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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 in vivo imaging.</td>
		</tr>
		<tr>
			<td>RCM-OCT</td>
			<td>Physical Science Inc.</td>
			<td>-</td>
			<td>A &#8220;multi-modal&#8221; 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 in vivo 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 in vivo 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

2.1K 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

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and invasive potential of cancer cells, and consequently the metastatic spread of disease, are being discovered and considered as complementary treatments in highly invasive cancers such as gliomas. Thus, generating data enabling the detailed analyses of cells in a 3D environment following the addition of a drug is required. The methodology described here, combining spheroid invasion assays with high-resolution image capture and data analysis by confocal laser scanning microscopy (CLSM), enabled detailed characterization of the effects of the potential anti-migratory inhibitor MI-192 on glioma cells. Spheroids were generated from cell lines for invasion assays in low adherent 96-well plates and then prepared for CLSM analysis. The described workflow was preferred over other commonly used spheroid-generating techniques due to both ease and reproducibility. This, combined with the enhanced image resolution attained by confocal microscopy compared to conventional wide-field approaches, allowed the identification and analysis of distinct morphological changes in migratory cells in a 3D environment following treatment with the migrastatic drug MI-192.

The effects of migrastatic inhibitors on glioma cancer cell migration in three-dimensional (3D) invasion assays using a histone deacetylase (HDAC) inhibitor are characterized by high-resolution confocal microscopy.

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, depending on the cancer type. To reduce the brain metastatic tumor burden, gaps in basic and translational knowledge need to be addressed. Major challenges include a paucity of reproducible preclinical models and associated tools. Three-dimensional models of brain metastasis can yield the relevant molecular and phenotypic data used to address these needs when combined with dedicated analysis tools. Moreover, compared to murine models, organ-on-a-chip models of patient tumor cells traversing the blood brain barrier into the brain microenvironment generate results rapidly and are more interpretable with quantitative methods, thus amenable to high throughput testing. Here we describe and demonstrate the use of a novel 3D microfluidic blood brain niche (&#181;mBBN) platform where multiple elements of the niche can be cultured for an extended period (several days), fluorescently imaged by confocal microscopy, and the images reconstructed using an innovative confocal tomography technique; all aimed to understand the development of micro-metastasis and changes to the tumor micro-environment (TME) in a repeatable and quantitative manner. We demonstrate how to fabricate, seed, image, and analyze the cancer cells and TME cellular and humoral components, using this platform. Moreover, we show how artificial intelligence (AI) is used to identify the intrinsic phenotypic differences of cancer cells that are capable of transit through a model &#181;mBBN and to assign them an objective index of brain metastatic potential. The data sets generated by this method can be used to answer basic and translational questions about metastasis, the efficacy of therapeutic strategies, and the role of the TME in both.

Here, we present a protocol for preparing and culturing a blood brain barrier metastatic tumor micro-environment and then quantifying its state using confocal imaging and artificial intelligence (machine learning).

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B cell malignancies or multiple myeloma (MM). However, numerous obstacles limit the efficacy and prohibit the widespread use of CAR T cell therapies due to poor trafficking and infiltration into tumor sites as well as lack of persistence in vivo. Moreover, life-threatening toxicities, such as cytokine release syndrome or neurotoxicity, are major concerns. Efficient and sensitive imaging and tracking of CAR T cells enables the evaluation of T cell trafficking, expansion, and in vivo characterization and allows the development of strategies to overcome the current limitations of CAR T cell therapy. This paper describes the methodology for incorporating the sodium iodide symporter (NIS) in CAR T cells and for CAR T cell imaging using [18F]tetrafluoroborate-positron emission tomography ([18F]TFB-PET) in preclinical models. The methods described in this protocol can be applied to other CAR constructs and target genes in addition to the ones used for this study.

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

This protocol describes the methodology for non-invasively tracking T cells genetically engineered to express chimeric antigen receptors in vivo with a clinically available platform.

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...

Three Differential Expression Analysis Methods for RNA Sequencing: limma, EdgeR, DESeq2

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration and complex biological processes and has great value in diagnostics, prognostics, and therapeutics of tumors. Differential analysis of RNA-seq data is crucial to identify aberrant transcriptions, and limma, EdgeR and DESeq2 are efficient tools for differential analysis. However, RNA-seq differential analysis requires certain skills with R language and the ability to choose an appropriate method, which is lacking in the curriculum of medical education.
Herein, we provide the detailed protocol to identify differentially expressed genes (DEGs) between cholangiocarcinoma (CHOL) and normal tissues through limma, DESeq2 and EdgeR, respectively, and the results are shown in volcano plots and Venn diagrams. The three protocols of limma, DESeq2 and EdgeR are similar but have different steps among the processes of the analysis. For example, a linear model is used for statistics in limma, while the negative binomial distribution is used in edgeR and DESeq2. Additionally, the normalized RNA-seq count data is necessary for EdgeR and limma but is not necessary for DESeq2.
Here, we provide a detailed protocol for three differential analysis methods: limma, EdgeR and DESeq2. The results of the three methods are partly overlapping. All three methods have their own advantages, and the choice of method only depends on the data.

A detailed protocol of differential expression analysis methods for RNA sequencing was provided: limma, EdgeR, DESeq2.

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration ...