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

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 (). 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 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 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 (). 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 (R_melanoma=0.42 × 10^-3) using the water/glass interface as a calibration standard (R_{water–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 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.

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.

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.

The C57/Black-6 mouse model (C57/B6) was developed at the National Institutes of Health (). 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 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 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.

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 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

and the total reflectance R is