Combining Reflectance Confocal Microscopy with Optical Coherence Tomography for Noninvasive Diagnosis of Skin Cancers via Image Acquisition

Summary

Here, we describe protocols for acquiring good-quality images using novel, noninvasive imaging devices of reflectance confocal microscopy (RCM) and combined RCM and optical coherence tomography (OCT). We also familiarize clinicians with their clinical applications so that they can integrate the techniques into regular clinical workflows to improve patient care.

Abstract

Skin cancer is one of the most common cancers worldwide. Diagnosis relies on visual inspection and dermoscopy followed by biopsy for histopathological confirmation. While the sensitivity of dermoscopy is high, the lower specificity results in 70%-80% of the biopsies being diagnosed as benign lesions on histopathology (false positives on dermoscopy).

Reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) imaging can noninvasively guide the diagnosis of skin cancers. RCM visualizes cellular morphology in en-face layers. It has doubled the diagnostic specificity for melanoma and pigmented keratinocytic skin cancers over dermoscopy, halving the number of biopsies of benign lesions. RCM acquired billing codes in the USA and is now being integrated into clinics.

However, limitations such as the shallow depth (~200 µm) of imaging, poor contrast for nonpigmented skin lesions, and imaging in en-face layers result in relatively lower specificity for the detection of nonpigmented basal cell carcinoma (BCCs) — superficial BCCs contiguous with the basal cell layer and deeper infiltrative BCCs. In contrast, OCT lacks cellular resolution but images tissue in vertical planes down to a depth of ~1 mm, which allows the detection of both superficial and deeper subtypes of BCCs. Thus, both techniques are essentially complementary.

A "multi-modal," combined RCM-OCT device simultaneously images skin lesions in both en-face and vertical modes. It is useful for the diagnosis and management of BCCs (nonsurgical treatment for superficial BCCs vs. surgical treatment for deeper lesions). A marked improvement in specificity is obtained for detecting small, nonpigmented BCCs over RCM alone. RCM and RCM-OCT devices are bringing a major paradigm shift in the diagnosis and management of skin cancers; however, their use is currently limited to academic tertiary care centers and some private clinics. This paper familiarizes clinicians with these devices and their applications, addressing translational barriers into routine clinical workflow.

Introduction

Traditionally, the diagnosis of skin cancer relies on visual inspection of the lesion followed by a closer look at suspicious lesions using a magnifying lens called a dermatoscope. A dermatoscope provides subsurface information that increases sensitivity and specificity over that of visual inspection for diagnosing skin cancers^1,2. However, dermoscopy lacks cellular detail, often leading to a biopsy for histopathological confirmation. The low and variable (67% to 97%) specificity of dermoscopy³ results in false positives and biopsies that turn out to show benign lesions on pathology. A biopsy is not only an invasive procedure that causes bleeding and pain⁴ but is also highly undesirable on cosmetically sensitive regions such as the face due to scarring.

To improve patient care by overcoming existing limitations, many noninvasive, in vivo imaging devices are being explored^{5,6,7,8,9,10,11,12,13,14,15,16,17,18}. RCM and OCT devices are the two main optical noninvasive devices that are used for diagnosing skin lesions, especially skin cancers. RCM has acquired Current Procedural Terminology (CPT) billing codes in the USA and is being increasingly used in academic tertiary care centers and some private clinics^7,8,19. RCM images lesions at near-histological (cellular) resolution. However, images are in the en-face plane (visualization of one layer of skin at a time), and the depth of imaging is limited to ~200 µm, sufficient to reach the superficial (papillary) dermis only. RCM imaging relies on the reflectance contrast from various structures in the skin. Melanin imparts the highest contrast, making pigmented lesions bright and easier to diagnose. Thus, RCM combined with dermoscopy has significantly improved diagnosis (sensitivity of 90% and specificity of 82%) over dermoscopy of pigmented lesions, including melanoma²⁰. However, due to a lack of melanin contrast in pink lesions, especially for BCCs, RCM has lower specificity (37.5%-75.5%)²¹. A conventional OCT device, another commonly used noninvasive device, images lesion up to 1 mm in depth within the skin and visualizes them in a vertical plane (similar to histopathology)⁹. However, OCT lacks cellular resolution. OCT is primarily used for diagnosing keratinocytic lesions, especially BCCs, but still has lower specificity⁹.

Thus, to overcome the existing limitations of these devices, a multi-modal RCM-OCT device has been built²². This device incorporates RCM and OCT within a single, handheld imaging probe, enabling the simultaneous acquisition of co-registered en-face RCM images and vertical OCT images of the lesion. OCT provides architectural detail of the lesions and can image deeper (up to a depth of ~1 mm) within the skin. It also has a larger field of view (FOV) of ~2 mm²² compared to the handheld RCM device (~0.75 mm x 0.75 mm). RCM images are used to provide cellular details of the lesion identified on OCT. This prototype is not yet commercialized and is being used as an investigational device in clinics^23,24,25.

Despite their success in improving the diagnosis and management of skin cancers (as supported by the literature), these devices are not yet widely used in clinics. This is mainly due to the paucity of experts who can read these images but is also due to the lack of trained technicians who can acquire diagnostic-quality images efficiently (within a clinical time frame) at the bedside⁸. In this manuscript, the goal is to facilitate the awareness and eventual adoption of these devices in clinics. To achieve this goal, we familiarize dermatologists, dermatopathologists, and Mohs surgeons with images of normal skin and skin cancers acquired with the RCM and RCM-OCT devices. We will also detail the utility of each device for the diagnosis of skin cancers. Most importantly, the focus of this manuscript is to provide step-by-step guidance for image acquisition using these devices, which will ensure good-quality images for clinical use.

Protocol

All the protocols described below follow the guidelines of the institutional human research ethics committee.

1. RCM device and imaging protocol

NOTE: There are two commercially available in vivo RCM devices: wide-probe RCM (WP-RCM) and handheld RCM (HH-RCM). The WP-RCM comes integrated with a digital dermatoscope. These two devices are available separately or as a combined unit. Below are the image acquisition protocols using the latest generation (Generation 4) of the WP-RCM and HH-RCM devices along with their clinical indications.

1. Lesion selection and clinical indications
   1. Look for the following types of lesions: dermoscopically equivocal pink (BCC, squamous cell carcinoma [SCC], actinic keratosis [AK], other benign lesions) or pigmented lesion (nevi and melanoma, pigmented keratinocytic lesions); a nevus that has recently changed on clinical or dermoscopy examination; inflammatory lesions to determine inflammatory patterns.
   2. Perform mapping for lentigo maligna (LM) margins to determine the extent of the lesion and mapping and selection of biopsy sites for disease with subclinical extension such as extramammary Paget's disease (EMPD) and LM.
   3. Carry out noninvasive monitoring of nonsurgical treatment such as topical drugs (imiquimod), radiation, photodynamic therapy, and laser ablation.
2. For device selection, use the WP-RCM device for lesions located on relatively flat surfaces of skin (the trunk and extremities) and the HH-RCM device for lesions on curved surfaces (the nose, earlobes, eyelids, and genitalia).
   NOTE: Selection of the imaging device will mainly depend on the location of the lesion.
3. For imaging, position the patient on a fully reclining chair or a flat examination table with pillows or an armrest for support and to achieve a flat imaging surface.
   NOTE: Older generation (Generation 3) WP-RCM devices took ~30 min per lesion. Imaging a single lesion may require ~15 min with the newer generation (Generation 4) WP-RCM device currently being used in clinics. Despite the improved acquisition time, positioning the patient comfortably will ensure minimum motion artifacts and aid the acquisition of superior quality images. The following steps may help with correctly positioning the patient:
4. To prepare for imaging, clean the lesion and surrounding skin with an alcohol wipe to eliminate any dirt, lotion, or make-up. Shave hairy skin surfaces before attaching the tissue window to avoid air bubbles that can hinder the visualization of tissue microstructures.
   NOTE: For removing heavy cosmetics or sunscreens, clean the site with a gentle soap and water prior to cleaning with alcohol.
5. Image acquisition using the WP-RCM device (Figure 1, Figure 2, Supplemental Figure S1, Supplemental Figure S2, and Supplemental Figure S3)
   ​NOTE: WP-RCM devices are capable of capturing stacks, mosaic, live single-framed videos, and single-framed images.
   1. To attach a disposable plastic window cap to the lesion (Figure 1), position the probe perpendicular to the lesion for the best images. Refer to Figure 1A-F for an example of attachment. Add a drop of mineral oil on the center of the plastic window, carefully spreading it across the window width (Figure 1A). Remove the paper backing from the adhesive side of the plastic window. Stretch the skin gently to avoid wrinkling and attach the window.
      NOTE: Use food-grade mineral oil that is safe and has a high viscosity. Ensure that the lesion is centered and covered in its entirety. For lesions larger than 8 mm x 8 mm, either image areas of concern based on dermoscopy or perform separate imaging sessions to cover the entire lesion.
   2. Acquiring dermoscopy images (Figure 1C,D)
      NOTE: A dermoscopy image is acquired to serve as a guide for navigating within the lesion. The following steps should be used to ensure perfect registration between the dermoscopy image and the confocal image.
      1. Hover the WP-RCM probe over the plastic window cap and approximate the best angle of insertion for the probe (Figure 1C). Locate the small, white arrow located on the side of the probe (Figure 1C) and align it with the arrow on the side of the dermoscopy camera (Figure 1C).
      2. Insert the dermoscopy camera into the plastic window cap (Figure 1D). Press the trigger on the camera to acquire an image. Remove the dermatoscope. Before starting the imaging session, ensure that the dermatoscope image covers the entire lesion surface.
   3. To attach the RCM probe to the plastic disposable cap (Figure 1E,F), place a pea-sized amount of ultrasound gel inside the disposable plastic window cap (Figure 1E). Insert the probe within the cap until a sharp click is heard (Figure 1F).
      NOTE: For the best images, insert the probe perpendicular (at a 90° angle) to the plastic window. The height of the examination chair can be raised to achieve a flatter surface, reduce motion artifacts, expel air bubbles (Figure 3 and Figure 4), and ensure secure attachment to the skin.
   4. Acquiring RCM images (Figure 2, Supplemental Figure S1, and Supplemental Figure S2)
      1. Use the dermoscopy image (step 5.2.) for guiding the RCM image acquisition (Supplemental Figure S1). Select the center of the lesion and identify the topmost (brightest) layer of the skin — the anucleate layer of the stratum corneum (Supplemental Figure S1).
      2. Set the imaging depth to zero at this level (Supplemental Figure S1).
         NOTE: This depth serves as a reference point for determining the actual z-depth of subsequent layers within the lesion.
      3. Acquire a stack in the lesion's center (Figure 2 and Supplemental Figure S1) by pressing the stack icon. Select an anatomical site from the drop-down menu: face or body. Set 4.5 µm step-size and 250 µm depth.
         NOTE: Begin the stacks from the stratum corneum and end at the deepest visible layers in the dermis. Supplemental Figure S1 shows an example of how to acquire a stack, while Figure 2 gives an example of a stack.
      4. Acquire a mosaic: take the first mosaic at the dermal-epidermal junction (DEJ) (Supplemental Figure S2). Identify the DEJ layer in the stack acquired and then use the mouse to select an 8 mm x 8 mm square to cover the entire lesion. Press the mosaic icon to complete the operation (Supplemental Figure S2). Acquire at least 5 mosaics at various depths: stratum corneum, stratum spinosum, suprabasal layer, DEJ, and superficial papillary dermis.
      5. Open the DEJ mosaic to guide the acquisition of the subsequent mosaics. Click on any structure on the DEJ mosaic to bring up that area on the live view imaging. Scroll down to acquire mosaics at the dermis and then up (from the DEJ) to take mosaics in the epidermis.
      6. Get the acquired mosaics evaluated by the expert RCM reader present at the bedside to identify the region of interest and take stacks. In the absence of an expert at the bedside, capture 5 stacks: one in each quadrant and one in the center of the lesion with a homogeneous pattern on dermoscopy (steps 1.5.2.). For heterogeneous lesions, acquire additional stacks to cover all the dermoscopy features.
         NOTE: A "stack" (Figure 2) is a sequential collection of high-resolution, single-frame, small field of view (FOV) images (0.5mm x 0.5 mm) acquired in depth starting from the topmost layer of the epidermis to the superficial dermis (~200 µm). A "mosaic" (Supplemental Figure S2) is a large FOV of images obtained by stitching individual 500 µm x 500 µm images together in "X-Y" (horizontal en face plane).
   5. Completing an imaging session
      1. Click on Done Imaging.
      2. Detach the microscope from the plastic window. Remove the plastic window by gently holding the patient's skin taut and dispose of it. Wipe off oil on the skin with an alcohol swab.
      3. Detach the protective cone surrounding the microscope lens. Clean the tip of the objective lens with an alcohol swab to remove the ultrasound gel. Dry the objective lens with a paper towel. Reattach the plastic cone to the microscope probe.
         NOTE: Images can be read, and a report can be generated and signed at the bedside by a trained physician. In the absence of an expert reader, a remote expert can be consulted either by transferring the images via the cloud or via a live teleconfocal session²⁶.
   6. Generating a Confocal Diagnostic Evaluation report (Supplemental Figure S3)
      1. Click on New Evaluation. Enter the diagnosis from the preselected options in the dropdown menu.
      2. If another imaging session is required, select images inadequate and need to be recaptured. If a descriptive diagnosis is needed, select other and describe in the free text box at the end of the form. Enter the CPT code for billing⁷ (Supplemental Figure S3A). Select the applicable features seen during imaging from the report checklist (Supplemental Figure S3B). Select the applicable management from the checklist.
         NOTE: No billing code is applicable for HH-RCM imaging.
      3. Click on Finish and Sign. Generate the report as a PDF and print. Get the report signed by a physician and add it to the patient's chart for billing.
6. Image acquisition using the HH-RCM device (Figure 5)
   NOTE: HH-RCM devices are capable of capturing stacks, live single-framed videos, and single-framed images.
   1. Encircle the lesion identified by the physician with a paper ring. Use steps detailed in section 3. for positioning the patient and cleaning the lesion site.
      NOTE: Select the size of the paper ring (5-15 mm) based on the lesion size to define the boundary of the lesion and ensure imaging is done within the lesion. If a paper ring is not available, use paper tape to define the lesion.
   2. Remove the plastic cap covering the microscope lens. Apply a pea-sized amount of ultrasound gel to the objective lens of the HH-RCM and cover it with the plastic cap (Supplemental Figure S3A). Add a generous drop of mineral oil to the side of the plastic cap that will be touching the skin.
      NOTE: Increase the amount of oil for very dry skin, if needed.
   3. Press the probe to the lesion site on the skin with firm pressure. Use the z-depth controls on the HH-RCM device to move up and down at various depths within the lesion (Supplemental Figure S3B). Acquire multiple single-frame images and stacks in the regions of interest. Take stacks as described in step 1.5.4.3.
   4. For large lesions where the WP-RCM device cannot be attached, take continuous videos at various layers by moving the HH-RCM probe over the entire lesion surface. Click on the video capture symbol to do so. Record the movement of blood cells within vessels, if needed.
      NOTE: These videos can be later stitched using software to provide large FOV images similar to mosaics.
   5. Press Done Imaging after the imaging session is complete. Clean the lesion with an alcohol swab to remove the oil. Remove the ultrasound gel from the objective lens of the probe by cleaning it with an alcohol wipe and reattaching the plastic cap.
      ​NOTE: Unlike the WP-RCM device, which can be operated by a technician, the HH-RCM should be operated by an RCM reader who can interpret images in real time to navigate within the lesion and arrive at a correct diagnosis.

2. Combined RCM-OCT device and imaging protocol

NOTE: There is only one prototype of the RCM-OCT device. This device has a handheld probe and can be used on all body surfaces, similar to the HH-RCM device. It acquires RCM stacks (similar to the RCM device) and OCT rasters (a video of sequential, cross-sectional images²²). Both RCM and OCT images are in gray scale. RCM images have an FOV of ~200 µm x 200 µm, while the OCT image has an FOV of 2 mm (in width) x 1 mm (in depth). Below is the image acquisition protocol using the RCM-OCT device, along with their clinical indications. Figure 6 shows an image of the RCM-OCT device, while Figure 7 shows the software system of the RCM-OCT device.

1. Lesion selection
   1. Look for dermoscopically equivocal pink or pigmented lesion to rule out BCC.
   2. Assess the depth of BCC for management, and assess the residual BCC post treatment.
2. Positioning the patient for imaging: Imaging a single lesion may require up to 20 minutes with the RCM-OCT device. The device is also a handheld probe similar to the HH-RCM device and, thus, can be moved freely over the lesion. For details of patient positioning, refer to section 1.4. above.
3. Preparation of the site for imaging: When using this probe, ensure that the boundary of the lesion is free of excessive hair and topical impurities and is clearly defined. Refer to step 1.4.1. above for more detail.
4. Image acquisition using the RCM-OCT device (Figure 6 and Figure 7)
   1. Prepare the probe similarly to the one used for the HH-RCM (steps 1.6.1-1.6.2.)
   2. Acquire images in line imaging mode and raster mode.
      1. Click on imaging settings (Figure 7A). Select the line imaging mode to acquire an RCM image (cellular resolution) (Figure 7B). Set the step size to 5 µm and the number of steps to 40 (Figure 7A).
      2. Click on Grab. Acquire stacks following step 1.5.4.3. Once complete, click on the Freeze button.
      3. Click on imaging settings. Select raster mode to acquire a correlative OCT video for the lesion architecture (Figure 7B). Switch to the technician tab (Figure 7C). Once complete, click on the Grab button (Figure 7A) and immediately press the save button.
      4. Acquire multiple stacks and videos based on the physician's interest.
      5. Clean the lesion and machine as described in step 1.6.5.

Representative Results

Reflectance confocal microscopy (RCM)
Image interpretation on RCM:
The RCM images are interpreted in a manner that mimics the evaluation of histopathology slides. Mosaics are evaluated first to get the overall architectural detail and identify areas of concern, akin to the evaluation of histology sections on scanning magnification (2x). This is followed by zooming in on the mosaic for evaluation of the cellular details, similar to evaluating slides at high magnification (20x). Figure 8 shows such a schema of the image analysis.

Image quality:
High-quality images without any significant artifacts, acquired at relevant depths in the skin, are essential for correct diagnosis. Figure 4A shows one such image. The main reason for uninterpretable images is related to artifacts or inexperience in acquiring images. Figure 3 and Figure 4B show images with artifacts such as air bubbles, surface debris, and motion artifacts, which hinder diagnostic evaluation. In addition to mastering the technical aspect of image acquisition, the RCM operator should be familiar with the morphology of the various skin layers to enable image acquisition at relevant depths.

Appearance of normal skin layers on RCM:
En-face (horizontal plane) "near-histology" quality images are acquired with the RCM device at varying depths starting from the topmost layer of the epidermis down to the superficial papillary dermis in the skin. RCM has its own terminology that enables the identification of various layers in the skin^5,27. Figure 2 shows five single-frame images acquired at different depths from a stack.

Appearance of various cells on RCM:
Images on RCM appear in gray scale, ranging from very bright structures to dark structures due to the variable sizes and refractive indices of different cells of the skin. Melanin, keratin, and collagen are the sources of highest reflectance in the skin^28,29. Thus, cells containing melanin such as melanocytes (banal and malignant), melanized keratinocytes, and melanophages appear bright. Likewise, cells rich in keratin such as stratum corneum and keratin cysts appear bright. Keratohyaline granules present in the keratinocytes of the stratum granulosum also appear bright. Another possible source of high reflectivity is the Birkbeck granules in Langerhans cells³⁰ and inflammatory cells^28,29. In contrast, intranuclear content lacks reflectance and appears dark on confocal³¹. This is also true for mucin secretions. Blood vessels are found in the papillary dermis. They appear as horizontal or vertical hyporeflective structures. Leukocytes appear as bright, hyperreflective, round, small cells within these hyporeflective blood vessels³². Leukocyte trafficking is prominent during live imaging. Figure 9 shows the appearance of normal skin layers on RCM. Video 1 shows an example of leukocyte trafficking on RCM.

Tumor-specific features on RCM:
Tumor-specific features are well established and aid in differentiating benign from malignant lesions. For example, tumor nodules with peripheral palisading and "cleft-like" space are specific features for BCC³³. Likewise, pagetoid nucleated cells in the epidermis, atypical cells at the DEJ, and a disarranged epidermal pattern suggest a diagnosis of melanoma³⁴. Atypical and disarranged honeycomb patterns are key features for diagnosing SCC³³ on RCM. Figure 10 shows an example of BCC, melanoma, and SCC as seen in RCM images.

Combined RCM-OCT device
Image interpretation on RCM-OCT:
For interpretation of RCM-OCT images, both stacks and rasters are evaluated. Stacks give information at the cellular level and at various depths of the lesion, while the raster provides a vertical view of the lesion and provides information on the overall architecture of the lesion. This vertical view is crucial for the detection of BCC, especially superficial BCCs, which, at times, appear as dark shadows on RCM and may be missed²⁵. In the vertical view of OCT images, BCC tumor nodule continuity with the overlying epidermis and separation from the dermis by a dark area of clefting is clearly discernible. Figure 11 shows an example of dermoscopy, RCM, OCT, and histology correlation of BCC.

Appearance of normal skin layers on RCM-OCT:
Skin layers appear similar to RCM images acquired with the HH-RCM device. More details are provided in the "appearance of various skin layers on confocal" and "appearance of various cells on confocal" sections and Figure 9.

Like RCM, OCT raster images are gray scale. However, OCT rasters show a vertical view similar to traditional histology slides but lack cellular resolution. OCT images have a similar appearance to the commercially available conventional OCT device images. The stratum corneum appears as a thin, bright (hyperreflective) line, with the underlying epidermis appearing grayish (hyporeflective) in color. The papillary dermis appears brighter than the epidermis, and the deepest part of the reticular dermis appears the darkest (nonreflective) due to loss of signal³⁵. The DEJ can be identified as a transitional zone between the grayish epidermis and the bright papillary dermis. Figure 12 shows RCM and OCT images acquired from normal skin on the hand of a healthy volunteer.

Although cellular resolution is not possible, many structures are visible on OCT. Blood vessels can be readily seen in the papillary dermis as reflective (signal-free), horizontal or vertical, tubular structures. Hair follicles are usually hyporeflective, round or tubular structures in the dermis. Their infundibulum (the topmost part of the hair follicle) is seen emerging from the dermis and protruding out of the epidermis at an angle during a live raster imaging session. They often cast a signal shadow on the surface of the epidermis³⁶. Sometimes, hair shafts can be seen emerging from the hair follicles, making their identification easy. Figure 11 provides a view of these structures.

Appearance of BCC on RCM-OCT:
The appearance of BCC in RCM is discussed in the "tumor-specific features" section of RCM. In OCT, BCC tumor nodules can be readily detected as grayish, round, hyporeflective nodules seen surrounded by a reflective, dark area of "clefting". This nodule can be seen attached with the overlying grayish band of epidermis in superficial BCC. The BCC tumor nodules are often surrounded by hyperreflective, white, thickened collagen bundles²³. Other subtypes, such as infiltrative or morpheaform BCCs, are challenging to diagnose with OCT. Figure 11 provides a view of BCC captured by OCT raster.

Figure 1: WP-RCM attachment: Generation 4 WP-RCM device. (A) Place a drop of mineral oil on the center of the plastic window. (B) Center the plastic window over the lesion. (C) Match the arrow on the microscope head (green dashed circle) with the arrow (yellow dashed circle) on the dermatoscope. (D) Insert the dermatoscope into the plastic window and click to take a dermatoscopic image with correct orientation. (E) Remove the dermatoscope and add ultrasound gel inside the plastic window. (F) Fully attach the microscope head to the plastic window at a 90° angle to the lesion. Abbreviation: WP-RCM = wide-probe reflectance confocal microscopy. Please click here to view a larger version of this figure.

Figure 2: An example of a stack. A stack showing a collection of single-frame images acquired in consecutive z-depths from normal skin. Please click here to view a larger version of this figure.

Figure 3: Low-quality image. (A) A low-quality image at the epidermal level showing a few air bubbles (yellow arrows), an external material (yellow circle), most likely a paper fiber, and the fringes of the plastic cap (red box), indicating improper attachment of the microscope to the skin. (B,C) Zoomed-in areas from panel A. Scale bars = 50 µm. Please click here to view a larger version of this figure.

Figure 4: Comparison of high-quality versus low-quality confocal images. (A) A high-quality mosaic (from Figure 6) at the level of the epidermis without any artifacts. (B) A low-quality mosaic at the epidermal level shows several large bubbles (blue arrows), which may impact evaluation. Scale bars = 50 µm. Please click here to view a larger version of this figure.

Figure 5: HH-RCM attachment using a generation 4 HH-RCM device. (A) Remove the plastic cap and add ultrasound gel on the top of the lens. (B) Reattach the plastic cap (green arrow) to the device and place it over the lesion for imaging. Abbreviation: HH-RCM = handheld reflectance confocal microscopy. Please click here to view a larger version of this figure.

Figure 6: RCM-OCT device. (A) The handheld probe (yellow arrow) of the combined RCM-OCT device. (B) The RCM-OCT device with a live imaging window showing an OCT image (black arrow) and an RCM image (green arrow) simultaneously. Abbreviations: RCM = reflectance confocal microscopy; OCT = optical coherence tomography. Please click here to view a larger version of this figure.

Figure 7: RCM-OCT software platform. Snapshots from live imaging windows simultaneously showing (A) an OCT image (blue diamond) and an RCM image with cellular resolution (yellow star). The step size, number of steps, and the z-depth are all controlled by the sliding scale systems (black box; black arrows). (B) Toggling between "line imaging" and "raster" modes (yellow arrows); (C) the button used to save raster images (black circle). Please click here to view a larger version of this figure.

Figure 8: Schema of image analysis at the level of the epidermis. (A) The image is analyzed first at the mosaic level (8 mm x 8 mm), which corresponds to approximately 4x magnification in histology. (B) Areas of interest can be then evaluated at the cellular level by zooming in to the live imaging window during image acquisition. This panel shows a submosaic zoomed-in view from the orange boxed area in panel A, which corresponds to approximately 20x magnification view on histology. Scale bar = (A) 50 µm. Please click here to view a larger version of this figure.

Figure 9: Appearance of normal skin layers on RCM. (A) Stratum corneum: the brightest and first layer of the skin composed of anucleated keratinocytes. (B) Stratum spinosum: composed of tightly packed, nucleated cells (nuclei are dark) with bright cytoplasm creating a typical "honeycomb pattern". (C) Stratum basale: identified by the characteristic "cobblestone pattern" (yellow circle) formed by the presence of the melanin cap of the basal keratinocytes. (D) DEJ: the interface between the stratum basale and the papillary dermis, which is characterized by the bright "ringed pattern" (yellow arrow). (E) Papillary dermis composed of bright collagen fibers (green arrow) and blood vessels. Scale bars = 50 µm. Abbreviations: RCM = reflectance confocal microscopy; DEJ = dermoepidermal junction. Please click here to view a larger version of this figure.

Figure 10: Confocal images from the most common skin cancers. (A) Basal cell carcinoma showing tumor nodules (yellow arrow) with clefting (blue arrow) and palisading. (B) Squamous cell carcinoma showing an atypical honeycomb pattern (yellow asterisks) and button-hole vessels (blue diamond). (C) Melanoma showing clusters of bright, large, round pagetoid cells (green arrows) in the epidermis. FOV = (A-C) 750 µm x 750 µm. Scale bars = 50 µm. Abbreviation: FOV = field of view. Please click here to view a larger version of this figure.

Figure 11: Dermoscopy, RCM, OCT, and histopathology correlation of BCC acquired with the RCM-OCT device. (A) A pink papule on chest post radiotherapy (yellow circle). (B) On RCM, basaloid tumor cords (blue stars) with palisading (red arrow) and clefting (yellow arrow) are seen at the DEJ along with thickened collagen (green star) without a definitive tumor nodule. (C) An OCT image of the same lesion captured with the RCM-OCT device. A distinct grey tumor nodule (blue star) is seen connected to the epidermis along with clefting (yellow arrow). Thickened collagen bundles are seen (green star). (D) H&E-stained biopsy confirmed superficial basal cell carcinoma diagnosis on H&E stain showing palisading (red arrow), clefting (yellow arrow), and thickened collagen bundles (green star) (10x magnification). Scale bars = 500 µm. Abbreviations: RCM = reflectance confocal microscopy; OCT = optical coherence tomography; BCC = basal cell carcinoma; DEJ = dermoepidermal junction; H&E = hematoxylin and eosin. Please click here to view a larger version of this figure.

Figure 12: RCM and OCT images from normal skin. These images were acquired from normal skin on the hand of a healthy volunteer. (A) Shows a single-frame en-face RCM image at the DEJ. (B) Shows a corresponding OCT image in a vertical view with all skin layers. FOV = (A) 750 µm x 750 µm; (B) 1.0 mm x 2.0 mm. Scale bar= 50 µm. Abbreviations: RCM = reflectance confocal microscopy; OCT = optical coherence tomography; DEJ = dermoepidermal junction. Please click here to view a larger version of this figure.

Video 1: RCM video of leukocyte trafficking acquired using an HH-RCM device. This video captured with an RCM device shows a dilated blood vessel with leukocyte trafficking. The surrounding dermis shows bright inflammatory cells. Abbreviation: HH-RCM = handheld reflectance confocal microscopy. Please click here to download this Video.

Supplemental Figure S1: Acquiring a "stack" using a generation 4 WP-RCM device. Select the center of the lesion (green diamond) and click on the stack option (orange box). Ensure that the stack starts from the stratum corneum (blue cross), the first and brightest layer of the skin. Set zero (orange star) depth where the first layer of the stack begins. Select the appropriate lesion site (white cross), spacing between two layers, and depth of imaging (yellow triangle). The blue box above the live view contains icons corresponding to the other functionalities of this system. Icons (blue arrows) from left to right: to capture a mosaic, to capture a cube, to capture a stack, to capture a single framed image, and to capture a video recording. Abbreviation: WP-RCM = wide-probe reflectance confocal microscopy. Please click here to download this File.

Supplemental Figure S2: Acquiring a "mosaic" using a generation 4 WP-RCM device. (A) Using the live view (blue cross), go to the desired lesion depth. Select the entire area of the lesion (if less than 8 mm) or the portion of entire lesion to be captured for imaging (green diamond). Select the mosaic option (orange box) to start the capture. (B) An example of a mosaic captured at the DEJ from the lesion in panel A. Abbreviations: WP-RCM = wide-probe reflectance confocal microscopy; DEJ = dermal-epithelial junction. Please click here to download this File.

Supplemental Figure S3: Example of a confocal diagnostic evaluation report. (A) Fill in the diagnosis (black arrow) by selecting from the dropdown menu (B), the CPT codes for billing (yellow arrow), and the relevant features seen during the confocal imaging session (blue star). Abbreviation: CPT = Current Procedural Terminology. Please click here to download this File.

Discussion

In this article, we have described protocols for image acquisition using in vivo RCM and RCM-OCT devices. Currently, there are two commercially available RCM devices: A wide-probe or arm-mounted RCM (WP-RCM) device and a handheld RCM (HH-RCM) device. It is crucial to understand when to use these devices in clinical settings. Cancer type and location are the main factors determining the selection of the device.

The WP-RCM device is well-suited for lesions on flat and gently undulating body surfaces, such as the trunk and extremities, since it requires contact with the skin. As the probe head is wide, it cannot be attached to narrow areas or corners of the body. However, HH-RCM is a more flexible device and has a narrower probe head. As a result, this device is frequently used to image lesions on curved and relatively undulating areas of the body, including the nose, eyelids, earlobes, and genitalia, where the WP-RCM cannot be attached.

Both the devices can acquire single-frame, cellular-resolution images, stacks, and videos and can be used to image all skin cancers. However, the WP-RCM device enables the visualization of an entire lesion measuring up to ~ 8 mm x 8 mm by acquiring mosaics. Mosaics provide an overview of the architectural details of the lesion (such as symmetry and circumscription). A WP-RCM device is also equipped with a digital dermatoscope camera to acquire dermoscopy images of the lesion, which guide RCM image acquisition throughout the imaging session. Both these unique features make the WP-RCM device preferable for the evaluation of melanocytic lesions for differentiating nevi from melanoma. In contrast, the handheld device is more suitable for keratinocytic lesions as these lesions typically do not require architectural evaluation but are more reliant on small-FOV, high-resolution images (0.75 mm x 0.75 mm). However, the HH-RCM device is very useful for imaging large lesions (measuring >8 mm) for tumor margin mapping for melanoma (lentigo maligna) and BCCs and for guiding biopsy site selection.

RCM is primarily used as a complementary tool to dermoscopy in triaging skin lesions that appear malignant and need a biopsy, while sparing biopsy for benign^7,19 lesions. Other indications include noninvasive monitoring of a suspicious lesion, assessing the response to topical or surgical treatment^19,37,38, delineating the surgical margins of large facial lesions of lentigo maligna (LM)^39,40,41, guiding targeted biopsies in large lesions of LM and EMPD⁴², and diagnosing inflammatory lesions^43,44. A major advantage of using RCM is the ability to render bedside diagnosis in vivo without any biopsy⁴⁵, facilitating immediate management. Furthermore, unlike histopathology evaluation, where only a small fraction of the lesion volume is analyzed, RCM enables the visualization of much larger volumes of the lesion in real time⁴⁵ and provides information on dynamic phenomena such as leukocyte trafficking^32,46.

RCM has some limitations. Unlike dermoscopy, RCM imaging requires ~15 min per lesion, which may disrupt the clinical workflow, and image evaluation requires pathologic expertise. It is not suited for evaluating lesions located deeper in the dermis or subcutis due to its limited depth of imaging (up to ~250 µm).

The "multi-modal" combined RCM-OCT device was built to overcome the limitations of RCM²². It provides the benefits of cellular-resolution imaging with RCM and the deeper and vertical images (similar to histopathology) of OCT. Initial studies have shown promising results for the use of RCM-OCT in the diagnosis and management of BCCs^23,24,25,47 (55 patients). RCM-OCT demonstrated a high accuracy (100% sensitivity, 75% specificity) in diagnosing BCCs in clinically suspicious, nonbiopsied lesions and accurately determined lesion depth for appropriate management. It also showed 100% sensitivity in detecting residual BCC in previously biopsied lesions²⁵. Recently, Monnier et al. used this device in real-world clinical settings for the evaluation of BCC in dermoscopically equivocal lesions (small, nonpigmented)²³ (18 patients). They compared the outcomes of the combined RCM-OCT device with the RCM-alone device on the same lesion. The study showed marked improvement in specificity from 62.5% to 100% and in sensitivity from 90% to 100% using the combined device over the RCM device alone, thus demonstrating the advantage and complementary nature of these two optical imaging devices. A study by Navarrete-Dechent et al. also proved the utility of the RCM-OCT device over the RCM device alone for the detection of residual BCC in "complex BCC" patients, which aided in their management and improved patient care²⁴ (10 patients). Outside dermatology clinics, RCM-OCT is being studied as a tool for the presurgical evaluation of BCC, where it has shown a high sensitivity of 82.6% and a high specificity of 93.8%, with a high correlation between the depth seen on OCT and the final depth on histopathology⁴⁷ (35 patients). Thus, this device has mostly been described for BCC diagnosis and management; its utility for melanoma and SCC is yet to be explored.

Beyond its use for BCC evaluation, Bang et al. also explored this device for the detection of cutaneous metastasis (CM) in breast cancer patients⁴⁸ (seven patients). They described the features of CM on RCM-OCT, for the first time, that would aid their diagnosis and management in the future. With the combination of high-resolution images and the ability to evaluate lesions in depth, they could detect CM in all six lesions imaged and could differentiate from a benign vascular ectatic lesion. Large-scale studies with more lesions are warranted to prove the diagnostic potential of the device for CM.

Irrespective of the device used, the following steps must be carefully executed to avoid artifacts and ensure high-quality images. To avoid motion artifacts, the patient should be positioned comfortably. Extra pillows or foot or armrests can be provided to support the imaging sites. Motion artifacts caused by breathing can be minimized by placing a firm hand on the probe during imaging. To reduce artifacts caused by any external material, clean the lesion site with an alcohol swab or soap and water before imaging. If necessary, trim the hair at the lesion site to prevent air bubble formation. All precautions should be taken to avoid cross-contamination. The disposable plastic window should be discarded after each use, and the imaging probe should be cleaned thoroughly with a disinfectant wipe after each use.

The advancements in noninvasive imaging are aimed at improving diagnostic accuracy and expanding their use worldwide. Additions to the existing HH-RCM device have been explored, such as the incorporation of a wide-field camera to enable a dual view of morphology of the lesion surface and the cellular details deeper within the lesion⁴⁹. Other additions to the HH-RCM include video mosaicking — a technique converting video into a mosaic image, thus expanding the FOV⁵⁰. To expand the use of these technologies, cheaper, smaller, and more portable devices are being developed^51,52,53, including a smaller, more flexible, handheld probe to be used for intraoral imaging⁵⁴. Furthermore, researchers are exploring targeted fluorescent probes to improve tumor detection sensitivity and specificity³¹. There are various artificial intelligence-based algorithms to assist with capturing imaging by automatically identifying the best depth to capture the DEJ⁵⁵ or removing artifacts⁵⁶. Additionally, certain algorithms are being developed to help clinicians detect skin cancers automatically^57,58. Lastly, using live, remote, in vivo RCM imaging²⁶, a remote, expert-guided technician can capture high-quality images and guide clinicians to make real-time diagnoses.

Commercially available competing devices are the line-field confocal OCT (LC-OCT)^15,16 and full-field OCT (FF-OCT)^17,18. These devices can generate images both in vertical (like OCT) and en-face planes (like RCM). The OCT images acquired with these devices have a higher lateral resolution of ~1-3 µm than the ~7 µm of OCT images of the RCM-OCT device²². However, this increase in resolution has come at the cost of a decreased imaging depth of ~300-500 µm and a smaller FOV of ~1-2mm to 500 µm x 500 µm compared to the RCM-OCT device. Thus, they are not ideal for providing any architectural detail. Their use has been described for imaging all skin cancers. In conclusion, both RCM and RCM-OCT devices are valuable noninvasive diagnostic tools and have unique clinical applications in dermatology. While RCM, as a stand-alone device (especially the WP-RCM device), is excellent for the evaluation of pigmented skin lesions, including melanoma, the RCM-OCT device is more valuable for BCC diagnosis and management. In the future, the integration of mosaicking capabilities to impart large FOV images (essential for the evaluation of melanoma) in the existing RCM-OCT device could be explored to provide one comprehensive multi-modal device for clinical use, which would be the "dream-machine" for the noninvasive imaging for all skin cancers.

Materials

Name	Company	Catalog Number	Comments
Crystal Plus 500FG mineral oil	STE Oil Company, Inc.		A food grade, high viscous mineral oil used with our various devices during in vivo imaging.
RCM-OCT	Physical Science Inc.	-	A “multi-modal” combined RCM-OCT device simultaneously images skin lesions in both horizonal and vertical modes.
Vivascope 1500	Caliber I.D.	-	A wide-probe RCM (WP-RCM) device that attaches to the skin to campture in vivo devices.
Vivascope 3000	Caliber I.D.	-	A hand-held RCM (HH-RCM) device that is moved across the skin to capture in vivo images.