Dual Mode Reflectance and Fluorescence Confocal Laser Scanning Microscopy for In Vivo Imaging Melanoma Progression in Murine Skin  Yanyun Li, Salvador Gonzalez, Theis H. Terwey, Jedd Wolchok, Yongbiao Li, Iana Aranda, Ricardo Toledo-Crow, Allan C. Halpern  Journal of Investigative Dermatology  Volume 125, Issue 4, Pages 798-804 (October 2005) DOI: 10.1111/j.0022-202X.2005.23786.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 In vivo imaging of normal green fluorescent protein mouse ear skin. The images in columns 1–4 correspond to in vivo fluorescence, in vivo reflectance, reflectance (red) and fluorescence (green) composite, and hematoxylin and eosin ex vivo images, respectively. The images in row (A) were obtained at the upper epidermis level. They show corneocytes (c), granular keratinocytes (g), hair follicles (hf), hair (h), and skin folds (f). In row (B), the images correspond to the dermis level showing hair follicles (arrowhead) and blood vessels (arrows). (B2) blood vessels (arrow) appear bright because of the reflectance from the blood flow in the vessels. The combined modality system provided single-cell resolution as seen by the melanocytes (arrows) in the figures of row (C). All scale bars correspond to 100 μm. Journal of Investigative Dermatology 2005 125, 798-804DOI: (10.1111/j.0022-202X.2005.23786.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 In vivo imaging of green fluorescent protein mouse ear skin after B16 cell inoculation. A population of fluorescent host-derived dendritic-shaped cells was observed 24 h after B16 cell inoculation in the mouse ear skin. (A1) In vivo fluorescence and (A2) hematoxylin and eosin (H&E). (B1) The ear epidermis in fluorescence mode 1 wk after the inoculation is shown, and (C1) is of the same location as (B1) but deeper in the dermis. (B2) and (C2) correspond to the correlated histology (H&E) images of (B1) and (C1). Arrows in these images point out the dendritic cells. Arrowheads point out the hair follicles. Double arrowhead stands for the blood vessel. All scale bars correspond to 100 μm. Journal of Investigative Dermatology 2005 125, 798-804DOI: (10.1111/j.0022-202X.2005.23786.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 In vivo imaging of B16 cell-induced tumor and blood vessels in green fluorescent protein (GFP) transgenic mouse skin. Images in row (A) correspond to the ear skin 1 wk after B16 cell inoculation. The tumor size was about 2.5 mm. In the fluorescent mode (A1), blood vessels (arrow) and hair follicles (arrowhead) can still be seen within the dark background of tumor cells. In the simultaneously acquired reflectance image (A2), melanocytes (arrows) could be detected around the hair follicles (arrowheads). In (B1) (fluorescent image), the border of a tumor on a GFP mouse haunch skin was acquired 14 d after B16 cell inoculation (tumor size was about 5 mm); the dark area corresponds to the tumor. Some residual structures, including keratinocytes (star), hair follicles (arrowheads), and dendritic cells (arrows), can be detected in the epidermal layer overlying the tumor. The same site, imaged with immunohistochemistry S-100 staining (image C2), showed the positive staining of tumor cells (dark brown). C1 is H&E staining and C3 shows CD34 staining of blood vessel in the tumor. All scale bars correspond to 100 μm. Journal of Investigative Dermatology 2005 125, 798-804DOI: (10.1111/j.0022-202X.2005.23786.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 In vivo imaging of C57BL/6 mouse haunch skin after B16-enhanced green fluorescent protein (EGFP) cell inoculation. GFP expressing B16-EGFP cells were intradermally inoculated into C57BL/6 non-GFP expressing mice. The induced tumor provided a bright fluorescence signal within the dark background of the mouse skin. (A) A 2 mm × 2 mm mosaic of in vivo fluorescence (green) and reflectance (red) composite images. In the center of (A), the fluorescence signal of the GFP expressing tumor is represented in green. The tumor border was clearly separated from the surrounding tissue. Individual images (440 μm × 330 μm) were taken at the center of the tumor (B). They show the tumor tissue in fluorescence mode (R1). The reflected light image of the tumor site (B2) shows coarse, fiber-like tissue instead of the normal morphology of healthy skin. The size of the tumor shown here was about 2.5 mm. Arrowheads in these images show hair follicles, and stars point to hairs. Journal of Investigative Dermatology 2005 125, 798-804DOI: (10.1111/j.0022-202X.2005.23786.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 In vivo images of blood vessels inside the green fluorescent protein (GFP) expressing tumor. In fluorescence mode, blood vessels (A, arrows) could be detected inside the B16-EGFP-induced melanoma. They appeared dark in contrast to the brightly fluorescent tumor tissue. (B, C) The corresponding reflectance and composite images to (A). The correlated hematoxylin and eosin staining (D) showed the distribution of blood vessels (arrows) inside and outside the tumor. All scale bars correspond to 100 μm. The concentric arcs seen in the fluorescence image are artifacts caused by interference between multiple reflections of the excitation light. Journal of Investigative Dermatology 2005 125, 798-804DOI: (10.1111/j.0022-202X.2005.23786.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Dual-contrast confocal microscope. The reflected light channel is formed by a laser diode (ld) aimed through a beamsplitter (bs) cube and onto a rotating polygonal mirror for fast scanning. The light then goes to a galvo scanner and the objective lens. The incident light is scattered by the tissue and the reflected light retraces the optical path. A bs diverts the reflected light to a confocal detector (RCM). The fluorescence channel has an argon laser (Ar+) coupled to the scanner; a dichroic mirror (dm) diverts the excitation light to a combining dichroic mirror (cd) that aligns the fluorescence excitation light (488 nm) to the reflectance light (830 nm). After the cd the two beams share the same optical path to the sample and back. The returning fluorescence signal is diverted by the combining dichroic (cd) through the dichroic mirror (dm), onto the barrier filter (bf) that eliminates any remaining excitation light, and to a second confocal detector (FCM). Journal of Investigative Dermatology 2005 125, 798-804DOI: (10.1111/j.0022-202X.2005.23786.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions