Confocal Reflectance Microscopy:
`Diagnosis of Skin Cancer Without Biopsy?
`
`MILIND RAJADHYAKSHA
`Lucid, Inc., Henrietta, New York
`and
`Massachusetts General Hospital, Boston
`
`INTRODUCTION
`
`X-ray tomography, magnetic resonance imaging, and ultrasound are non-
`invasive biomedical imaging modalities commonly used in the clinic. These
`imaging modalities have resolutions of 10 to 1,000 µm, which allows assessment
`of the gross (macro) structure of living tissue but not its detailed cellular and
`nuclear microstructures. Clinical assessment of cellular and nuclear microstruc-
`ture (histology) requires a resolution of 0.1 to 10 µm and is performed by con-
`ventional optical microscopy. Conventional microscopy is invasive: one must
`remove (biopsy) the tissue, fix or freeze, excise into thin sections (typically
`5-µm slices), and stain with dyes to enhance contrast. Biopsies destroy the site
`being investigated and prevent subsequent imaging of dynamic events. Tissue
`processing introduces artifacts and is expensive and time consuming. An alter-
`native technique that potentially avoids biopsies or tissue processing is confocal
`reflectance microscopy. A confocal microscope can noninvasively image cellu-
`lar and nuclear microstructures in thin sections within living tissue with high
`resolution and contrast (Pawley, 1995; Webb, 1996).
`Confocal reflectance microscopy offers a noninvasive window into living
`human tissue for basic and clinical research. No biopsy, processing, or staining
`of tissue is necessary. Imaging is based on the detection of backscattered light
`with contrast due to naturally occurring refractive index variations of tissue
`microstructures. Between 1991 and 1996, scientists at L’Oreal in France dem-
`onstrated confocal imaging of living human skin with a white-light tandem scan-
`ning microscope (Bertrand and Corcuff, 1994; Corcuff et al., 1993, 1996; Corcuff
`and Leveque, 1993; New et al., 1991). In 1995 we developed a confocal scan-
`ning laser microscope for real-time imaging of human tissues (Rajadhyaksha et
`3SHAPE EXHIBIT 1063
`3Shape v. Align
`IPR2019-00160
`National, Academy of Engineering. Frontiers of Engineering : Reports on Leading Edge Engineering from the 1998 NAE
` Symposium on Frontiers of Engineering, National Academies Press, 1999. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/csusm/detail.action?docID=3375614.
`Created from csusm on 2018-10-10 10:20:25.
`
`24
`
`Copyright © 1999. National Academies Press. All rights reserved.
`
`

`

`Confocal Reflectance Microscopy: Diagnosis of Skin Cancer Without Biopsy?
`
`25
`
`al., 1995). Cellular and nuclear microstructures in normal human skin and skin
`cancers, and dynamic events such as circulating blood flow, the response of skin
`to ultraviolet light, and wound healing were investigated (Rajadhyaksha and
`Zavislan, 1998).
`
`MOTIVATION
`
`Dermatologists spend a large portion of their time diagnosing skin lesions or
`“moles” (see Figure 1). These lesions are of various shapes, sizes, and colors.
`Clinical screening is initially performed with either the naked eye or a low-
`power microscope (i.e., magnifying glass). Often the initial screening is not
`reliable because different cancers may look alike on the skin surface, and clinical
`pictures (such as Figure 1) do not reveal the subsurface tissue cellular and nucle-
`ar microstructures. Consequently, the accuracy of clinical screening of skin
`cancers is low; for example, the success rate for detecting melanomas (the most
`serious and potentially fatal skin cancer) is only 60 to 90 percent, depending on
`the dermatologist’s expertise. Biopsies are almost always required for an accu-
`rate diagnosis. Of the approximately 3 million biopsies performed annually in
`the United States, 60 to 80 percent turn out to be noncancerous and therefore
`could have been avoided. Confocal reflectance microscopy offers dermatolo-
`gists a noninvasive real-time imaging diagnostic tool that may potentially be
`useful for either screening lesions prior to biopsy or for diagnosis without biopsy.
`
`PRINCIPLES OF CONFOCAL MICROSCOPY
`
`Confocal microscopy is one of those wonderful ideas that seem obvious but
`only after someone else (Minsky, 1957) has intuitively figured it out. A confocal
`
`FIGURE 1 Clinical appearance of a melanoma, which is the most serious and potentially
`fatal skin cancer. Clinical screening is based on this low-magnification, low-resolution
`photograph, which does not reveal subsurface tissue cellular and nuclear microstructures.
`By comparison, the microstructure of this melanoma can be noninvasively visualized
`with confocal microscopy (Figure 6b); thus, confocal imaging can potentially improve the
`accuracy of clinical screening.
`
`Copyright © 1999. National Academies Press. All rights reserved.
`
`National, Academy of Engineering. Frontiers of Engineering : Reports on Leading Edge Engineering from the 1998 NAE
` Symposium on Frontiers of Engineering, National Academies Press, 1999. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/csusm/detail.action?docID=3375614.
`Created from csusm on 2018-10-10 10:20:25.
`
`

`

`26
`
`BIOMATERIALS AND OPTICAL IMAGING FOR BIOMEDICINE
`
`FIGURE 2 A confocal microscope can noninvasively image a thin plane (section) that is
`in focus in a scattering object. This is called optical sectioning. The small aperture in
`front of the detector collects only the light that is in focus (solid lines) while spatially
`filtering light that is out of focus (dotted lines). Although the light is shown to penetrate
`the object, imaging of living tissue is based on the detection of backscattered light (Fig-
`ure 3). SOURCE: Reprinted with permission from Mediscript Ltd. (Rajadhyaksha and
`Zavislan, 1998).
`
`microscope (see Figure 2) consists of a “point” or small source of light that
`illuminates a “point” or small spot within an object, and the illuminated spot is
`then imaged onto a detector through a “point” or small aperture. The source,
`illuminated spot, and detector aperture are placed in optically conjugate focal
`planes, so we say they are “confocal” to each other. The detector aperture size is
`matched to the illuminated spot size through the intermediate optics. Because
`we illuminate a small spot and detect through a small aperture, we image only
`the plane that is in focus within the object. Light originating in planes that are
`out of focus is spatially filtered from entering the detector. A confocal micro-
`scope thus allows noninvasive imaging of a thin plane (section) within a scatter-
`ing object with high axial (and also lateral) resolution; because we reject all the
`light that is not in focus, the image has high contrast. This is known as “optical
`sectioning.”
`The arrangement in Figure 2 shows that only a single spot may be illuminat-
`ed and imaged at a time. Imaging a single point is often not useful in medicine.
`To view the whole object, one must then scan the illuminated spot over the
`desired field of view. We illuminate the object point by point in a two-dimen-
`sional raster and then create the image correspondingly point by point. Scanning
`may be done by either moving the specimen relative to a stationary illumination
`beam (object scanning) or by moving the beam relative to a stationary specimen
`(beam scanning). For imaging of human beings, beam scanning is obviously
`easier than object scanning. Although the configuration in Figure 2 shows the
`light transmitting through the object, imaging of living human tissue is based on
`
`Copyright © 1999. National Academies Press. All rights reserved.
`
`National, Academy of Engineering. Frontiers of Engineering : Reports on Leading Edge Engineering from the 1998 NAE
` Symposium on Frontiers of Engineering, National Academies Press, 1999. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/csusm/detail.action?docID=3375614.
`Created from csusm on 2018-10-10 10:20:25.
`
`

`

`Confocal Reflectance Microscopy: Diagnosis of Skin Cancer Without Biopsy?
`
`27
`
`FIGURE 3 Confocal imaging is based on the detection of backscattered light, with the
`illumination source and detector being on the same side of the human being. SOURCE:
`Reprinted with permission from Mediscript Ltd. (Rajadhyaksha and Zavislan, 1998).
`
`the detection of backscattered light (see Figure 3) such that the illumination
`source and the detector are on the same side of the human being.
`
`DEVELOPMENT OF CONFOCAL SCANNING LASER MICROSCOPES
`
`Laboratory Prototype
`
`At Wellman Laboratories, Massachusetts General Hospital (MGH), we built
`a video-rate confocal scanning laser microscope (CSLM) for noninvasive imag-
`ing of living human tissue (Rajadhyaksha et al., 1995). Figure 4 shows the
`optical design of our prototype CSLM. Any laser and wavelength may be used
`for illumination; we use near-infrared 800- to 1,064-nm wavelengths. Near-
`infrared wavelengths are preferred to visible wavelengths because of reduced
`scattering and absorption and hence deeper penetration into tissue (Anderson
`and Parrish, 1981). The collimated laser beam is scanned in the fast (X) direc-
`tion with a rotating polygon mirror and in the slow (Y) direction with an oscillat-
`ing galvanometric mirror. The scanning is at standard video rates of 15.734 kHz
`along X and 60 Hz along Y, so that the images can be displayed in real time on a
`television monitor. Two fields at 60 Hz are interlaced to produce a frame rate of
`30 Hz, which is the standard for television in the United States. The X and Y
`scanning produces a raster of laser beam spots in the back focal plane of the
`objective lens, which, when demagnified by the objective lens, defines the field
`of view at the tissue. For 20× to 100× objective lenses that we usually use, the
`field of view is 800 to 160 µm. The raster illuminates an XY plane within the
`tissue through a standard microscope objective lens. The XY plane is a horizon-
`tal plane parallel to the surface of the tissue, and the Z axis (optical axis of the
`
`Copyright © 1999. National Academies Press. All rights reserved.
`
`National, Academy of Engineering. Frontiers of Engineering : Reports on Leading Edge Engineering from the 1998 NAE
` Symposium on Frontiers of Engineering, National Academies Press, 1999. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/csusm/detail.action?docID=3375614.
`Created from csusm on 2018-10-10 10:20:25.
`
`

`

`28
`
`BIOMATERIALS AND OPTICAL IMAGING FOR BIOMEDICINE
`
`FIGURE 4 Laboratory-prototype confocal scanning laser microscope for imaging living
`human skin. The illumination source is the collimated output beam from a near-infrared
`800- to 1,064-nm laser (L). The collimated laser beam is scanned in the fast direction
`with a rotating polygon mirror (P) and in the slow direction with an oscillating galvano-
`metric mirror (G). The scanning produces a two-dimensional raster of laser beam spots
`that illuminate the skin through an objective lens (O). Light that is backscattered from the
`tissue retraces its path through the objective lens and the two scanners. The returned light
`is descanned at the two scanners and then separates from the illumination path at the
`beamsplitter (BS). Beyond the beamsplitter, the backscattered light is detected through a
`pinhole (P) with a silicon avalanche photodiode (D). The detector output is sent to a
`television monitor (TV). The control and video-timing electronics is not shown.
`
`CSLM) is perpendicular to it. The intermediate optics consist of folding mirrors
`and achromatic lenses. Light that is backscattered from the tissue retraces its
`path through the objective lens and the two scanners. The returned light is
`descanned at the two scanners and then separates from the illumination path at
`the beamsplitter. Beyond the beamsplitter, the backscattered light is detected
`through a pinhole by a silicon avalanche photodiode. The detector output is sent
`to a television monitor and storage devices such as a super-VHS videotape re-
`corder and an 8-bits/pixel frame grabber. We built the control and video-timing
`electronics using well-known designs from the confocal scanning laser ophthal-
`moscope (Webb and Hughes, 1981; Webb et al., 1987).
`Confocal imaging of living tissue is most useful if the resolution is similar
`to that of conventional microscopy (histology), so that cellular and nuclear micro-
`structures can be seen. Histology involves viewing of typically 5-µm thin sec-
`tions with lateral resolution of 1 µm. We optimized the CSLM design and
`operating parameters to achieve lateral resolution of 0.5 to 1 µm and axial reso-
`
`Copyright © 1999. National Academies Press. All rights reserved.
`
`National, Academy of Engineering. Frontiers of Engineering : Reports on Leading Edge Engineering from the 1998 NAE
` Symposium on Frontiers of Engineering, National Academies Press, 1999. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/csusm/detail.action?docID=3375614.
`Created from csusm on 2018-10-10 10:20:25.
`
`

`

`Confocal Reflectance Microscopy: Diagnosis of Skin Cancer Without Biopsy?
`
`29
`
`lution (section thickness) of 2 to 5 µm. Confocal microscopy provides the highest
`resolution yet of any noninvasive imaging modality, and this resolution compares
`very well to that of histology.
`
`Commercial Product
`
`Our research at MGH-Wellman Laboratories with the prototype CSLM dem-
`onstrated that video-rate confocal imaging of cellular and nuclear microstruc-
`tures with near-infrared light is possible noninvasively in living human skin
`(Rajadhyaksha et al., 1995; Rajadhyaksha and Zavislan, 1997, 1998). In 1997,
`Lucid, Inc. (Henrietta, N.Y.) entered into a five-year partnership with MGH-
`Wellman Labs to commercialize this technology for basic and clinical skin re-
`search. Engineers at Lucid, in collaboration with MGH scientists, reengineered
`the CSLM prototype into a turnkey user-friendly confocal imaging system called
`the VivaScope™, with flexible user-controlled operating parameters (see Fig-
`ure 5). The VivaScope™ is much smaller than the prototype and portable, so we
`can move it easily between different labs and clinics. The optics and their
`mounts are robust, so alignment is not necessary, and setup time is 15 minutes
`when we move it to a new location. It is supported on a stand that can be raised
`or lowered relative to the skin site on the human subject to be imaged. We have
`built an arm and rotatable head that allow easy interfacing to different sites such
`as arms, legs, back, torso, face, and scalp. Stable imaging at different sites on
`the body is possible when using a specially designed skin-to-CSLM contact
`device.
`
`Copyright © 1999. National Academies Press. All rights reserved.
`
`FIGURE 5 Commercial confocal scanning laser microscope (VivaScope™) for imaging
`living human skin.
`
`National, Academy of Engineering. Frontiers of Engineering : Reports on Leading Edge Engineering from the 1998 NAE
` Symposium on Frontiers of Engineering, National Academies Press, 1999. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/csusm/detail.action?docID=3375614.
`Created from csusm on 2018-10-10 10:20:25.
`
`

`

`30
`
`BIOMATERIALS AND OPTICAL IMAGING FOR BIOMEDICINE
`
`IMAGING OF LIVING HUMAN SKIN
`
`Clinical Applications: Normal Skin Versus Skin Cancers
`
`Confocal microscopy of normal human skin reveals cellular and nuclear
`microstructures in the stratum corneum and epidermis, and collagen and blood
`flow in the underlying dermis (Rajadhyaksha et al., 1995). We can image the
`superficial 100-µm thin epidermis and to a maximum depth of 350 µm in the
`dermis. Epidermal and dermal features in the confocal images were qualitatively
`and quantitatively analyzed; these correlated well to the corresponding histology.
`The epidermal features were cellular and nuclear shape and size, internucleus
`spacing, nuclear/cytoplasm ratio, cellular density, and spatial distribution of
`melanin (pigment that gives color to our skin). The shape, size, and density of
`circulating blood cells in the superficial dermal capillaries were measured. Mor-
`phological features included thickness of stratum corneum and epidermis and
`modulation depth of the epidermal-dermal junction. A group at L’Oreal has
`demonstrated similar imaging in normal human skin with their white light tan-
`dem scanning confocal microscope (Bertrand and Corcuff, 1994; Corcuff and
`Leveque, 1993; Corcuff et al., 1993, 1996; New et al., 1991). Two other groups
`have more recently reported images of skin (Masters et al., 1997) and skin struc-
`tures such as nails (Kaufman et al., 1995).
`Skin cancers appear different from normal skin (Figure 6). They have patho-
`logical differences such as atypical cells and nuclei that have abnormal shapes
`and sizes, lateral and vertical spreading of abnormal cells and formation of
`clusters of these cells, increase in melanin content and changes in its spatial
`distribution, formation of star-shaped projections (dendrites), loss of epidermal
`structure, elongation in the epidermal-dermal junction, and increased number of
`blood vessels and blood flow. In early clinical studies we have characterized
`melanomas and basal and squamous cell cancers. The control images were those
`of normal skin adjacent to the lesions. Preliminary analysis showed reasonably
`good qualitative correlation between the confocal images and the histology. Clin-
`ical research is currently progressing in the characterization of skin cancers as
`well as benign (noncancerous) lesions and other types of disorders, including
`detection of margins between lesions and surrounding normal tissue.
`
`Basic Research Applications
`
`Confocal microscopy is an excellent imaging modality for basic skin (and
`other tissue) research. We can study living skin in its native state, without the
`artifacts of biopsy and histological processing. Because it is noninvasive, dy-
`namic changes can be imaged over hours, days, weeks, and months. For exam-
`ple, we have investigated the response of the skin to ultraviolet-B (UVB) irradi-
`ation from sunlight, the process of wound healing, inflammatory responses to
`allergic or irritant agents, and delivery of drugs through blood vessels. Other
`
`Copyright © 1999. National Academies Press. All rights reserved.
`
`National, Academy of Engineering. Frontiers of Engineering : Reports on Leading Edge Engineering from the 1998 NAE
` Symposium on Frontiers of Engineering, National Academies Press, 1999. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/csusm/detail.action?docID=3375614.
`Created from csusm on 2018-10-10 10:20:25.
`
`

`

`Confocal Reflectance Microscopy: Diagnosis of Skin Cancer Without Biopsy?
`
`31
`
`FIGURE 6 Pathological differences in cellular and nuclear microstructures between
`normal human skin (a) and skin cancers (b-d) can be noninvasively visualized with
`confocal microscopy. The skin cancers shown are melanoma (b), basal cell carcinoma
`(c), and squamous cell carcinoma (d). Normal skin epidermis contains a regular chicken
`wire-mesh network of circular, oval, or polygonal cells. By comparison, the skin cancers
`show distinct differences: (b) melanoma cells develop star-shaped projections called
`dendrites (arrow), (c) basal cell carcinomas show increases in size and migration of basal
`cells from the superficial epidermis into the underlying dermis (arrow), (d) squamous cell
`carcinomas show changes in the shape and size of squamous cells in the superficial
`epidermis (arrow). Scale bar = 25 µm.
`
`exciting applications may include cell-to-cell interactions, microcirculation, drug
`delivery through the skin, photoaging, and artificial tissue.
`
`FUTURE
`
`Confocal imaging of living tissue is a new imaging modality. Our research
`effort will increase understanding of the morphology of normal versus abnormal
`skin. Other tissue types to which we have applied confocal microscopy are oral
`(lip and tongue) mucosa and bladder. When we compare confocal images to
`histology slides (i.e., the gold standard), two limitations are obvious: (1) confocal
`microscopy can probe only the upper 0.5 mm of tissue over fields of view limit-
`ed to less than 1 mm, whereas histology probes down to depths of 2 to 3 mm
`over large fields of view, typically 5 mm; and (2) the use of dyes to stain specific
`cell types enhances tissue contrast, so that critical information necessary for
`diagnosis can be easily read in histology, whereas confocal microscopy relies on
`the natural (low) reflectance contrast of tissue without the advantages of stains.
`At present we are not certain whether diagnosis of skin cancers would be possi-
`
`Copyright © 1999. National Academies Press. All rights reserved.
`
`National, Academy of Engineering. Frontiers of Engineering : Reports on Leading Edge Engineering from the 1998 NAE
` Symposium on Frontiers of Engineering, National Academies Press, 1999. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/csusm/detail.action?docID=3375614.
`Created from csusm on 2018-10-10 10:20:25.
`
`

`

`32
`
`BIOMATERIALS AND OPTICAL IMAGING FOR BIOMEDICINE
`
`ble with confocal reflectance microscopy. However, results from our clinical
`studies strongly suggest that confocal microscopy may potentially be useful for
`screening skin cancers versus benign (noncancerous) lesions and normal skin,
`including detection of margins between the lesions and surrounding normal skin.
`Confocal images may thus provide a useful adjunct to clinical screening and
`histology by helping a dermatologist make decisions such as whether, where, or
`when to biopsy a lesion.
`Further development of confocal scanning microscopes to make them highly
`effective for basic research and clinical applications includes several scientific
`and instrumentation challenges. The main scientific challenge is to understand
`light-tissue interaction and mechanisms of contrast and to determine techniques
`to enhance tissue contrast by staining cells types with reflectance microparticles.
`The instrumentation challenges are to make confocal imaging as similar to his-
`tology as possible through vertical sectioning, increasing the depth of imaging,
`and enlarging the field of view. As with any new imaging modality, we must
`learn to interpret, analyze, and extract meaningful information from the confocal
`images. This will involve an extensive correlation of confocal images to con-
`ventional (gold standard) histology.
`The future will see the development of inexpensive handheld confocal
`microscopes that will become commonplace in the clinic and linked to each
`other in a telemedicine network. Although this is being initially applied to easily
`accessible tissues (skin, oral), the combination of confocal microscopy with other
`techniques such as laparoscopy should enable imaging of internal organs. Ulti-
`mately, optical imaging must be combined with nonoptical imaging modalities
`to create a noninvasive diagnostic tool kit for the medical profession.
`
`ACKNOWLEDGMENTS
`
`Instrumentation development was funded in part by the U. S. Department of
`Energy and a Whitaker Foundation grant to M. Rajadhyaksha when he was at
`MGH-Wellman Labs. Clinical and basic research in skin, oral mucosa, and
`bladder was funded by Lucid, Inc. Credit for research goes to Richard Langley
`and Melanie Grossman (pigmented skin lesions), Steve Ugent (UVB effects),
`Salvador Gonzalez (tumor margins, proliferative and inflammatory disorders),
`Frank Koenig (bladder), and W. Matthew White (oral tissue) at MGH-Wellman
`Labs.
`
`REFERENCES
`
`Anderson, R. R., and J. A. Parrish. 1981. The optics of human skin. Journal of Investigative
`Dermatology 77(1):13–19.
`Bertrand, C., and P. Corcuff. 1994. In vivo spatio-temporal visualization of the human skin by real-
`time confocal microscopy. Scanning 16(3):150–154.
`
`Copyright © 1999. National Academies Press. All rights reserved.
`
`National, Academy of Engineering. Frontiers of Engineering : Reports on Leading Edge Engineering from the 1998 NAE
` Symposium on Frontiers of Engineering, National Academies Press, 1999. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/csusm/detail.action?docID=3375614.
`Created from csusm on 2018-10-10 10:20:25.
`
`

`

`Confocal Reflectance Microscopy: Diagnosis of Skin Cancer Without Biopsy?
`
`33
`
`Corcuff, P., and. J. L. Leveque. 1993. In vivo vision of the human skin with the tandem scanning
`microscope. Dermatology 186(1):50–54.
`Corcuff, P., C. Bertrand, and J. L. Leveque. 1993. Morphometry of human epidermis in vivo by
`real-time confocal microscopy. Archives of Dermatology Research 285(8):475–481.
`Corcuff, P., G. Gonnord, G. E. Pierard, and J. L. Leveque. 1996. In vivo confocal microscopy of
`human skin: A new design for cosmetology and dermatology. Scanning 18(5):351–355.
`Kaufman, S. C., R. W. Beuerman, and D. L. Greer. 1995. Confocal microscopy: A new tool for the
`study of the nail unit. Journal of the American Academy of Dermatology 32(4):668–670.
`Masters, B. R., G. Gonnord, and P. Corcuff. 1997. Three-dimensional microscopic biopsy of in vivo
`human skin: A new technique based on a flexible confocal microscope. Journal of Microscopy
`185(Part 3):329–338.
`Minsky, M. November 7, 1957. Microscopy Apparatus. U.S. Patent No. 3013467.
`New, K. C., W. M. Petroll, A. Boyde, L. Martin, P. Corcuff, J. L. Leveque, M. A. Lemp, H. D.
`Cavanagh, and J. V. Jester. 1991. In vivo imaging of human teeth and skin using real-time
`confocal microscopy. Scanning 13(5):369–372.
`Pawley, J. B., ed. 1995. Handbook of Biological Confocal Microscopy, 2nd ed. New York:
`Plenum Press.
`Rajadhyaksha, M., and J. M. Zavislan. 1997. Confocal laser microscope images tissue in vivo.
`Laser Focus World 33(2):119–127.
`Rajadhyaksha, M., and J. M. Zavislan. 1998. Confocal reflectance microscopy of unstained tissue in
`vivo. Retinoids & Lipid-Soluble Vitamins in Clinical Practice 14(1):26–30.
`Rajadhyaksha, M., M. Grossman, D. Esterowitz, R. H. Webb, and R. R. Anderson. 1995. In vivo
`confocal scanning laser microscopy of human skin: Melanin provides strong contrast. Journal
`of Investigative Dermatology 104(6):946–952.
`Webb, R. H. 1996. Confocal optical microscopy. Reports on Progress in Physics 59(3):427–471.
`Webb, R. H., and G. W. Hughes. 1981. Scanning laser ophthalmoscope. IEEE Transactions on
`Biomedical Engineering 28(7):488–492.
`Webb, R. H., G. W. Hughes, and F. C. Delori. 1987. Confocal scanning laser ophthalmoscope.
`Applied Optics 26(8):1492–1499.
`
`Copyright © 1999. National Academies Press. All rights reserved.
`
`National, Academy of Engineering. Frontiers of Engineering : Reports on Leading Edge Engineering from the 1998 NAE
` Symposium on Frontiers of Engineering, National Academies Press, 1999. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/csusm/detail.action?docID=3375614.
`Created from csusm on 2018-10-10 10:20:25.
`
`

`

`Copyright © 1999. National Academies Press. All rights reserved.
`
`National, Academy of Engineering. Frontiers of Engineering : Reports on Leading Edge Engineering from the 1998 NAE
` Symposium on Frontiers of Engineering, National Academies Press, 1999. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/csusm/detail.action?docID=3375614.
`Created from csusm on 2018-10-10 10:20:25.
`
`

`

`ADVANCED MATERIALS
`
`Copyright © 1999. National Academies Press. All rights reserved.
`
`National, Academy of Engineering. Frontiers of Engineering : Reports on Leading Edge Engineering from the 1998 NAE
` Symposium on Frontiers of Engineering, National Academies Press, 1999. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/csusm/detail.action?docID=3375614.
`Created from csusm on 2018-10-10 10:20:25.
`
`

`

`Copyright © 1999. National Academies Press. All rights reserved.
`
`National, Academy of Engineering. Frontiers of Engineering : Reports on Leading Edge Engineering from the 1998 NAE
` Symposium on Frontiers of Engineering, National Academies Press, 1999. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/csusm/detail.action?docID=3375614.
`Created from csusm on 2018-10-10 10:20:25.
`
`