Name: combining reflectance confocal microscopy with optical coherence tomography for noninvasive diagnosis of skin cancers via image acquisition
Uploaded: 2022-08-18T12:30:00.000+00:00
Duration: 9 min 37 s
Description: Watch this Scientific Journal Video about Combining Reflectance Confocal Microscopy with Optical Coherence Tomography for Noninvasive Diagnosis of Skin Cancers via Image Acquisition at JoVE.com

A special thank you is given to Kwami Ketosugbo and Emily Cowen for being volunteers for imaging.This research is funded by a grant from the National Cancer Institute /National Institutes of Health (P30-CA008748) made to the Memorial Sloan Kettering Cancer Center.

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.
<ol>
	<li>Lesion selection and clinical indications
	<ol>
		<li>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.</li>
		<li>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&#39;s disease (EMPD) and LM.</li>
		<li>Carry out noninvasive monitoring of nonsurgical treatment such as topical drugs (imiquimod), radiation, photodynamic therapy, and laser ablation.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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:</li>
	<li>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.</li>
	<li>Image acquisition using the WP-RCM device (Figure 1, Figure 2, Supplemental Figure S1, Supplemental Figure S2, and Supplemental Figure S3) 
	&#8203;NOTE: WP-RCM devices are capable of capturing stacks, mosaic, live single-framed videos, and single-framed images.
	<ol>
		<li>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.</li>
		<li>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.
		<ol>
			<li>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).</li>
			<li>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.</li>
		</ol>
		</li>
		<li>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&#176; 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.</li>
		<li>Acquiring RCM images (Figure 2, Supplemental Figure S1, and Supplemental Figure S2)
		<ol>
			<li>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 &#8212; the anucleate layer of the stratum corneum (Supplemental Figure S1).</li>
			<li>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.</li>
			<li>Acquire a stack in the lesion&#39;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 &#181;m step-size and 250 &#181;m 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.</li>
			<li>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.</li>
			<li>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.</li>
			<li>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 &#34;stack&#34; (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 &#181;m). A &#34;mosaic&#34; (Supplemental Figure S2) is a large FOV of images obtained by stitching individual 500 &#181;m x 500 &#181;m images together in &#34;X-Y&#34; (horizontal en face plane).</li>
		</ol>
		</li>
		<li>Completing an imaging session
		<ol>
			<li>Click on Done Imaging.</li>
			<li>Detach the microscope from the plastic window. Remove the plastic window by gently holding the patient&#39;s skin taut and dispose of it. Wipe off oil on the skin with an alcohol swab.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Generating a Confocal Diagnostic Evaluation report (Supplemental Figure S3)
		<ol>
			<li>Click on New Evaluation. Enter the diagnosis from the preselected options in the dropdown menu.</li>
			<li>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&#160;(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.</li>
			<li>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&#39;s chart for billing.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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. 
		&#8203;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.</li>
	</ol>
	</li>
</ol>
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 &#181;m x 200 &#181;m, 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.
<ol>
	<li>Lesion selection
	<ol>
		<li>Look for dermoscopically equivocal pink or pigmented lesion to rule out BCC.</li>
		<li>Assess the depth of BCC for management, and assess the residual BCC post treatment.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>Image acquisition using the RCM-OCT device (Figure 6 and Figure 7)
	<ol>
		<li>Prepare the probe similarly to the one used for the HH-RCM (steps 1.6.1-1.6.2.)</li>
		<li>Acquire images in line imaging mode and raster mode.
		<ol>
			<li>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 &#181;m and the number of steps to 40 (Figure 7A).</li>
			<li>Click on Grab. Acquire stacks following step 1.5.4.3. Once complete, click on the Freeze button.</li>
			<li>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.</li>
			<li>Acquire multiple stacks and videos based on the physician&#39;s interest.</li>
			<li>Clean the lesion and machine as described in step 1.6.5.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Ucalene Harris has no competing financial interest. Dr. Jain is a consultant on Enspectra Health Inc. Dr. Milind Rajadhyaksha is a former employee of and owns equity in Caliber ID (formerly, Lucid Inc.), the company that manufactures and sells the VivaScope confocal microscope. The VivaScope is the commercial version of an original laboratory prototype that was developed by Dr. Rajadhyaksha when he was at Massachusetts General Hospital, Harvard Medical School.

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 &#62;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 benign7,19 lesions. Other indications include noninvasive monitoring of a suspicious lesion, assessing the response to topical or surgical treatment19,37,38, 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 lesions43,44. 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 trafficking32,46.
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 &#181;m).
The &#34;multi-modal&#34; 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 BCCs23,24,25,47 (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 &#34;complex BCC&#34; 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 &#8212; 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 developed51,52,53, 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 automatically57,58. 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 &#181;m than the ~7 &#181;m 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 &#181;m and a smaller FOV of ~1-2mm to 500 &#181;m x 500 &#181;m 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 &#34;dream-machine&#34; for the noninvasive imaging for all skin cancers.

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 cancers1,2. 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 explored5,6,7,8,9,10,11,12,13,14,15,16,17,18. 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 clinics7,8,19. 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 &#181;m, 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 clinics23,24,25.
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.

<table><tbody><tr><td>Crystal Plus 500FG mineral oil</td><td>STE Oil Company, Inc.</td><td></td><td>A food grade, high viscous mineral oil used with our various devices during &lt;em&gt;in vivo &lt;/em&gt;imaging.</td></tr><tr><td>RCM-OCT</td><td>Physical Science Inc.</td><td>-</td><td>A &amp;ldquo;multi-modal&amp;rdquo; combined RCM-OCT device simultaneously images skin lesions in both horizonal and vertical modes.</td></tr><tr><td>Vivascope 1500</td><td>Caliber I.D.</td><td>-</td><td>A wide-probe RCM (WP-RCM) device that attaches to the skin to campture &lt;em&gt;in vivo&lt;/em&gt; devices.</td></tr><tr><td>Vivascope 3000</td><td>Caliber I.D.</td><td>-</td><td>A hand-held RCM (HH-RCM) device that is moved across the skin to capture &lt;em&gt;in vivo&lt;/em&gt; images.</td></tr></tbody></table>

combining reflectance confocal microscopy with optical coherence tomography for noninvasive diagnosis of skin cancers via image acquisition

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 &#181;m) 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) &#8212; 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 &#34;multi-modal,&#34; 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.

Watch this Scientific Journal Video about Combining Reflectance Confocal Microscopy with Optical Coherence Tomography for Noninvasive Diagnosis of Skin Cancers via Image Acquisition at JoVE.com

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

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.

Skin cancer is one of the most common cancers worldwide. Diagnosis relies on visual inspection and dermoscopy followed by biopsy for histopathological ...

combining-reflectance-confocal-microscopy-with-optical-coherence

Nanyang Technological University

Research

JoVE Journal

Cancer Research

2.2K Views.  Memorial Sloan Kettering Cancer Center. 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.

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

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

Medicine

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

Bioengineering

Guided Tumor Inoculation and Multimodal Imaging in Mice

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

Author Spotlight: Integrating High-Resolution Intravital Imaging and MRI to Enhance Stereotactic Body Radiation Therapy Planning

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

Evaluating Ocular Surface Squamous Neoplasia Using Anterior Segment HR-OCT

Anterior High-Resolution Optical Coherence Tomography in the Diagnosis and Therapeutic Monitoring of Ocular Surface Squamous Neoplasia

Ocular surface squamous neoplasia (OSSN) is the most common tumor of the ocular surface, ranging from mild dysplasia to invasive squamous carcinoma. Traditionally, the diagnosis of OSSN relies on histopathological confirmation followed by a full-thickness biopsy. However, in the past two decades, the therapeutic approach to OSSN has shifted from surgical intervention to topical chemotherapy regimens in clinical settings. This shift emphasizes the need for less invasive or non-invasive methods to diagnose ocular surface pathologies. Among various imaging devices, commercially available high-resolution optical coherence tomography (HR-OCT) has emerged as a powerful tool for the characterization of OSSN. HR-OCT provides an in vivo, cross-sectional view of ocular surface lesions, offering an "optical biopsy" for OSSN with high sensitivity and specificity. It provides valuable information in differentiating intraepithelial or invasive OSSN from other benign lesions. Additionally, HR-OCT can be used to monitor the response to topical chemotherapy and to detect subclinical OSSN during follow-up visits. In this article, the scanning protocol for image acquisition is presented, and image interpretation for OSSN is outlined. This standardized, practical, and reproducible approach is recommended in clinical workflows and is expected to assist clinicians in the management of OSSN.

Author Spotlight: Anterior HR-OCT as a Non-Invasive Tool for Characterizing Ocular Surface Squamous Neoplasia

Anterior segment high-resolution optical coherence tomography (HR-OCT) is a promising non-invasive modality for the diagnosis and therapeutic evaluation of ocular surface squamous neoplasia (OSSN). Here, the system setup, scanning technique, and representative diagnostic results are presented.

Ocular surface squamous neoplasia (OSSN) is the most common tumor of the ocular surface, ranging from mild dysplasia to invasive squamous carcinoma. ...

Engineering

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

We describe use of full-field optical coherence microscopy as a method for high quality assessment of corneal donor stroma. This protocol can be used to identify features indicative of health or disease, and is aimed at improving the screening and selection of donor tissues, and hence the results of keratoplasty.

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting ...

Immunology and Infection

Mouse Retina OCT Fibergram Alignment with Confocal Images

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

Author Spotlight: Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

Neuroscience

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...

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

Summary

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Chapters in this video

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Anterior High-Resolution Optical Coherence Tomography in the Diagnosis and Therapeutic Monitoring of Ocular Surface Squamous Neoplasia

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy