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Lasers for Dermatology and Skin Biology

      Popular media initially portrayed lasers, the brightest light sources in the known universe, as a "death ray". Instead, lasers became minimally destructive, precise, versatile surgical tools that also enabled a revolution in biological sciences through live-tissue microscopy. How did that happen, and what might come next? It turns out that laser capabilities-impressive optical power density, collimation, coherence, well-defined wavelength, and impressively brief pulses-are better suited for gentle precision than for wanton destruction. This trend has been going strong for 30 years and shows no signs of slowing down.
      The need for a better treatment of port wine stains (PWS) led us to hatch the concept of "selective photothermolysis" (SP) (
      • Anderson R.R.
      • Parrish J.A.
      Selective photothermolysis: precise microsurgery by selective absorption of pulsed radiation.
      ), which became the basis for many new therapeutic lasers. Light passes harmlessly through space and matter until it is absorbed. It stands to reason that heating (and other events caused by light) begins when and where the light is absorbed. Skin contains specific structures with a high concentration of light-absorbing molecules (chromophores), such as the abnormal blood vessels of PWS. Intense pulses of light at wavelengths preferentially absorbed in these "target" structures can cause selective thermal damage. We showed this for microvessels in skin using yellow light pulses and for melanin-pigmented cells using UV light pulses. For the best selectivity, heat should be confined to the target during the light pulse. In other words, pulse duration should be about equal to the time it takes for cooling the target. Thermal relaxation time turns out to vary with the square of target size. For targets ranging from subcellular particles (nm scale) to multicellular structures (mm scale), thermal relaxation time varies a million-fold, from nanoseconds to milliseconds.
      These simple ideas led to a burst of new applications for nonscarring, pulsed lasers over a wide range of pulse duration and wavelength, including lasers that did not previously exist. Millisecond pulsed dye lasers (PDLs) for treating microvascular malformations are the first example of any laser specifically invented for a medical need, and they remain the treatment of choice for neonatal and childhood PWS (
      • Chapas A.M.
      • Eikhorst K.
      • Geronemus R.G.
      Efficacy of early treatment of facial port wine stains in newborns: a review of 49 cases.
      ). The latest generation of PDLs has variable pulse duration to match various target vessel sizes, and uses dynamic cryogen spray cooling for epidermal protection in pigmented skin. However, PWS treatment remains challenging because a residual vascular lesion almost always persists. Three reasons are hypothesized to account for persistence of PWS-(a) angiogenesis produces new PWS vessels after each laser treatment, (b) insufficient depth of PDL treatment, and (c) an intrinsic lack of sympathetic innervation. These might be overcome. The first report of combined PDL plus the angiogenesis inhibitor drug rapamycin (
      • Nelson J.S.
      • Wangcun J.
      • Phung T.L.
      • et al.
      Observations on enhanced portwine stain blanching induced by combined pulsed dye laser and rapamycin administration.
      )-and for adults with hypertrophic PWS, the use of deeply penetrating near-infrared wavelengthsis highly effective (
      • Izikson L.
      • Nelson J.S.
      • Anderson R.R.
      Treatment of hypertrophic and resistant port wine stains with a 755?nm laser: a case series of 20 patients.
      ). PWS being microvenous lesions, in theory a source could be made that targets de-oxyhemoglobin, sparing the arterial circulation (
      • Rubin I.K.
      • Farinelli W.A.
      • Doukas A.
      • et al.
      Optimal wavelengths for vein-selective photothermolysis.
      ). Other indications for PDLs and their cousin technologies include rosacea, scars, ulcerated infantile hemangioma, warts, and laryngeal lesions including carcinomas.
      The precision with which very short (nanosecond 10-9s, or shorter) pulses can target individual pigmented cells or organelles is impressive. In our initial investigation of SP, we found that even Langerhans cells adjacent to pigmented epidermal cells were unaffected. Ophthalmology subsequently developed this as a successful treatment for early-stage glaucoma, targeting pigmented cells in the trabecular meshwork (
      • Latina M.A.
      • Sibayan S.A.
      • Shin D.H.
      • et al.
      Q-switched 532-nm Nd:YAG laser trabeculoplasty (selective laser trabeculoplasty): a multicenter, pilot, clinical study.
      ). In dermatology, Q-switched red and near-infrared lasers proved to be excellent for treating some melanocytoses such as nevus of Ota, useful for cafe- au-lait macules and lentigines, but generally a failure for other conditions such as Becker's nevi. As one might expect, the biology of particular pigmented skin lesions critically affects their response to laser treatment. The pigmented, well-differentiated dermal melanocytes of nevus of Ota are easily removed by Q-switched ruby, alexandrite, or Nd:YAG laser treatment (
      • Taylor C.R.
      • Anderson R.R.
      • Gange R.W.
      • et al.
      Light and electron microscopic analysis of tattoos treated by Q-switched ruby laser.
      ). In contrast, con-genital melanocytic nevi include both pigmented and nonpigmented melano-cytes at various levels of differentiation. Surgical excision is the preferred treatment, and lasers offer a useful alternative only in some cases. Other fascinating aspects of laser targeting of melanocytes have never been developed. For example, sublethal fluences from these Q-switched lasers stimulate melanogenesis and tanning of the skin.
      Tattoos are a poorly regulated form of ancient nanotechnology. Insoluble, indigestible ink particles ranging from about 50 to 2,000 nm in size are permanently trapped in phagocytic dermal cells. Tattoo "removal" uses Q-switched lasers to selectively rupture these ink-containing cells. Some of the liberated ink is shed from the skin surface, appears in lymph nodes, or remains in the skin after each treatment. (
      • Taylor C.R.
      • Anderson R.R.
      • Gange R.W.
      • et al.
      Light and electron microscopic analysis of tattoos treated by Q-switched ruby laser.
      ) Almost half of US young adults have a tattoo, and many of them come to regret it, but very few seek laser tattoo removal (
      • Laumann A.E.
      • Derick A.J.
      Tattoos and body piercings in the United States: a national data set.
      ). After nearly a year of monthly Q-switched laser treatments, about 70% of tattoos are removed without side effects, and 30% are not. Recent work suggests that repeated laser exposures given 20min apart (
      • Kossida T.
      • Rigopoulos D.
      • Katsambas A.
      • et al.
      Optimal tattoo removal in a single laser session based on the method of repeated exposures.
      ) and the use of picosecond (10-12 s) laser pulses are more effective (
      • Ross V.
      • Naseef G.
      • Lin G.
      • et al.
      Comparison of responses of tattoos to picosecond and nanosecond Q-switched neodymium: YAG lasers.
      ;
      • Brauer J.A.
      • Reddy K.K.
      • Anolik R.
      • et al.
      Successful and rapid treatment of blue and green tattoo pigment with a novel picosecond laser.
      ). We are getting close to being able to remove tattoos in a day's work, much as they are created. If so, would that capability be wisely used, or would tattoos become even more common? A modern version of tattoos is being developed for cancer therapy. Systemically delivered gold nanoparticles coated with molecules that bind to tumors are preferentially retained, after which laser exposure can be used to kill the tumor (
      • Von Maltzahn G.
      • Park J.H.
      • Agrawal A.
      • et al.
      Computationally guided photothermal tumor therapy using long-circulating gold nanorod antennas.
      ).
      Sometimes the biological target is not itself pigmented, but there is a chromophore nearby. An "extended theory" of SP describes this situation (
      • Altshuler G.B.
      • Anderson R.R.
      • Manstein D.
      • et al.
      Extended theory of selective photothermolysis.
      ). Permanent laser hair reduction is a good example. Melanin normally exists in the epidermis, hair shafts, and matrix at the bottom of the hair follicles. Follicular epithelial stem cells appear to be an important target for permanent hair loss. These nonpigmented stem cells are located in the outer root sheath, at some distance from the hair shaft (
      • Lyle S.
      • Christofidou-Solomidou M.
      • Liu Y.
      • et al.
      The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells.
      ). Early attempts at laser hair removal failed because they used short, Q-switched laser pulses that vaporized the pigmented hair shafts, but did not allow sufficient time for heat conduction to the outer root sheath. Therefore, we tested whether millisecond-domain pulses of red light could permanently remove pigmented hair (
      • Dierickx C.C.
      • Grossman M.C.
      • Farinelli W.A.
      • et al.
      Permanent hair removal by normal-mode ruby laser.
      ), using a ruby laser similar to the first-ever laser built by Ted Maiman in 1960. At present, permanent hair reduction is the most popular biomedical use of lasers. Why did it take more than 30 years to come up with this application, using the oldest laser in world? Alexandrite, diode, Nd:YAG lasers, and IPLs have since replaced ruby lasers for pigmented hair removal. Enigmas remain, such as the side effect of permanently stimulated hair growth, which occurs on the face in Mediterranean women.
      The concept of thermal confinement was extended to surgical CO2 lasers, which can precisely ablate (remove) tissue, leaving a very thin layer of residual thermal injury. Richard Fitzpatrick introduced laser resurfacing as an alternative to dermabrasion and deep chemical peels for photoaged skin (
      • Fitzpatrick R.E.
      • Goldman M.P.
      • Satur N.M.
      • et al.
      Pulsed carbon dioxide laser resurfacing of photo-aged facial skin.
      ). The entire epidermis and papillary dermis are removed. Re-epithelialization and dermal remodeling thereafter leaves a more youthful appearance, and pro-foundly decreases the rate of skin cancer in high-risk patients. Delayed, permanent hypopigmentation after CO2 laser resurfacing in Caucasians led to a search for alternatives, although the procedure remains very useful for selected patients. Nonablative, mid-infrared lasers (diode and 1326 nm Nd:YAG lasers) were developed that preserve the epidermis while heating the upper and mid-dermis. Despite minimal efficacy for photoaging, these lasers entered dermatology to stay, as the first example of "fractional" laser treatment.
      Fractional photothermolysis (
      • Manstein D.
      • Herron G.S.
      • Sink R.K.
      • et al.
      Fractional photothermolysis: a new concept for cutaneous remodeling using microscopic patterns of thermal injury.
      ) is a nonselective cousin of SP, which uses focused laser microbeams to make hundreds to thousands of small, vertical columns of thermal injury per cm2 of skin. Each microthermal damage zone is about the size of a hair follicle and heals by local remodeling rather than by scar tissue (
      • Laubach H.J.
      • Tannous Z.
      • Anderson R.R.
      • et al.
      Skin responses to fractional photothermolysis.
      ;
      • Hantash B.M.
      • Bedi V.P.
      • Kapadia B.
      • et al.
      In vivo histological evaluation of a novel ablative fractional resurfacing device.
      ). This fact poses a basic, unanswered question for skin biology-what are the criteria and mechanisms for which a small wound stimulates tissue remodeling rather than replacement with scar tissue? In practice, the laser technology previously used for skin resurfacing was reborn. Fractional ablative CO2 and erbium lasers are now popular, and a non-ablative fractional laser is even approved for consumer use at home. Looking forward, fractional laser treatment will probably find a number of unexpected uses. This is the first laser treatment that accurately controls both anatomic depth and the percentage (fraction) of skin treated. Deep channels created by ablative fractional lasers can deliver topically applied drugs and macromolecules into skin (
      • Haedersdal M.
      • Sakamoto F.H.
      • Farinelli W.A.
      • et al.
      Fractional CO2 laser-assisted drug delivery.
      ). Surprisingly, even dense scars heal by remodeling after fractional laser treatment, with the creation of normal elastic fibers, and in some cases new hair follicles. Still, the present generation of fractional lasers is "dumb"-unlike SP, they are not targeted. Potentially, imaging could be used to make "smart" fractional microbeam lasers, i.e., treatment devices that offer software-programmable targeting.
      In vivo laser microscopy has already revolutionized biological research, and it could eventually change dermato- pathology. There are several distinct strategies. Confocal scanning laser microscopy captures reflected or fluorescent light from a thin (~4 mm) plane inside the tissue, corresponding to a scanned laser focal spot. Resolution is equal to conventional light microscopy, revealing cells and extracellular matrix architecture. Pigmented lesions, capillary blood flow, inflammatory cell migration, melanoma, and non-mela-noma skin cancer margins can be seen, without harming the tissue (
      • Rajadhyaksha M.
      • Grossman M.
      • Esterowitz D.
      • et al.
      In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast.
      ). However, depth of imaging is limited to the superficial dermis, and there are no stains-confocal scanning laser microscopy is an exercise in microscopic morphology per se. In contrast, optical coherence tomography is the optical equivalent of ultrasound, using an interferometer to detect the depth from which light is back-scattered inside the tissue (
      • Huang D.
      • Swanson E.A.
      • Lin C.P.
      • et al.
      Optical Coherence Tomography.
      ). Optical coherence tomography is widely used in ophthalmology, cardiology, and (emerging) gastroenterology for highspeed microscopy of retina, coronary arteries, and esophagus, respectively. Recent versions of optical coherence tomography can produce the deep vertical sections familiar to dermato- pathologists, show 3-D microvasculature of carcinomas in exquisite detail, and combine cellular resolution, high speed, depth, and large field of view. (
      • Vakoc B.J.
      • Lanning R.M.
      • Tyrrell J.A.
      • et al.
      Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging.
      )
      The advent of femtosecond (10-15 s) lasers made it possible to achieve very high power density at a focal spot, without depositing much energy. When power density exceeds about 109W/cm2, more than one photon is absorbed simultaneously by a chromophore molecule. Multiphoton microscopy stimulates fluorescence in a thin image plane within live tissue. Multiphoton laser microscopy is widely used in the biological sciences, to image fluorescent probes and optical reporter genes such as green fluorescent protein. In skin, the endogenous fluoro- phores of tryptophan, NADH, and elastin provide detailed views of cells, extracellular matrix, and eosinophils (
      • Masters B.R.
      • So P.T.
      • Gratton E.
      Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin.
      ). A radically different form of laser microscopy is photoacoustic imaging, in which a laser pulse is used to excite acoustic waves that are detected at the tissue surface. Photoacoustic imaging combines the excellent contrast of light, with the excellent depth of ultrasound imaging. Pigmented melanoma and other tumors can be imaged in detail, up to several centimeter deep into tissue (
      • Wang L.V.
      • Hu S.
      Photoacoustic tomography: in vivo imaging from organelles to organs.
      ). There is an unprecedented opportunity now to "translate" these imaging technologies into clinical use for dermatopathology and dermatology.

      Conflict of Interest

      The author states no conflict of interest.

      Acknowledgements

      The author receives a portion of royalties from patents owned and licensed by Massachusetts General Hospital, related to laser hair removal.

      To Cite this Article

      Anderson RR (2013) Lasers for dermatology and skin biology. J Invest Dermatol 133: E21-E23.

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