Gregg M. Menaker, M. D.* Peter J. Dwyer, M. S. ‡ Milind Rajadhyaksha, Ph. D. ‡ Salvador González, M. D., Ph. D.** James M. Zavislan, Ph.D. ‡ Confocal Microscopy for Mohs Surgery An Atlas of Images * Massachusetts General Hospital, Harvard Medical School, Boston, MA ** Massachusetts General Hospital, Wellman Laboratories of Photomedicine, Boston, MA ‡ Lucid, Inc., Rochester, NY © Copyright Lucid, Inc. ENTER

Introduction Near infrared confocal microscopy has been used to image the morphology of in- vivo and ex vivo tissues. We are currently studying the use of confocal microscopy to screen margins in Mohs specimens. Confocal microscopy is unique because it can rapidly produce images of cellular morphology without the need to process the tissue (i.e., without freezing, sectioning and staining). A confocal microscope images native refractive index variation within the epithelial and stromal compartments of the tissue. These refractive index variations are due to the chemical variations within the tissue. Structures that backscatter more light appear brighter than less scattering structures. Because the source of image contrast is not due to exogenous stains, confocal images appear different than those from tissue that has been histologically processed and stained. The purpose of the atlas is to introduce the clinician to interpretation of confocal pathology by comparing confocal images to frozen section hematoxylin and eosin- stained histopathology. By comparing the similarities and noting the differences between confocal images and standard histopathology, we hope to familiarize Mohs surgeons with this new imaging modality. NextPrevious EXIT

Materials & Methods Included in this mini-version of the Mohs Micrographic Surgery Atlas are images of normal skin as well as basal cell carcinomas and squamous cell carcinomas. The images illustrate the comparison of confocal reflectance images to H&E and describe different features unique to confocal reflectance images. The confocal images of Mohs specimens were prepared as follows. After the standard frozen histopathology sections were prepared, the remaining (frozen) tissue was thawed and confocally imaged. The thawed tissue specimen was washed in phosphate buffered saline and 5% acetic acid (3 minutes each solution) prior to confocal imaging. The frozen H&E stained sections were then compared to the confocal images. The acetic acid causes the aggregation of chromatin within the cell nuclei and enhances contrast in confocal images. NextPrevious EXIT

Table of Images Normal Skin Epidermis Stratum Corneum Stratum Granulosum Stratum Spinosum Stratum Germinativum Tissue Edge Dermis Dermal Appendages Hair Follicles Sebaceous Gland Eccrine Glands Basal Cell Carcinoma Nodular BCC Infiltrative/Sclerosing BCC Squamous Cell Carcinoma Bowen’s Disease (SCC In-situ) Invasive SCC Miscellaneous Inflammatory Cells Solar ElastosisNextPrevious EXIT

The stratum corneum on the lower lip of a 63 year old woman illustrating the large, flattened corneocytes(C). The absence of nuclei suggests this is a fully differentiated layer. There are wrinkles (W) and lines in the stratum corneum. Normal Skin - Stratum Corneum Next Image Previous Image Table of Images EXIT

The Granular layer of the epidermis has large keratinocytes with large nuclei (G) that appear round and as bright dots in confocal images. The cytoplasm appears gray around each nucleus. A hair follicle (HF) containing a hair shaft is also present. The tissue edge (TE) of the specimen is in the lower left corner. Normal Skin - Stratum Granulosum Next Image Previous Image Table of Images EXIT

The spinous layer of normal epidermis illustrating the slightly smaller nuclei of the spinous cells (S) compared to the granular layer. The bright dots are the nuclei and the cytoplasm appears gray around the nuclei. The same hair follicle (HF) can be seen in both images of the granular and spinous layers. Normal Skin - Stratum Spinosum Next Image Previous Image Table of Images EXIT

The basal layer of normal epidermis showing horizontal (en face) sections of rete ridges, with the papillary dermis (PD) in the center of the rete. The basal cells (B) line the rete ridges and differentiate toward the stratum corneum. The basal cell nuclei appear bright due to the acetic acid preparation of the tissue. Normal Skin - Stratum Germinativum Next Image Previous Image Table of Images EXIT

A cross section through a tissue edge of a Mohs specimen illustrating the entire thickness of the epidermis (EP) along with hair follicles (HF) and inflammatory cells (IC) within the dermis. Normal Skin - Tissue Edge Next Image Previous Image Table of Images EXIT

A cross section of a terminal hair on the face using confocal microscopy and the same specimen stained with Hematoxylin and Eosin (H&E). The hair cortex (HC), inner root sheath (IRS) and outer root sheath (ORS) are illustrated on both images. Notice the nuclei of the pale cells (P) of the outer root sheath in the confocal image appears bright. The hair cortex and inner root sheath appear dark on the confocal image. The collagen (Co) and fibroblasts (FB) are also illustrated. Normal Skin - Hair Follicle Next Image Previous Image Table of Images EXIT

A sebaceous gland from the nose of a 53 year-old woman. In the confocal image, the sebocytes (SB) are located near the center of the sebaceous gland and the undifferentiated precursor cells (UC) are along the periphery of the gland. Notice the inflammatory cells (IC) surrounding the sebaceous gland. In the confocal image, hair follicles (HF) and another nearby sebaceous gland (SG) are well demonstrated. Normal Skin - Sebaceous Gland Next Image Previous Image Table of Images EXIT

An eccrine gland in a Mohs section from the chin. The images show the tubular nature of the gland. Also evident are cuboidal cells (Cu) two or more layers thick that line the eccrine ducts. The lumen of the duct (L) is visible as a dark/gray region in the center of the tubular gland in the confocal image. Normal Skin - Eccrine Gland Next Image Previous Image Table of Images EXIT

Nodular basal cell carcinoma in a Mohs section. The confocal image shows the bright basaloid nuclei (B) crowded within the tumor nests. Common characteristics of basal cell carcinoma seen in these images are peripheral palisading of nuclei (Pa) and retraction clefting (R). Also note the asymmetrical angular configuration of the tumor nest distinguishing it from other dermal appendages. Basal Cell Carcinoma - Nodular Next Image Previous Image Table of Images EXIT

Infiltrative/sclerosing basal cell carcinoma exhibiting invasive narrow strands of tumor cells within a fibrotic stroma. Note the differences in the visibility of the two and three-cell layer thick basaloid tumor strands (B) in the H&E image. The H&E image is dominated by basophilic staining of the neoplastic cells. In the confocal image the bright tumor strands with high nuclear to cytoplasmic ratio are surrounded by the dark collagen (Co). Basal Cell Carcinoma - Infiltrative Next Image Previous Image Table of Images EXIT

Squamous cell carcinoma in-situ of the right periocular region. Full thickness atypia is noted in the epidermis with pleomorphic nuclei and nuclear crowding (NC). The normal polarity of the basal layer (BL) is lost on both confocal and H&E stained images. NC BL NC BL Squamous Cell Carcinoma - In Situ Next Image Previous Image Table of Images EXIT

Superficially invasive well differentiated SCC with prominent dermal papillae (DP) in both confocal and H&E sections. The keratinocyte nuclei are pleomorphic (PC). Overabundance/crowding of nuclei is visible in both the confocal and H&E images. Squamous Cell Carcinoma - Invasive Next Image Previous Image Table of Images EXIT

Inflammatory cells appear as round basophilic cells in the dermis. In the confocal image, note the size of the cells is smaller than cancerous, infiltrative basal cells. Also note the diffuse pattern of the bright nuclei (IC) within the dark collagen (Co). Miscellaneous - Inflammatory Cells Next Image Previous Image Table of Images EXIT

Solar elastosis in a Mohs section from the cheek of a 54- year-old woman. In the confocal image, the denatured collagen (SE) appears bright likely due to the altered structure of the collagen and elastin which changes the index of refraction. The blue elastoic material is clearly visible in the H&E stained section. Parallel orientation on the normal collagen (Co) fibers is lost in the area of solar elastosis (SE). Miscellaneous - Solar Elastosis Next Image Previous Image Table of Images EXIT