the advanced lesions had a central region surrounded by an outer rim or shoulder-region of the plaque, which is considered a weak spot in vulnerable lesions. These features could be identified in both the white-light and fluorescence data. Hyperspectral imaging was shown to be a promising tool for detection and characterization of advanced atherosclerotic plaques in vitro. Hyperspectral imaging provides more diagnostic information about the heterogeneity of the lesions than conventional single point spectroscopic measurements.

Tissue responses to hexyl 5-aminolevulinate-induced photodynamic treatment in syngeneic orthotopic rat bladder cancer model: possible pathways of action.

Orthotopic bladder cancer model in rats mimics human bladder cancer with respect to urothelial tumorigenesis and progression. Utilizing this model at pT1 (superficial stage), we analyze the tissue responses to hexyl 5-aminolevulinate-induced photodynamic therapy (HAL-PDT). In comparison to untreated rats, HAL-PDT causes little change in tumor-free rat bladder but induces inflammatory changes with increased lymphocytes and mononuclear cell infiltration in rat bladders with tumor. Immunohistochemistry reveals that HAL-PDT is without effect on proliferating cell nuclear antigen expression within the tumor and increases caspase-3 expression in both normal urothelium and the tumor. Transmission electron microscopy reveals severe mitochondrial damage, formations of apoptotic bodies, vacuoles, and lipofuscin bodies, but no microvillus-formed niches in HAL-PDT-treated bladder cancer rats. Bioinformatics analysis of the gene expression profile indicates an activation of T-cell receptor signaling pathway in bladder cancer rats without PDT. HAL-PDT increases the expression of CD3 and CD45RA in the tumor (determined by immunohistochemistry). We suggest that pathways of action of HAL-PDT may include, at least, activations of mitochondrial apoptosis and autophagy, breakdown of cancer stem cell niches, and importantly, enhancement of T-cell activation.

Remittance at a single wavelength of 390 nm to quantify epidermal melanin concentration.

Objective quantification of epidermal melanin concentration (EMC) should be useful in laser dermatology to determine the individual maximum safe radiant exposure (IMSRE). We propose a single-wavelength remittance measurement at 390 nm as an alternative optical method to determine EMC and IMSRE. Remittance spectra (360 to 740 nm), melanin index (MI) measurements and the transient radiometric temperature increase, DeltaT(t), upon skin irradiation with an Alexandrite laser (755 nm, 3-ms pulse duration, 6 Jcm(2)) were measured on 749 skin spots (arm and calf) on 23 volunteers (skin phototypes I to IV). Due to the shallow penetration depth and independence of blood oxygen saturation (isosbestic point), remittance at 390 nm appears to provide better estimates for EMC and IMSRE than MI.

Remote plethysmographic imaging using ambient light.

Plethysmographic signals were measured remotely (> 1m) using ambient light and a simple consumer level digital camera in movie mode. Heart and respiration rates could be quantified up to several harmonics. Although the green channel featuring the strongest plethysmographic signal, corresponding to an absorption peak by (oxy-) hemoglobin, the red and blue channels also contained plethysmographic information. The results show that ambient light photo-plethysmography may be useful for medical purposes such as characterization of vascular skin lesions (e.g., port wine stains) and remote sensing of vital signs (e.g., heart and respiration rates) for triage or sports purposes.

Monitoring of hexyl 5-aminolevulinate-induced photodynamic therapy in rat bladder cancer by optical spectroscopy.

Monitoring of the tissue response to photodynamic therapy (PDT) can provide important information to help optimize treatment variables such as drug and light dose, and possibly predict treatment outcome. A urinary bladder cancer cell line (AY-27) was used to induce orthotopic transitional cell carcinomas (TCC) in female Fischer rats, and hexyl 5-aminolevulinate (HAL, 8 mM, 1 h)-induced PDT was performed on day 14 after instillation of the cancer cells (20 J/cm(2) fluence at 635 nm). In vivo optical reflectance and fluorescence spectra were recorded from bladders before and after laser treatment with a fiberoptic probe. Calculated fluorescence bleaching and oxygen saturation in the bladder wall were examined and correlated to histology results. Reflectance spectra were analyzed using a three-layer optical photon transport model. Animals with TCC treated with PDT showed a clear treatment response; decreased tissue oxygenation and protoporphyrin IX (PpIX) fluorescence photobleaching were observed. Histology demonstrated that 3 of 6 animals with treatment had no sign of the tumor 7 days after PDT treatment. The other 3 animals had significantly reduced the tumor size. The most treatment-responsive animals had the highest PpIX fluorescence prior to light irradiation. Thus, optical spectroscopy can provide useful information for PDT. The model has proved to be very suitable for bladder cancer studies.

Dynamic skin cooling with an environmentally compatible alternative cryogen during laser surgery.

BACKGROUND AND OBJECTIVES: Cryogen spray cooling with tetrafluoroethane (R134a) has been used to enhance epidermal protection during dermatologic laser surgery. However, R134a has a very high global warming potential (GWP = 1300). Our objective was to evaluate the cooling effectiveness of an alternative cryogen with a much lower GWP, namely liquid carbon dioxide (CO(2), GWP = 1). STUDY DESIGN/MATERIALS AND METHODS: A thin-film thermocouple deposited on an epoxy skin phantom was used to measure surface temperature (T(s)) variations induced by R134a or CO(2) sprays. The temperature distribution in the skin phantom was estimated using T(s) and Duhamel's method. Impact pressure and noise level of both cryogen sprays were measured with a dynamic sensor and sound meter, respectively. Consumption of both cryogens was also evaluated. RESULTS: For R134a sprays, T(s) was kept almost constant after 15 milliseconds. For CO(2) sprays, T(s) decreased continuously during the entire spurt of 50 milliseconds. The minimum T(s) induced by the CO(2) sprays was lower than that induced by R134a when the spurt duration was longer than 35 milliseconds. Numerical simulation shows that CO(2) sprays were able to induce very similar temperature reductions in the skin phantom as compared to R134a sprays when the spurt duration and delay time were selected appropriately. R134a sprays induced an impact pressure of 3.6 kPa, as compared to 43.1 kPa for CO(2) sprays. The maximum noise level for R134a sprays was 109 dBA as compared to 135 dBA for the CO(2) sprays. The R134a consumption for a 50 milliseconds spurt is 67 mg as compared to 225 mg for a CO(2) spurt of the same duration. CONCLUSIONS: CO(2) sprays are expected to have similar skin cooling efficacy as R134a sprays. Although the CO(2) consumption is higher than R134a, its contribution to global warming is still much less than R134a. The effects of varying spurt durations on in vivo human skin and the impact on cutaneous blood flow require further investigation.

Skin changes following minor trauma.

BACKGROUND AND OBJECTIVE: Bruises are currently evaluated by visual inspection, and little is known about the first phase after injury. The temporal development of fresh injuries must be accurately described to be able to age bruises in a reliable manner. Color changes in a bruise caused by hemoglobin breakdown products will depend on the severity of the trauma, and thus on the local immune response in the skin. It is therefore important to relate the nature of the impact to the temporal tissue responses. MATERIALS AND METHODS: Controlled injuries were inflicted on anesthetized domestic pigs. Trauma was induced either by a pendulum device, or by paintballs released using pressurized air. The speed of the projectiles was recorded using a high speed camera. Biopsies and reflection spectra (400-850 nm) were collected from normal and bruised skin. The experiments were approved by the national animal research authority. RESULTS: The temporal development of the injury was found to depend strongly on the weight and speed of the object. Low speed, blunt objects did not cause persistent skin changes. However, deep muscular bleeding could be found in most cases. High speed, light weight objects caused a rapidly developing bruise. These bruises were fully developed within 15-20 minutes. No deep muscular hemorrhages were observed in those cases. White blood cells (neutrophilic granulocytes) could be found in biopsies from high speed injuries. The amount of white blood cells depended on the time between injury and collection of the biopsies. CONCLUSION: Further investigations utilizing a larger range of object weight and velocities are required to be able to fully classify minor traumatic injuries. Preliminary results indicate that this can be achieved by controlled experiments using a porcine model. Reflectance spectroscopy was found to be a useful tool to study immediate skin reactions to the trauma.

Reflection spectroscopy of atherosclerotic plaque.

Heart disease is the primary cause of death in the western world. Many of these deaths are caused by the rupture of vulnerable plaque. Vulnerable plaques are characterized by a large lipid core covered by a thin fibrous cap. One method for detecting these plaques is reflection spectroscopy. Several studies have investigated this method using statistical methods. A more analytic and quantitative study might yield more insight into the sensitivity of this detection modality. This is the approach taken in this work. Reflectance spectra in the spectral region from 400 to 1700 nm are collected from 77 measurement points from 23 human aortas. A measure of lipid content in a plaque based on reflection spectra is presented. The measure of lipid content is compared with the thickness of the lipid core, determined from histology. Defining vulnerable plaque as having a lipid core >500 microm and fibrous cap <500 microm, vulnerable plaques are detected with a sensitivity of 88% and a specificity of 94%. Although the method can detect lipid content, it is not very sensitive to the thickness of the fibrous cap. Another detection modality is necessary to detect this feature.

Measurement of dye diffusion in scattering tissue phantoms using dual-wavelength low-coherence interferometry.

We demonstrate low-coherence interferometry (LCI) for dye diffusion measurements in scattering tissue phantoms. The diffusion coefficient of a phthalocyanine dye in 1.5% agar gel containing scattering Intralipid was measured using a dual-wavelength interfero-meter. One wavelength was matched to the absorption peak of the dye at 675 nm. The other, 805 nm, was not affected by the dye, and was used to correct for varying sample scattering as a function of depth, assuming a constant ratio between scattering at the two wavelengths. The same wavelength dependence of scattering is assumed for the entire sample, but no a priori knowledge about the amount of scattering is needed. The dye diffusion coefficient was estimated by fitting a mathematical model of the interferometer signal to the measured LCI envelope. We compare results obtained using both a constant-scattering and a depth-resolved-scattering approach to determine the sample scattering. The presented method provides robust estimation of the diffusion coefficient when spatial resolution in determining the depth-resolved scattering is varied. Results indicate that the method is valid for samples having continuous spatial variations in the scattering coefficient over lengths as short as the coherence length of the probing light. The method allows in situ characterization of diffusion in scattering media.

A novel approach to age determination of traumatic injuries by reflectance spectroscopy.

BACKGROUND AND OBJECTIVES: Aging of injuries on a victim's body is an important aspect of forensic medicine. Currently, visual assessment and colorimetry based on empirical criteria are the most common techniques for this task, although the results are uncertain. A trauma causing localized vessel damage will rapidly result in a pool of blood in subcutaneous tissues. The color of the bruise is, however, primarily due to hemoglobin transport into dermis and secondarily to its breakdown products. This transport is analyzed in terms of hemoglobin diffusion followed by clearance by macrophage activity, lymphatic flow, and conversion to breakdown products such as bilirubin. The color of a bruise is caused by hemoglobin and hemoglobin breakdown products. The color will change with time, and such color changes can be recorded using reflectance spectroscopy. The aim of this study was to develop a mathematical model to describe blood diffusion within bruised skin, and to use this method to retrieve the age of a bruise from measured skin reflectance. STUDY DESIGN/MATERIALS AND METHODS: An analytic model was established to describe the development and fading of bruise color. The model, which is based on Darcy's law of convection flow and Fick's law of diffusion, describes the distribution of blood and hemoglobin breakdown products within a hematoma as a function of time after injury. The initial phase after injury is described by a convective extravascular blood flow in subcutaneous tissues, and further development of the bruise is described by diffusion and breakdown of whole erythrocytes and hemoglobin in dermis. Experimental data were used to verify the model. Reflection spectra in the 400-850 nm wavelength range were collected from normal and bruised skin using an integrating sphere setup. The subjects were adult patients admitted to the Department of cardiothoracic surgery, St. Olav's Hospital, Trondheim, Norway. The skin hematomas were caused by external trauma, cardiothoracic examinations, or surgery. RESULTS: Preliminary results show that measured and simulated skin reflectance agrees well. The model predicts the age of a hematoma with an accuracy of approximately 1 day. The accuracy of the method depends on precise information of skin thickness in the injured area. The quality of the estimates from the model will thus be enhanced if a reliable measure of skin thickness is collected concurrently with the reflection measurement. CONCLUSIONS: The time development of a skin hematoma is described with good accuracy by the implemented model. The analytic method provides a theoretical basis for developing an apparatus to determine the age of injuries in forensic medicine.

Determination of human skin optical properties from spectrophotometric measurements based on optimization by genetic algorithms.

We present an initial study on applying genetic algorithms (GA) to retrieve human skin optical properties using visual reflectance spectroscopy (VRS). A three-layered skin model consisting of 13 parameters is first used to simulate skin and, through an analytical model based on optical diffusion theory, we study their independent effects on the reflectance spectra. Based on a preliminary analysis, nine skin parameters are chosen to be fitted by GA. The fitting procedure is applied first on simulated reflectance spectra with added white noise, and then on measured spectra from normal and port wine stain (PWS) human skin. A normalized residue of less than 0.005 is achieved for simulated spectra. In the case of measured spectra from human skin, the normalized residue is less than 0.01. Comparisons between applying GA and manual iteration (MI) fitting show that GA performed much better than the MI fitting method and can easily distinguish melanin concentrations for different skin types. Furthermore, the GA approach can lead to a reasonable understanding of the blood volume fraction and other skin properties, provided that the applicability of the diffusion approximation is satisfied.

Functional imaging of dye concentration in tissue phantoms by spectroscopic optical coherence tomography.

We present functional imaging of the concentration of a photodynamic therapy (PDT)-related dye in scattering tissue phantoms based on spatially resolved measurements of optical properties through spectroscopic optical coherence tomography (OCT). Expressions for the OCT signal are developed, enabling estimation of depth-resolved sample optical properties. Based on these expressions, we discuss speckle statistics and speckle correlations of the OCT signal. Speckle noise reduction is performed by spatial filtering and is used to improve accuracy in the estimated optical properties at the expense of spatial resolution. An analytic expression for the precision in the estimated optical properties is derived. This expression shows that axial filtering, and thereby a reduction of axial resolution, gives a larger improvement in precision compared to the same filtering and reduction in the transversal resolution. It also shows that imaging with a shorter coherence length, or a larger numerical aperture, improves precision when the filter length determines the spatial resolution. Good agreement is obtained between experimentally determined and theoretically predicted variance in the estimated attenuation coefficients and dye concentration. Finally, we present guidelines for spectroscopic OCT systems for concentration imaging and discuss application of the method to more realistic phantoms and tissue.

Influence of laser wavelength and pulse duration on gas bubble formation in blood filled glass capillaries.

BACKGROUND AND OBJECTIVES: Hypervascular skin lesions (HVSL) are treated with medical lasers characterized by a variety of parameters such as wavelength lambda, pulse duration t(p), and radiant exposure E that can be adjusted for different pathology and blood vessel size. Treatment parameters have been optimized assuming constant optical properties of blood during laser photocoagulation. However, recent studies suggest that this assumption may not always be true. Our objective was to quantify thermally induced changes in blood that occur during irradiation using standard laser parameters. STUDY DESIGN/MATERIALS AND METHODS: Glass capillary tubes (diameter D = 100, 200, and 337 microm) filled with fresh or hemolyzed rabbit blood were irradiated once at lambda = 585, 595, or 600 nm, t(p) = 1.5 milliseconds; and also at lambda = 585 nm, t(p) = 0.45 milliseconds. E was increased until blood ablation caused formation of permanent gas bubbles. In a corroborative study, human blood was heated at 50 degrees C and absorbance spectra were measured as a function of time. RESULTS: Threshold radiant exposure, E(thresh), for gas bubble formation was found not to depend on lambda, which might be surprising in view of the 10-fold lower absorption coefficient at 600 nm as compared to 585 nm. The spectroscopic study revealed heat-induced changes in blood constituent composition of hemoglobins (Hb) from initially 100% oxyhemoglobin (HbO2) to deoxyhemoglobin (HHb) and, ultimately, methemoglobin (metHb) as the major constituent. Model calculations of E(thresh)(lambda,D) based on changing constituent blood composition during heating with milliseconds lasers were found to correlate with experimental results. CONCLUSIONS: For laser treatment of HVSL it appears that lambda is of secondary importance and that the choice of t(p) is a more important factor.

Effects of hypobaric pressure on human skin: feasibility study for port wine stain laser therapy (part I).

BACKGROUND AND OBJECTIVES: Since the development of laser-induced photothermolysis for the therapy of port wine stain (PWS) birthmarks, clinical results have shown that dark purple lesions usually respond well to the first three to five treatments. However, for most PWS, complete blanching is never achieved, and the lesion stabilizes at a red-pink color. The aim of this feasibility study is to demonstrate that with the aid of a local vacuum applied to the lesion site prior to laser exposure, photocoagulation of the smaller PWS blood vessels may be successfully achieved. STUDY DESIGN/MATERIALS AND METHODS: Suction cups were designed to fit onto the hand pieces of commercial laser devices used for PWS laser therapy. One subject with normal skin and another with PWS skin were recruited for this study. Laser pulses of various fluences were applied at atmospheric pressure or shortly after (5-15 seconds) hypobaric pressures (17-51 kPa) were placed as test sites on the forearm of both subjects. The laser-induced purpura at the test sites was documented over the course of 1 week on both subjects and the resulting PWS blanching was optically quantified by visible reflectance spectrometry 7 months after therapy. RESULTS: For the subject with normal skin, the laser fluence needed with hypobaric pressure (51 kPa) to induce similar purpura intensity to that observed with atmospheric pressure was approximately 35% lower. For PWS skin, all suction application times (5-15 seconds) and hypobaric pressures (17-51 kPa) resulted in more intense purpura and the PWS blanching 7 months after treatment was clinically significant for test sites treated with hypobaric pressures ranging from 17 to 34 kPa. CONCLUSIONS: The temporary and controlled dilation of the targeted blood vessels achieved with a local vacuum can significantly reduce the "small-vessel-limitation" in the treatment of PWS without increasing the risk of epidermal damage.

Effects of hypobaric pressure on human skin: implications for cryogen spray cooling (part II).

BACKGROUND AND OBJECTIVES: Clinical results have demonstrated that dark purple port wine stain (PWS) birthmarks respond favorably to laser induced photothermolysis after the first three to five treatments. Nevertheless, complete blanching is rarely achieved and the lesions stabilize at a red-pink color. In a feasibility study (Part I), we showed that local hypobaric pressure on PWS human skin prior to laser irradiation induced significant lesion blanching. The objective of the present study (Part II) is to investigate the effects of hypobaric pressures on the efficiency of cryogen spray cooling (CSC), a technique that assists laser therapy of PWS and other dermatoses. STUDY DESIGN/MATERIALS AND METHODS: Experiments were carried out within a suction cup and vacuum chamber to study the effect of hypobaric pressure on the: (1) interaction of cryogen sprays with human skin; (2) spray atomization; and (3) thermal response of a model skin phantom. A high-speed camera was used to acquire digital images of spray impingement on in vivo human skin and spray cones generated at different hypobaric pressures. Subsequently, liquid cryogen was sprayed onto a skin phantom at atmospheric and 17, 34, 51, and 68 kPa (5, 10, 15, and 20 in Hg) hypobaric pressures. A fast-response temperature sensor measured sub-surface phantom temperature as a function of time. Measurements were used to solve an inverse heat conduction problem to calculate surface temperatures, heat flux, and overall heat extraction at the skin phantom surface. RESULTS: Under hypobaric pressures, cryogen spurts did not produce skin indentation and only minimal frost formation. Sprays also showed shorter jet lengths and better atomization. Lower minimum surface temperatures and higher overall heat extraction from skin phantoms were reached. CONCLUSIONS: The combined effects of hypobaric pressure result in more efficient cryogen evaporation that enhances heat extraction and, therefore, improves the epidermal protection provided by CSC.

A library based fitting method for visual reflectance spectroscopy of human skin.

The diffuse reflectance spectrum of human skin in the visible region (400-800 nm) contains information on the concentrations of chromophores such as melanin and haemoglobin. This information may be extracted by fitting the reflectance spectrum with an optical diffusion based analytical expression applied to a layered skin model. With the use of the analytical expression, it is assumed that light transport is dominated by scattering. For port wine stain (PWS) and highly pigmented human skin, however, this assumption may not be valid resulting in a potentially large error in visual reflectance spectroscopy (VRS). Monte Carlo based techniques can overcome this problem but are currently too computationally intensive to be combined with previously used fitting procedures. The fitting procedure presented herein is based on a library search which enables the use of accurate reflectance spectra based on forward Monte Carlo simulations or diffusion theory. This allows for accurate VRS to characterize chromophore concentrations in PWS and highly pigmented human skin. The method is demonstrated using both simulated and measured reflectance spectra. An additional advantage of the method is that the fitting procedure is very fast.

A comparative study of photoacoustic and reflectance methods for determination of epidermal melanin content.

Although epidermal melanin content has been quantified non-invasively using visible reflectance spectroscopy (VRS), there is currently no way to determine melanin distribution in the epidermis. We have developed a photoacoustic probe that uses a Q-switched, frequency-doubled Nd:YAG (neodymium, yttrium, aluminum, garnet) laser operating at 532 nm to generate acoustic pulses in skin in vivo. The probe contained a piezoelectric element that detected photoacoustic waves that were then analyzed for epidermal melanin content using a photoacoustic melanin index (PAMI). Melanin content was compared between results of photoacoustics and VRS. Spectra from human skin were fitted to a model based on diffusion theory that included parameters for epidermal thickness, melanin content, hair color and density, and dermal blood content. Ten human subjects with skin phototypes I-VI were tested using the photoacoustic probe and VRS. A plot of PAMI v. VRS showed a good linear fit with r2=0.85. Photoacoustic and VRS measurements are shown for a human subject with vitiligo, indicating that melanin was almost completely absent. We present preliminary modeling for photoacoustic probe design and analysis necessary for depth profiling of epidermal melanin.

Combined photodynamic and photothermal induced injury enhances damage to in vivo model blood vessels.

BACKGROUND AND OBJECTIVES: The degree of port wine stain (PWS) blanching following pulsed dye laser (PDL) therapy remains variable and unpredictable. Because of the limitations of current PDL therapy, alternative treatment approaches should be explored. The objective was to evaluate a novel methodology for selective vascular damage, combined photodynamic (PDT) and photothermal (PDL) treatment, using the in vivo chick chorioallantoic membrane (CAM) model. STUDY DESIGN/MATERIALS AND METHODS: Thirty microliters of benzoporphyrin derivative monoacid ring A (BPD) solution was administered intraperitoneally into chick embryos at day 12 of development. Study groups were: (1) control (no BPD, no light); (2) BPD alone; (3) continuous wave irradiation (CW) alone (576 nm, 60 mW/cm2, 125 seconds); (4) CW + PDL; (5) BPD+PDL; (6) PDT (BPD+CW); (7) PDL alone (585 nm, 4 J/cm(2)); and (8) PDT+PDL (BPD + CW followed immediately by PDL). Vessels were videotaped prior to, and at 1 hour post-intervention and then assessed for damage based on the following scale: 0, no damage; 1, coagulation; 1.5, vasoconstriction; 2.0, coagulation+vasoconstriction; 2.5, angiostasis; 3.0, hemorrhage. Damage scores were weighted by vessel "order." RESULTS: PDT + PDL resulted in significantly (P < 0.01) more severe vascular damage than was observed in any other study group: 127% more than PDT, 47% more than PDL alone. CONCLUSIONS: PDT + PDL is a novel and promising approach for selective vascular damage and may offer a more effective method for treatment of PWS and other vascular skin lesions.

On the physics of laser-induced selective photothermolysis of hair follicles: Influence of wavelength, pulse duration, and epidermal cooling.

The physical basis for optimization of wavelength, pulse duration, and cooling for laser-induced selective photothermolysis of hair follicles in human skin is discussed. The results indicate that the most important optimization parameter is the cooling efficiency of the technique utilized for epidermal protection. The optical penetration is approximately the same for lasers at 694, 755, and 800 nm. The penetration of radiation from Nd:yttrium-aluminum-garnet lasers at 1064 nm is, however, somewhat larger. Photothermal damage to the follicle is shown to be almost independent of laser pulse duration up to 100 ms. The results reveal that epidermal cooling by a 30-80-ms-long cryogen spurt immediately before laser exposure is the only efficient technique for laser pulse durations less than 10 ms. For longer pulse durations in the 30-100 ms range, protection can be done efficiently by skin cooling during laser exposure. For laser pulses of 100 ms, an extended precooling period, e.g., by bringing a cold object into good thermal contact with the skin for about 1 s, can be of value. Thermal quenching of laser induced epidermal temperature rise after pulsed exposure can most efficiently be done with a 20 ms cryogen spurt applied immediately after irradiation.