Confocal microscopy – Working principle and applications in dermatology

In vivo CFM

In vivo CFM [Figure 3] predominantly works in reflectance mode (reflectance confocal microscope, RCM). The image contrast is provided by the differential refractive properties of the subcellular structures. No special staining is required in reflectance mode, though contrast agents such as aluminum chloride, citric acid, or acetic acid can accentuate the nuclei.^[9,10] Keratin, melanin, and collagen have greater refractive indices than water and act as endogenous chromophores in the skin.

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Figure 3:

In vivo confocal laser scanning microscope (830 nm), the wide-probe confocal device (VivaScope^®1500, Image courtesy – VivaScope GmbH, Munich, Germany).

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They appear brighter, and hence whiter, compared to the rest of the structures which are darker and hence appear black/ grey.^[2] This produces an image contrast in grayscale.^[2] In vivo RCM is used to examine directly accessible tissues such as skin, mucosa, and eyes and endoscopically viewed structures such as the gastrointestinal tract and genitourinary tract.^[11-13] There are two different models of RCM for in vivo examination of skin – a large device called a wide-probe confocal device having one articulated arm and a handheld device.^[4] Both models use diode laser at 830nm wavelength.^[1,5] With in vivo CFM, one can explore the living tissue at a near histological resolution, visualizing the epidermis, dermo-epidermal junction, and upper dermis up to a depth of 200–250 µm.^[13-15]

Normal skin under in vivo RCM

The appearance of the normal skin under in vivo RCM has been described.^[5,14] Shahriari et al. elaborately defined terminologies used in RCM and elucidated the same with detailed images.^[15] As the RCM images are generated from top to bottom, in normal skin, the first layer encountered is the stratum corneum. The keratinocytes are polygonal cells with decreasing size from stratum corneum to stratum basale.^[5,15] The stratum corneum [Figure 4a] appears bright and characterized by flat, polygonal, and anucleate cells.^[5] The flattened granular keratinocytes have dark central nuclei with a surrounding rim of refractile and granular cytoplasm [Figure 4b].^[5] The granular layer manifests a honeycomb pattern. The pattern is imparted by the polygonal, bright cellular outlines of keratinocytes that are arranged back to back.^[5,15] The honeycomb pattern is seen in stratum spinosum [Figure 4c] also.^[5] The keratinocytes of the basal layer [Figure 4d] are brighter at the center in comparison to the peripheral cytoplasm.^[5] This is attributed to the melanin caps that enclose the nuclei.^[5] The bright, round basal keratinocytes above dermal papillae give a cobblestone pattern.^[5] The characteristic feature of the dermo-epidermal junction is referred to as “edged papilla.”^[5] The appearance is produced by rings of basal keratinocytes that surround dark circular structures (dermal papillae) with collagen and blood capillaries within it. The melanocytes, pigmented keratinocytes, and Langerhans cells (when metabolically active in the inflammatory phase) strongly reflect the 830 nm laser light and appear bright.^[5] However, in normal human skin, it is difficult to distinguish between melanocytes and pigmented keratinocytes. Furthermore, in their quiescent state, it is difficult to visualize Langerhans cells clearly.^15]

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Figure 4:

(a): In vivo reflectance confocal microscopy image of stratum corneum – corneocytes appear as large, polygonal cells without a visible nucleus. Islands of keratinocytes are separated by linear, dark fissures that represent dermatoglyphics (Image courtesy – VivaScope GmbH, Munich, Germany); (b): In vivo reflectance confocal microscopy image of stratum granulosum showing keratinocytes (central dark nuclei and surrounding refractile granular cytoplasm) with well-demarcated outlines giving a honeycomb pattern (Image courtesy – VivaScope GmbH, Munich, Germany); (c): In vivo reflectance confocal microscopy image of stratum spinosum showing smaller, polygonal cells with thin, white cytoplasm and oval, dark nuclei. Cells of stratum spinosum also form a honeycomb pattern (Image courtesy – VivaScope GmbH, Munich, Germany); (d): In vivo reflectance confocal microscopy image of stratum basale showing small basal cells with uniform size and shape. The cells in this layer appear brighter in the center due to the presence of melanin caps encasing the nuclei (Image courtesy – VivaScope GmbH, Munich, Germany).

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Collagen fibers are seen in a reticulated pattern in the superficial dermis, while they appear as thick bundles arranged parallelly in the deep, reticular dermis.^[5]

Ex vivo CFM

Ex vivo CFM has both reflectance and fluorescence modes.^[10,13,14] The former uses a laser wavelength of 830 nm and the latter uses a laser wavelength of 488 nm or 658 nm.^[13,14] The fluorescence mode confocal microscope (FCM) uses exogenous fluorochromes to enhance the image contrast. Acridine orange is the most widely used agent, which fluorescently stains the nuclei of cells white.^[8,13,14] The other fluorochromes used are methylene blue, toluidine blue, Nile blue, fluorescein, and patent blue V.^[8,13,14] Acridine orange does not alter the tissue specimens. Therefore, these specimens can be taken up for a further frozen section or conventional histopathology.^[13]

In ex vivo RCM, the specimen is placed en masse between two thick glass slides, which are glued together with silicone glue or modeling clay. To ensure adequate contact between the specimen and the glass slide, the viewing side is smeared with aqueous gel or silicone. Furthermore, the specimen is flattened using a tissue press for better contact. This can then be placed on the specimen stage for viewing in RCM/FCM modes.^[13] The ex vivo CFM can image up to a depth of 200 µm corresponding to the upper dermis, producing horizontal images of 750 × 750 µm, which are combined to produce an image mosaic of 20 × 20 mm.^[10,14]

The latest generation FCM employs two wavelengths of laser (488 nm blue and 758 nm infrared).^[16] In fluorescent mode, dyes such as acridine orange are excited by the blue laser and the nuclear structures stained by them appear green, while the infrared laser highlights melanin and keratin in reflectance mode, which appear in different shades of grey.^[16]

One limitation encountered in CFM in comparison to routine histopathology was that fluorescence mode showed only nuclear detail and did not show collagen or cytoplasm.^[17] This was comparable to the role of hematoxylin in histopathology. The contrast provided by reflectance mode (without staining) was sensitive only to collagen and cytoplasm, which was comparable to eosin in histopathology.^[17] Gareau described “digitally stained confocal mosaics (DSCMs) that combined confocal fluorescence and reflectance images in a multimodal pseudo-color image which simulated staining with hematoxylin and eosin” and improved the sensitivity and specificity of CFM.^[16,17] Mu et al. reported that DSCMs improved the diagnostic accuracy of physicians (with training) to detect non-melanoma skin cancers.^[18]

In vivo CFM

Other applications of in vivo RCM

Applications of ex vivo CFM

The utility of ex vivo CFM is in the immediate perioperative examination of an excised tissue in its entirety, thus bypassing the need for tissue processing. Bennàssar et al. proposed FCM criteria for diagnosis of BCC (peripheral palisading, clefting, nuclear pleomorphism, and presence of stroma).^[9] Stroma refers to the modified dermis that surrounds the tumor mass. In FCM, it gives the characteristic “starry sky” appearance.^[9] “Inflammatory cells and activated fibroblast nuclei” correspond to the “stars.”^[9] Authors identified certain features as characteristics of specific subtypes [Table 3].^[9]

Ruini et al., in a study on “frozen sections of BCCs, stained with acridine orange and digitally colored to simulate hematoxylin and eosin dyes,” found FCM to have a sensitivity and specificity of 92% and 91%, respectively, to identify tumor-free margins (in comparison to conventional histopathology).^[16] A concordance of 88% was noted between the two techniques in the subtype classification of BCC.^[16]

Besides its use in assessing tumor margins in BCC, ex vivo CFM has various other indications including identification and grading of the degree of differentiation/invasiveness of SCC, diagnosis of melanoma, and measurement of tumor thickness in melanoma.^[10] This may help to determine the disease-free tumor margins during excision.

Ex vivo RCM helps to confirm nail tumors intraoperatively, which, in turn, makes immediate excision possible without tissue wastage.^[10]

Limitations

The greatest disadvantage of CFM is the shallow depth of imaging due to the limited depth of optical penetration. As a result, the mid and deep dermis cannot be visualized.^[34] Hence, the depth of the lesion cannot be assessed and in doubtful cases, lesions are better biopsied and subjected to histopathology evaluation to rule out an invasive process. Unlike conventional histology where vertical sections are the norm, here, horizontal sections are analyzed.^[14] CFM has a steep learning curve in image interpretation for practitioners in clinical practice.^[35] Another major concern of CFM is the high cost compared to conventional microscopy/dermoscopy.

RCM has increased the diagnostic accuracy in skin cancers, thus reducing the number of invasive biopsies.^[13] However, histopathology still remains the gold standard in the diagnosis of skin cancers. RCM produces grayscale images which may be difficult to interpret unlike conventional hematoxylin and eosin stained specimens, where good image contrast is obtained.^[13] RCM produces poor quality pictures on the acral skin due to increased thickness of stratum corneum.^[31]

Photobleaching (chemical alterations on exposure to light that result in a molecule losing its ability to fluoresce) of fluorescent probes and phototoxicity of live samples – the limitations of conventional fluorescence microscopy – are the limitations of FCM also.