Replica Microscopy and Scanning Electron Microscopy of Laser Impacts on the Skin

Abstract

Reactions following the impact of the laser beam on the skin surface are complex including significant thermal mechanisms with elastic recoil and pressure phenomena. Such forces then can produce tissue changes about the target area. Elastic recoil and pressure phenomena are found especially with pulsed-laser systems and not as much with the continuous-wave lasers which are used as optical knives. Because of the availability and relative ease of examination, the skin serves as an excellent test model system for the study of the laser reaction in living tissue. Direct examination of the skin surface, fixed tissue sections for both light microscopy, including histochemistry, and transmission electron microscopy have given many details of the coagulation necrosis in target areas induced by the laser impact, but very little detail of changes in the adjacent areas and practically none for surface changes. MATERIALS AND METHODS To study the surface topography of the target and adjacent areas of living skin, the following techniques were used : 1. Before laser impact a. Direct examination of the skin with skin microscopes (1). b. Photography of the skin in color at IX, and microphotography with the skin microscope at 20 to 50X. c. Replica microscopy of the skin surface with silicone-rubber negatives and various materials as positive replicas. d. Skin biopsies with routine histologic and histochemical studies, scanning electron microscopy (Stereoscan) and transmission electron microscopy (Elmskop I). 2. Pulsed and continuous-wave laser systems were used for testing experimental subjects on the flexor surface of the forearm in normal skin and for the treatment of portwine lesions and tattoos. Tattoos included also puncture spots of India ink in the skin. a. After laser impacts, skin microscopy and photography in color at IX and 20-50X. b. After laser impacts, repeat of replica microscopy, biopsies, scanning electron microscopy and transmission electron microscopy. Portable skin microscopies (20X) and large flexible stereobinocular skin microscopies 50-80X were used. Microphotography was done in black and white and in color at 20 and 50X. Replicas of the skin surface were done with silicone rubber technique of Sampson (2), Sarkany (3), and Facq (4). Positive replicas were made of silicone rubber, paraffin, wax, polyvinyl alcohol, latex rubber, Alginats after the technique of Cselpak and Martin (5), plaster of Paris and acrylic resin. The scanning electron microscope used was the Stereo-scan. Skin specimens for scanning electron microscopy were fixed in formaldehyde and also liquid nitrogen, then freeze dried. Skin tissues and replicas were coated in vacuo with copper and gold to make it conductive. A fine electron probe with beam diameters of the order of 100 A° scans the surface. Resolution of the scanning electron microscope is 10X the light microscope, the depth of field 500-1000X the light microscope. In these studies, magnifications of 60 to 5000X were used. Transmission electron microscopy was done for the treated areas and controls after glutaraldehyde and osmium tetroxide fixation. RESULTS Skin microscopy revealed the local charring reaction and the development of smooth surfaces of erythema-edema (Fig. 1). Occasionally after laser impacts of darkly pigmented tattoos, radiating stress lines about the target area could be seen. 50X represented the limit of good definition of skin microscopy, with skin 18 REPLICA MICROSCOPY AND SCANNING ELECTRON MICROSCOPY 19 Fig. 1. Negro skin, forearm, showing small crater and charring from low-output pulsed-ruby laser impact, which had no effect on Caucasian skin. 20X 5). In some specimens, striking pictures were evident about the definite craters of laser impacts. With tattoo spots, deeper and broader craters were observed. For some distance about the crater, irregular folds, fissures, heavy crusting and scaling could be observed. In general, more detailed topographical changes could be seen than were observed with the biopsy studies from the light microscope. Artifacts were usually more extensive with the replicas. These ; were in the form of crystals with plaster of Paris, granular surfaces with the Alginat, bubbles with polyvinyl alcohol, and pits with paraffin sections. In our experience, thick, smooth, preparations of liquid latex gave the least num- '. ber of artifacts (Figs. 6A&6B). In some good replicas, isolated areas of scaling of the surface of normal skin was observed in great de- i tail. In replicas of laser impact specimens, dis- ; tortion about target area was extensive. Again, f this was more extensive than could be detected -with light microscopy of the replicas. With the instrumentation now available, direct examination of the skin surface after laser i impacts can be done only up to magnifications ; of 50X. This does give some information of immediate and delaved erythema-edema reac-tions about the calculated target area. Light surface photography 25X was the usual limit of good pictures, occasionally 50X. Studies are under way with illumination of the skin surface and trans-illumination skin micro-photography with helium-neon and krypton laser beams to improve depth of focus and resolution. Routine histological studies showed coagulation necrosis of the target area (6). (Figs. 2A&2B). With Q-switched ruby laser impacts on normal skin, microscopic sections revealed minimal intra-epidermal “steam-bubble” areas not detected on skin microscopy. PAS, Feulgen and glycogen studies of areas adjacent to the target showed no changes. In 8 specimens, transmission electron microscopy showed fragmentation and distortion of epidermis and collagen fibers especially limited to the target area (Fig. 3). Twenty-one specimens were studied with scanning electron microscopy. As Loomans has indicated, it was difficult to establish patterns of normal skin even on the relatively “stable” area as the flexor surface of the forearm (Fig. 4). Few specimens revealed smooth surfaces; many areas of normal skin showed lines and folds. These were evident even on control specimens of skin taken from a bald head (Fig. 20 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY SSSS'J-Mfe 7>- *. r sp>../-- mP.'sj.?,' t*- '.-.---.?.. . ' -./' '>- K'.'ft:v> sp>' ''• '- : sp>-' - '-'-; * T SB>: ” : >. ..-.v- \t ' ' j.- ?.. ' ; Fig. 2. A. Microscopic section of normal skin showing slight epidermal changes after impact of Q-switched ruby laser, 25 megawatts peak power output, 10 nanoseconds on flexor surface of forearm of Caucasian adult, hematoxylin-eosin 200X B. Microscopic section of ink-spot area of the same forearm showing localized intra-epidermal vesicle formation, hematoxylin-eosin 200X REPLICA MICROSCOPY AND SCANNING ELECTRON MICROSCOPY 21 f*f . J; i-- -J* . “ . . .' * ** JJV- . - f#'. j Si Fio. 3. Edge of dark tattoo on flexor surface of forearm immediately after laser impact of ruby laser 75 joules/cm2 sp>. Fig. 4. Replica microscopy of colored plaster of Paris positive of silicone rubber negative of normal skin of forearm. 50X 22 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Fig. 5. Scanning electron microscopy of skin of bald scalp showing bizarre folds (artifacts?) A. 124X B. 376X microscopy and even transmission electron microscopy, as yet, does not offer much for detail other than in the target area. Replica microscopy for direct observation with our available instrumentation also does not give data much beyond 80X. The vast amount of artifacts with many diverse types of positive replicas made it difficult and hazardous to interpret changes about the craters produced by lasers. The value of the scanning electron microscope in the study of laser impacts on the teeth has been shown by Vahl (7). In this hard tissue, the characteristics of the crater and adjacent changes can be observed in great detail. Also, in the study of bone impacts by the laser, scanning electron microscopy can reveal many details not evident on sections studies under light microscopy. Since scanning electron microscopy has been used in examination of insects, cerebral cortex and also the hair (8), then it can be used also for studies of soft tissues. In our studies occasional areas, free from artifacts, have revealed more topographical detail about the laser impact than was observed with the other techniques. Improvement in technique for replica microscopy and for preparation of skin specimens for scanning electron microscopy should help in determining the true value of this in laser research to find the minimal reactive dose for the skin and the presence or absence of tissue change about the target area. Also, scanning electron microscopy should be of great interest and value in investigative dermatology to bridge the vast area between light and transmission electron microscopy. CONCLUSION To attempt to define precisely the minimal reactions of the laser on living skin and the changes about the target areas, various techniques were used. These included skin microscopy of living skin, replica microscopy, light microscopy of fixed tissues, scanning and electron microscopy. Topographical details of living tissue can be observed satisfactorily with skin microscopies up to 50X. More topographic details can be found with replica microscopy and then scanning electron microscopy. The high percentage of artifacts both with replica microscopy and scanning electron microscopy makes interpretation difficult. More studies are needed to improve these techniques for research in topography of the skin at high magnifications. Fig. 6. Scanning electron microscopy of positive polyvinyl alcohol replica of normal skin of forearm showing one small area of scaling, ridges and furrows, and numerous artifacts. A. 115X B. 344X Wypsavz, 24 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY