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Imaging Melanoma in a Murine Model Using Reflectance-Mode Confocal Scanning Laser Microscopy and Polarized Light Imaging

      The light-scattering properties of cutaneous tissues provide optical contrast for imaging the presence and depth of pigmented melanoma in a highly pigmented murine model, the C57/B6 mouse. Early lesions are difficult to identify when viewing black lesions on a black mouse. Two methods were used to image early lesions in this model. (1) A reflectance-mode confocal scanning laser microscope (rCSLM) was built to provide horizontal images (x–y at depth z) and transverse images (x–z at position y) non-invasively in the living mouse. (2) A polarized light imaging (PLI) camera was built using a linearly polarized white light source that viewed the skin through an analyzing linear polarizer oriented either parallel or perpendicular to the illumination's polarization to yield two images, “PAR” and “PER,” respectively. The difference image, PAR–PER, eliminated multiply scattered light and yielded an image of the superficial but subsurface tissues based only on photons scattered once or a few times so as to retain their polarization. rCSLM could image melanoma lesions developing below the epidermis. PLI could distinguish superficial from deeper melanoma lesions because the melanin of the superficial lesions attenuated the PAR–PER image, whereas deeper lesions failed to attenuate the PAR–PER image.

      Keywords

      rCSLM
      reflectance-mode confocal scanning laser microscope
      PLI
      polarized light imaging
      This report summarizes a presentation at the 53rd annual Montagna Symposium on Skin Biology, Salishan Lodge, Gleneden, Oregon, October 15–19, 2004, which described the use of two novel optical imaging techniques to monitor the onset and progression of melanoma lesions in a highly pigmented murine model, the C57/Black-6 mouse (C57/B6) (
      • Jhappan C.
      • Noonan F.P.
      • Merlino G.
      Ultraviolet radiation and cutaneous malignant melanoma.
      ). The two techniques are (1) reflectance-mode confocal scanning laser microscopy (rCSLM) (
      • Rajadhyaksha M.
      • Grossman M.
      • Esterowitz D.
      • Webb R.H.
      • Anderson R.R.
      Video-rate confocal scanning laser microscopy for human skin: Melanin provides strong contrast.
      ), and (2) polarized light imaging (PLI) with a CCD camera, which was introduced by
      • Jacques S.L.
      • Roman J.
      • Lee K.
      Imaging superficial tissues with polarized light.
      ,
      • Jacques S.L.
      • Ramella-Roman J.C.
      • Lee K.
      Imaging skin pathology with polarized light.
      .
      The ability of the eye to detect early cancer lesions is limited. In human tissues, this task is comparable with discerning a drop of milk on a white plate. There is no contrast based on color. But, there is a difference in light scattering. Techniques such as rCSLM and PLI provide contrast based largely on photon scattering and can discern early changes that appear colorless but influence photon scattering. Imaging early lesions in a highly pigmented skin like the C57/B6 mouse offers a similar challenge, comparable with discerning a drop of black ink on a black plate. Again, the difference in light scattering offers a mechanism of contrast.
      In this report, the ability of rCSLM and PLI to image early melanoma lesions in the C57/B6 mouse is demonstrated. The significance of such imaging is that early lesions can be detected for biopsy, and that lesions can be followed with non-invasive imaging in longitudinal studies to monitor the progression of cancer in this model.
      The rCSLM follows the work of
      • Rajadhyaksha M.
      • Grossman M.
      • Esterowitz D.
      • Webb R.H.
      • Anderson R.R.
      Video-rate confocal scanning laser microscopy for human skin: Melanin provides strong contrast.
      , but uses the short wavelength of an argon ion laser (blue light, 488 nm) to optimize the reflectivity from the very superficial epidermis and sub-epidermal layers of mouse skin. The mouse skin is very thin, e.g., the epidermis varies between 10 and 20 μm in thickness, and imaging must be able to perform well in this superficial region.
      The PLI at first glance would appear to be similar to the current common practice of dermatoscopy, which often illuminates with linearly polarized light and views through a “parallel” polarizer to accent the surface glare or views through a “perpendicular” polarizer to reject the surface glare and accent the multiply scattered light. The latter image back-illuminates blood vessels and melanin and provides very good images based on absorption of light by these structures. But, the image does not offer contrast based on scattering of light by the superficial layers. In contrast, the PLI is designed to reject multiply scattered light and generate an image based only on photons scattered from the superficial tissues, thereby imaging the fabric pattern of the superficial dermis whose disruption by cancer growth becomes discernable. The PLI illuminates the skin from an oblique angle through a glass plate that is optically coupled to the skin by a gel or drop of water, so that surface glare does not enter the camera. Then the difference image, “parallel”–“perpendicular,” subtracts the multiply scattered light that constitutes most of the backscattered light and obscures details of the superficial tissues. Hence, the PLI image might be called an image based on the “subsurface glare” of the superficial tissues, excluding the glare from the skin surface, and the images are not at all like dermatoscopy images. Application of PLI to the black mouse of this study differed from our previous work with PLI on human skin, because the strong absorption by the superficial melanin of this mouse influenced the images and allowed discrimination of superficial versus deeper melanoma.
      The long-term goal of this work is to contribute to on-going efforts to “humanize” the mouse melanoma model so that basic science on melanoma onset and progression can be conducted. Such “humanization” involves developing melanoma models in which melanoma originates in the epidermis rather than in the deeper dermis as in current mouse models. Our rCSLM images can detect the early progression of melanoma in the sub-epidermal layer and its violation of the epidermal–dermal junction. Our PLI images can survey the entire back of a mouse and discriminate early lesions that are superficial versus deeper.

      Results

      rCLSM

      Figure 1 shows examples of rCSLM images. Figure 1a shows a horizontal x–y image at a particular depth z. The x–y plane cuts through the irregular surface of the skin such that the surface of the skin presents as a ring of bright reflectance from the stratum corneum, labeled “S” in the figure. The center of the image is at a depth of 19 μm. The keratinocytes of the viable epidermis present as a pattern of dark regions surrounded by brighter material because nuclear chromatin filaments scatter less than cytoplasm and cell membranes (
      • Rajadhyaksha M.M.
      • Gonzalez S.
      • Zavislan J.M.
      Detectability of contrast agents for confocal reflectance imaging of skin and microcirculation.
      ). Two melanocytes are labeled as “M1” and “M2,” and appear brighter than the surrounding epidermis because of the strong photon scattering from the melanosomes.
      Figure thumbnail gr1
      Figure 1Horizontal images using reflectance-mode confocal scanning laser microscopy for an in vivo mouse dorsal skin site. (a) The stratum corneum (S) is at the skin surface. The depth of the center of image is 19 μm below the skin surface. A keratinocyte (K) shows a typical dark nuclear region surrounded by a brighter cytoplasm and cellular membrane. Two melanocytes (M1, M2) are shown, displaying an increased brightness because of scattering by melanosomes. (b) An 8 μm deeper image. The depth of the center of image is 27 μm below skin surface. The two positions below the melanocytes (M1, M2) now present dark regions because the overlying melanocytes scatter photons that attempt to penetrate into and reflect from the region below each melanocyte.
      Figure 1b shows the same view as Figure 1a; however, the depth of the image is 8 μm deeper. Now, the regions underlying the two melanocytes M1 and M2 appear dark. Apparently, the photons are scattered by the melanocyte in their effort to penetrate into and scatter from the region below the melanocyte. Hence, the photon intensity is strongly attenuated and this region presents as a dark region.
      Figure 2 shows a transverse x–z plane located at one y position. The stratum corneum (SC) strongly scatters light and appears bright. The epidermis (epi) is less strongly scattering and appears as a darker layer. The dermal–epidermal junction (dej) and the underlying dermis are more strongly scattering. The dej includes bright melanocytic cells that strongly scatter light. In the center of the image below the dej there is a tumor (T) roughly 20 μm × 50 μm in size. The tumor scatters light rather strongly relative to the surrounding dermis. A patch of bright melanin-containing cells populates the upper portion of the tumor.
      Figure thumbnail gr2
      Figure 2Transverse images using reflectance-mode confocal scanning laser microscopy. The water/skin surface at the stratum corneum (sc) appears bright. The epidermis (epi) has lower scattering and presents a darker layer. The dermis is strongly scattering and presents a brighter layer. The melanoma appears bright where light first enters the lesion and melanosomes scatter strongly, and appears dark where melanin absorption prevents efficient penetration and escape of photons. In the center of this image, a 50-μm-wide × 20-μm-thick melanoma lesion is centered at a depth of 15 μm below the epidermis. Bright melanocytes are seen at the dermal–epidermal junction (dej).
      Figure 3 shows axial scans through a cultured melanoma cell on a glass cover slip and through a melanocyte within the epidermis of the in vivo mouse skin site. The cultured cells were 1984-1 melanoma cells derived from TP-Ras mice treated with DMBA (
      • Broome P.M.
      • Gause P.R.
      • Hyman P.
      • Gregus J.
      • Lluria-Prevatt M.
      • Nagle R.
      • Bowden G.T.
      Induction of melanoma in TPras transgenic mice.
      ). Figure 3a shows an axial scan through one melanoma cell and the underlying glass slide. This axial scan allows a quantitative assessment of the reflectivity of the melanoma cell (Rmelanoma=0.42 × 10-3) using the water/glass interface as a calibration standard (Rwater–glass=4.4 × 10-3). Figure 3b shows an axial scan through the melanocyte labeled M1 in Figure 1a, illustrating the magnitude of melanocyte reflectance relative to that of the surrounding epidermal cells. The melanocyte reflectance of Figure 3b was tentatively equated with that of the melanoma cell in Figure 3a to achieve calibration, implying that the background reflectance of the epidermis is about 1.5 × 10-4.
      Figure thumbnail gr3
      Figure 3Reflectivity of melanoma cells in reflectance-mode confocal scanning laser microscope images. (a) Axial scan of melanoma cells cultured on a glass cover slip, showing reflectance signal through one melanoma cell and the underlying glass plate. The reflectance of the water/glass interface (4.4 × 10-3) is used as a calibration to allow specification of the reflectance of the melanoma cell (0.42 × 10-3). The scans signals were averaged over the pixels corresponding to one cell. (b) Axial scan of reflectance through the melanocyte labeled M1 in , tentatively calibrated as being similar to the 0.42 × 10-3 cell reflectance of , which implies a background reflectance for the epidermis of 0.15 × 10-3.

      PLI

      Figure 4 shows PER and PER–PAR images for the green channel of the color camera. The pixels values were normalized by the total reflectance from a 100% white reflectance standard, so the values are in fractional units of reflectance, 0–1.00. For example, a value of 0.08 implies that the reflectance was 8%. The PER image Figure 4a consists of D, where D is half of the multiply scattered escaping light. The PAR image (not shown) is brighter because it consists of S+D, where S is the subsurface glare because of single or few scatterings of photons such that the original polarization of the illumination is retained. The difference image, PAR–PER Figure 4b, consists of (S+D)–D=S, which isolates the superficial scattering, S.
      Figure thumbnail gr4
      Figure 4Polarized light images of C57/B6 mouse with melanoma lesions, using the green channel of the color camera. (a) PER image corresponds to deeply multiply scattered light, D, which has randomized the polarization. (b) PAR–PER image isolates the photons that have undergone a single or few scatterings, S, which retains the polarization of the illumination light. The color bar is in reflectance units, where 1.00 indicates the pixel values from a 100% diffuse reflectance standard. Six lesion sites are indicated by labels. A label “A” denotes a superficial melanoma that appears dark in both the PER and PAR–PER images, and a label “B” denotes a deeper melanoma that appears dark in the PER image but lighter in the PAR–PER image because of scattering by the superficial tissues overlying the melanoma.
      Two categories of melanoma structure were observed and labeled A and B in Figure 4: (A) Superficial melanoma lesions, characterized by a low value of PER and a low value of PAR–PER. Multiply scattered light penetrates to the lesion where the melanin absorbs photons, so PER is low. Because the lesion is superficial, even superficially scattered photons are attenuated by the melanoma's melanin, so PAR–PER is low too.
      (B) Deep melanoma lesions, characterized by a low value of PER, but a higher value of PAR–PER. As before, the multiply scattered light penetrates to the lesion and is attenuated, so PER is low. But, the superficially scattered photons scatter off the epidermis and upper dermis and do not reach the deeper melanoma, so PAR–PER is higher.
      Values of PAR (i.e., S+D), PER (i.e., D), and PAR–PER (i.e., S) for the lesions indicated in Figure 4 are summarized in Table I as the mean±standard deviation values for nine pixels centered at the position indicated in Figure 4.
      Table IPixel values of PER, PAR, and PAR–PER for superficial (A) and deeper (B) melanoma lesions
      LesionPER (=D)PAR (=S+D)PAR–PER (=S)
      1A0.0008±0.00020.0045±0.00030.0037±0.0003
      2B0.0016±0.00020.0146±0.00100.0129±0.0010
      3A0.0012±0.00050.0071±0.00080.0058±0.0008
      4B0.0021±0.00020.0139±0.00080.0117±0.0007
      5A0.0006±0.00030.0059±0.00030.0052±0.0003
      6B0.0010±0.00030.0145±0.00090.0135±0.0009
      Mean±SD, n=9 pixels, corresponding to the labeled lesions in Figure 4.
      Not shown in this report are the color images of PAR, PER, and PAR–PER. Although the color PAR and PER images look similar to the gray–black image of Figure 4a, the color PAR–PER images show an interesting distribution of colors, where shifts in color toward red or blue indicate how the skin's architecture (i.e., the depth of the melanoma) and ultrastructure (i.e., the diameter of collagen fiber bundles) shift the balance of red versus blue photon reflectance.

      Discussion

      These preliminary results were shown at the 53rd annual Montagna Symposium on Skin Biology to illustrate the opportunity for novel optical imaging to assist the early detection of skin pathology in murine models. The early detection of the lesions such as skin cancer facilitates acquisition by biopsy of early stages of disease. Non-invasive imaging allows the study of the time course of cancer progression. Our current imaging can visualize the onset of melanoma in sub-epidermal locations and its progression as it compromises the epidermis.
      The rCSLM studies continue on quantitative assessment of the reflectivity of various cell types as part of an effort to investigate the underlying mechanisms of optical contrast based on photon scattering. The magnitude of photon scattering and the angle and wavelength dependence of photon scattering offer a fingerprint that characterizes the architecture and ultrastructure of the skin, a fingerprint that may provide a characterization of the progression of a disease. This work is exploring the threshold optical change that allows detection of an early lesion.
      The PLI studies continue to explore the mechanisms of contrast available from superficially but subsurface-scattered photons. The work of this report is exploring the opportunity for PLI to distinguish superficial versus deep melanoma lesions in the C57/B6 murine model.
      In non-melanoma skin pathology, the PAR–PER image presents a complex pattern of reflectivity because of the structure of the superficial papillary dermis, similar to a textured fabric. Pathology disrupts this textured pattern allowing the eye of the doctor to discern the margins of the lesions. This clinical work is exploring the use of PLI to guide surgical excision of skin cancers in the dermatology clinic.

      Materials and Methods

      Animal model

      The C57/Black-6 mouse model (C57/B6) was developed at the National Institutes of Health (
      • Jhappan C.
      • Noonan F.P.
      • Merlino G.
      Ultraviolet radiation and cutaneous malignant melanoma.
      ). The model develops melanoma in sub-epidermal locations, as illustrated in Figure 5 showing a histological preparation of formalin-fixed tissue that was prepared using melanin bleach with a nuclear fast red counter stain. All animal studies were approved by the Oregon Health & Science University Institutional Review Board.
      Figure thumbnail gr5
      Figure 5Histopathology of melanoma lesions in the C57/B6 mouse. The formalin-fixed specimen was prepared using a melanin bleach with a nuclear fast red counter stain. The melanoma lesions originated in the dermis (Scale bar=50 μm).

      rCSLM

      Figure 6a shows the basic design of the rCSLM system. A collimated argon ion laser operating at 488 nm wavelength, 10 mW power, was sent to the sample via an optical scanning assembly and an objective lens (NA=0.90, water-dipping lens, × 60 magnification, Olympus America, Melville, New York). The beam was directed upward toward the animal by a mirror and focused into the mouse through a droplet of water (normal saline) that coupled the objective lens to the mouse skin through a 4 mm diameter. aperture in the stage that held the animal. The x–y scanning assembly consisted of two galvanometer mirrors and a pair of relay lenses that directed the laser beam into the objective lens at slightly varying angles such that the focus was translated in an x–y plane within the tissue. The laser beam reflected by the tissue from the focus of the lens was recollimated and returned through the optical train until a portion of the beam was re-directed by a beam splitter toward a lens/pinhole/photodetector assembly. Only photons scattered from the focus in the tissue could refocus through the pinhole to reach the photodetector, thereby achieving confocal detection. A normalized pinhole radius of 1.3 (pinhole radius=1.3 × Airy disk radius) was used trading reduced z-axis resolution for increased light collection. The stage was controlled by a computer-controlled z-axis micrometer that allowed 1 μm steps. For each z-axis step of the stage over a 60 μm range, an x–y horizontal image was acquired by the system. Data were acquired using an A/D converter controlled by Labview software (National Instruments Corp., Austin, Texas), and image reconstruction was conducted using MATLAB software (The Mathworks Inc., Natick, Massachusetts).
      Figure thumbnail gr6
      Figure 6Reflectance-mode confocal scanning laser microscopy. (a) The reflectance of a blue laser focused on the tissue by a × 6x objective lens is collected by a photodetector as scanning mirrors move the focus over an x–y plane of tissue. A z-axis microscope stage moves the stage holding the animal in 1 μm steps, and x–y images are acquired at a series of tissue depths. (b) The two types of images produced are the horizontal image (x–y at a depth z) and the transverse image (x–z image at position y).
      Figure 6b depicts the two types of images that were generated. A horizontal image portrayed an x–y plane at a single depth z. A transverse image portrayed an x–z plane at a single lateral position y. Because the skin is not flat, each horizontal image cuts an x–y plane through the tissue, such that the surface reflectance at the stratum corneum appears as a circle of high reflectivity and the image within the circle is at some depth within the tissue.

      PLI

      Figure 7a shows the basic design of the polarized light camera. A white light source was passed through a linear polarizer that was aligned so that the transmitted electric field was parallel to the scattering plane, defined as the source/tissue/camera triangle. The illumination was delivered at an oblique angle (45°) onto a glass plate contacting the skin of the animal such that glare from the air/glass and glass/skin interfaces was directed away from the camera. Only light that entered the skin could scatter toward the camera and be collected. The animal was coupled to the glass plate by a drop of clear gel (ultrasound coupling gel, ESC Medical Systems Inc., Lumenis Inc. USA Operations, Santa Clara, California). The light passing to the camera passed through an analyzing polarization assembly consisting of an electronically controlled Faraday rotator (Displaytech Inc., Longmont, Colorado) that either did or did not rotate the orientation of the polarized light by 90° before the light passed through a second linear polarizer then entered the camera. As the Faraday rotator was switched on/off the camera received light that was oriented either parallel to or perpendicular to the orientation of the illumination light, yielding two images called “PAR” and “PER,” respectively. A color CCD camera (Micropublisher, QImaging Inc., Burnaby, BC) acquired the images as 384 × 512 pixel images for the red, green, and blue channels of the camera. For this paper, only the green channel images were used. A DARK image was acquired with the camera aperture closed, and this image was subtracted from the PAR and PER images before they were processed.
      Figure thumbnail gr7
      Figure 7Polarized light imaging (PLI). (a) The basic setup is a linearly polarized white-light source that illuminates from an angle of about 45°. The tissue is coupled by a drop of clear gel to a glass plate, such that surface glare from the air/glass and glass/skin surfaces is reflected obliquely away from the camera. Only photons that enter the skin are scattered toward the camera. An analyzing linear polarizer in front of the camera is electronically rotated and aligned either parallel to the illumination or perpendicular to the illumination, yielding two images called PAR and PER, respectively. (b) The photons that scatter from the subsurface but superficial tissue layers retain the polarization of the illumination light (labeled S). The photons that penetrate more deeply and are multiply scattered become randomly polarized (labeled D). Therefore, PER=D, PAR=S+D, and total reflectance R=PAR+PER=S+2D.
      Figure 7b illustrates the different fates of photons propagating in the system. About 5% of the delivered photons were deflected as surface glare from the air/glass and glass/tissue interfaces. About 6% of the photons were scattered by the superficial skin layers involving a single or few scattering events such that the photons still retained the polarization of the illumination light, contributing only to the PAR image. About 4% of the photons penetrated more deeply into the skin and were multiply scattered such that their polarization was randomized, yielding equal contributions (2% each) to the PAR and PER images. About 85% of the photons were absorbed by skin largely because of the melanin of the C57/B6 mouse. These % values are only approximate and pertain only to these highly pigmented mouse skin sites. In less pigmented skin sites, the distribution of photons among these different pathways is different, with the multiply scattered escaping light approaching 40%, the absorbed fraction dropping to 50%, and the superficial scattering about the same at 5–10%.
      The fraction of photons that is superficially scattered and retains its polarization may be denoted by “S,” whereas the fraction multiply scattered may be denoted by “D,” such that
      MathJax Formula fx1


      and the total reflectance R is
      MathJax Formula fx2


      ACKNOWLEDGMENTS

      This work was supported by the National Institutes of Health, EB000224 (S. L. J.), CA98893 (M. K. M.), and CA69533 (OHSU Cancer Institute).

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