2.1.

Study Participants

Forty individuals aged between 37 and $67\text{years}$ were asked to participate in this study. All patients were recruited from the Department of Dermatology at the University Hospital of the Charité, Berlin. Written consent was obtained prior to investigation. All research was conducted according to the Declaration of Helsinki principle.

2.2.

Fluorescence Confocal Laser Scanning Microscope

A commercially available FLSM (OptiScan Ltd., Melbourne, Australia) was used in this study. A comprehensive review of the optical principles has been published elsewhere.^{12, 13}

The system is equipped with a single-moded optical fiber incorporated into a handheld scanning device, which is used for both illumination of the tissue and detection of fluorescence signals. A coherent argon-ion laser light source at a wavelength of $488\mathrm{nm}$ is used to illuminate the tissue, thereby exciting the fluorescent molecules within the specimen to a singlet state. Upon returning to their respective ground states, the wavelengths emitted by these fluorophores are longer than illumination wavelengths, permitting them to be propagated through the optical fiber. A fiber-optic coupler separates the shorter wavelengths used for illumination from the longer wavelengths returning from the focal point within the tissue, and signals are then propagated to the photomultiplier tube (PMT) for detection. Light coming from out-of-focus planes will not be propagated, thus enabling the high resolution of FLSM images. Similarly, reflected or backscattered laser light is eliminated by a fluorescence barrier filter, only permitting longer wavelengths corresponding to fluorescence signals to pass through to the filter. Tissue emitted fluorescence is then focused onto a PMT for detection, generating images of a lateral resolution of approximately $0.5\text{to}1\mu \mathrm{m}$ and an axial resolution (section thickness) of $3\text{to}5\mu \mathrm{m}$ . Images are resolved in $12\text{-}\text{bit}$ gray scale and the scanned field of view (FOV) is $250\times 250\mu \mathrm{m}$ producing images of $1024\times 1024\text{pixels}$ . Based on a $250\text{-}\mu \mathrm{m}$ FOV, when this is displayed on the standard $19\text{-}\mathrm{in}$ monitor on the Stratum used in the present study, the displayed image is approximately $25\times 25\mathrm{cm}$ , which equates to approximately $1000\times$ magnification. The numerical aperture (NA) of the system was 0.5, illumination power on tissue level was less then $0.5\mathrm{mW}$ . Figure 1 illustrates the optical scheme of FLSM.

Briefly, after intraepidermal injection of 0.2% sodium fluorescein ( $0.02\text{to}0.05\mathrm{ml}$ total volume), the optical fiber is placed onto the skin using a handheld contact device without fixing the optical system to the patient’s skin. By moving the objective lens in the $z$ (vertical) direction with respect to the skin surface, it is possible to image at different horizontal planes within the tissue. During the imaging process, the depth level being imaged was ascertained by the morphological appearance of tissue or by using a micrometer attached to the $z$ stage of the objective lens. By zeroing the imaging depth at the level of the most superficial layer before going vertically into the tissue, depth measurements can be obtained. By moving the tip of the scanning device in the $x$ and $y$ directions, different areas within the specimen are evaluated. To allow an evaluation of corresponding skin sites over time, skin markings were made using permanent marker on the day of the baseline evaluation, whereby skin sites were determined for all followup evaluations. Additionally, a schematic depiction of the lesion and its outline was obtained on transparencies, such that by localizing anatomic hallmarks, relatively precise colocalization was possible over extended periods of time. Furthermore, four to six representative FLSM images of each level are obtained to demonstrate the reproducibility of each parameter and correct for potential variations with the tissue under investigation.

2.3.

Application of the Fluorescent Dye

A 0.2% solution of sodium fluorescein (Fluorescein SE Thilo, Producer Alcon, France) was used as fluorescent dye and contrast agent for labeling of skin structures. The solution was administered under standardized conditions using a $1\text{-}\mathrm{ml}$ tuberculin syringe fitted with a 32-gauge needle^{13, 14} for superficial, intraepidermal injection. The injected volume did not exceed a total volume of $10\text{to}20\mu \mathrm{l}$ and a $3\text{to}5\text{-}\mathrm{mm}$ diameter area was infiltrated with fluorescein sodium. In addition, one drop of sodium fluorescein was applied onto the site of FLSM evaluation for labeling of skin surface structures. Exact placement of the syringe within the epidermal compartment was ensured by using a $5\text{to}10\mathrm{deg}$ angle with respect to the surface. Once intraepidermally injected, fluorescein is readily distributed within the skin. There is minimal pain associated with the insertion of the needle. In concordance with previous publications,^{13, 14} the administration of fluorescein was performed at approximately $5\text{to}10\min$ prior to the evaluation. The fluorophore is first detectable in the extracellular space, contrasting the intercellular compartment resulting in a bright white-gray contrast. It then diffuses into the cytoplasm and nuclei of epidermal keratinocytes, to finally be redistributed into the cytoplasm. Following fluorescein injection, a drop of immersion oil was applied to the sites selected for imaging, matching the refraction index of the stratum corneum $(n=1.55)$ to reduce backscatter and reflectance from surface structures.

2.4.

FLSM Measurements

2.5.

Routine Histology

We obtained $4\text{-}\mathrm{mm}$ punch skin biopsies from selected skin sites suspicious for AK or BCC upon clinical evaluation for correlation with FLSM. After fixation with formalin, the $4\text{-}\mu \mathrm{m}$ , paraffin-embedded sections were stained with H&E for histological characterization of the tumor. All cases were examined by a board-certified dermatopathologist. No biopsies were obtained from control sites. Histological evaluation criteria for AK included the presence of parakeratosis (presence of nuclei in the stratum corneum) and hyperkeratosis (increased thickening of the stratum corneum), both of which indicate epidermal hyperproliferation and aberrant epidermal differentiation. Nuclear pleomorphism and cellular atypia appear as variable sizes of keratinocyte nuclei, the eccentric placement of the nuclei, and an aberrant nuclei-to-cytoplasm ratio with atypically enlarged nuclei. The loss of normal epidermal architecture is indicative of aberrant keratinocyte differentiation and notably refers to the loss of the characteristic layering of the epidermis due to the replacement by aberrant, neoplastic keratinocytes. Histological evaluation criteria for BCC include the formation of tumor nests consisting of aberrant basal cells. The polarization of cells gives a monomorphic appearance to the tumor nests, and a characteristic cleft formation may be observed surrounding these tumor lobules. The pleomorphism of the epidermal keratinocytes surrounding BCC nests reflects an aberrant maturation and differentiation of keratinocytes and corresponds to the long-standing actinic damage that has occurred in the affected skin sites. Unspecific criteria of both malignancies include the presence of inflammatory cells, spongiosis (inter- and intracellular edema), and increased vascularity in the superficial dermal layer, all of which reflect an unspecific response to the presence of tumor tissue and neovascularization.

3.1.

Fluorescence Confocal Microscopy (FLSM) Findings of Normal Skin

We have based our analysis on observations previously made by Swindle ^{13, 14} and our findings were in agreement with those obtained in other experiments.^{13, 18} When imaging the skin in real time starting from the surface and progressing deeper, the most superficial images obtained are from the stratum corneum. The morphological appearance is that of polygonal cells measuring approximately $25\text{to}40\mu \mathrm{m}$ in size, grouped in “islands” separated by skin folds, which appear very dark [Fig. 2a ]. The stratum granulosum consists of two to four layers of cells, each cell measuring $20\text{to}25\mu \mathrm{m}$ in size. By using FLSM, nuclei can be appreciated as either bright or dark, round to oval structure placed centrally within the cell depending on the distribution of fluorescein as a function of time [Fig. 2b]. The cells of the stratum spinosum are arranged in a tight honeycombed pattern of smaller cells, each cell measuring $15\text{to}20\mu \mathrm{m}$ in size, with well-demarcated cell borders.

Fig. 2

Illustration of the horizontal FLSM morphology of normal skin. (a) FLSM morphology of stratum corneum (SC) shown. Corneocytes appear as bright structures of polygonal shape, $25\text{to}40\mu \mathrm{m}$ in diameter. Due to the anucleated nature of corneocytes, no significant uptake of fluorescein is observed, leaving the surrounding intercellular spaces to appear as a bright rim due to accumulated fluorescein (black arrow). (b) Representative image of the stratum spinosum (SS) as obtained by FLSM. Spinous keratinocytes are seen at $25\text{to}50\mu \mathrm{m}$ below the stratum corneum. The dark oval structures correspond to the spinous keratinocytes (SS), and intercellular spaces appear bright. Rhomboidal image artifact corresponds to accumulation of fluorescein in overlying skin folds. (c) Basal keratinocytes in the stratum basale (SB), surrounding each papillary structure (PS); basal keratinocytes appear darker than surrounding keratinocytes (white arrows). The center of each papilla appears bright due to the abundance of collagen (PS). All images were obtained approximately $10\text{to}15\min$ after injection of fluorescein. All images resolved in gray scale; respective $\mathrm{FOV}=250\times 250\mu \mathrm{m}$ (scale bar, $50\mu \mathrm{m}$ ).

The deepest layer of epidermis, the basal layer, is seen as dark clusters of cells, each cell measuring about $10\text{to}12\mu \mathrm{m}$ . When imaging at the suprapapillary epidermal plate, the dermoepidermal junction appears as round to oval rings of dark basal cells surrounding dermal papillae, which appear bright due to the homogenous distribution of fluorescein in this layer. A central area of blood flow corresponds to the presence of superficial dermal capillary loops [Fig. 2c]. Other features that can be observed in normal skin include hair shafts with pilosebaceous units [Figs. 3a and 3b ]; the latter appear as whorled centrally hollow structures with elliptically elongated cells at the circumference and a central hair shaft. Similarly, eccrine ducts, which appear as bright centrally hollow structures that spiral through epidermis and dermis, can be visualized [Figs. 3c and 3d].

Fig. 3

FLSM morphology of adnexal structures in normal skin. Panels (a) and (b) correspond to hairshafts with associated pilosebaceous structures. The white arrow indicates hair appearing as a dark longitudinal structure within the anatomic outline of hairshaft, which appears as a dark, hollow, canalicular structure (white arrowheads). (b) Asterisks $\left({}^{*}\right)$ correspond to areas of associated sebaceous glands. Panels (c) and (d) represent FLSM images of eccrine ducts imaged on the palm. Panel (c) shows the most superficial aspect of the eccrine duct with the narrow palmar opening indicated by a white arrow. Panel (d) corresponds to the FLSM image of an eccrine gland obtained from palmar skin; black arrow indicates the wide lumen of the eccrine gland, lined circumferentially by eccrine epithelial cells. All images resolved in gray scale; respective $\mathrm{FOV}=250\times 250\mu \mathrm{m}$ (scale bar, $50\mu \mathrm{m}$ ).

When looking at the topographical variations of normal skin, patterns vary depending on the site and skin color being evaluated. Chronically sun-exposed skin also demonstrates a thicker and more fissured or wrinkled stratum corneum and more randomly arranged and irregularly shaped dermal papillae. Furthermore, bright reticulated patterns consistent with collagen and irregular elastin fibers may be visualized. According to our preliminary findings, there may be significant numerical variations of epidermal keratinocytes such that the number of keratinocytes is greater in sun-protected sites. The palms and soles are characterized for having an extremely thick stratum corneum and a greater number of eccrine ducts. Similarly, dry skin exhibits more superficial disruptive features in the stratum corneum compared to healthy skin [Figs. 4a, 4b, 4c, 4d ].

Fig. 4

FLSM images of different physiologic and pathophysiologic states of the stratum corneum (SC). Panels (a) and (b) show corresponding images of SC in normal skin (a) and dry skin (b), respectively. Black arrows denote individual detached corneocytes. Normal skin (a) exhibits a homogenous, cohesive pattern of polygonal cells with regular openings of the eccrine ducts. Intercellular brightness relates to intercellular or cytoplasmatic fluorescein accumulation. No detached cells can be appreciated. Dry skin (b) shows decreased cohesiveness of the SC with individual detached corneocytes and partial widening of the eccrine openings. Similarly, (c) and (d) show the SC of a study participant with dry skin before (c) and after (d) the application of moisturizer. The black arrow in (c) indicates the presence of detached corneocytes as a sign of superficial SC disruption. After the application of moisturizer, (d), increased homogeneity and cohesiveness of the SC layer were noted (black arrows). All images resolved in gray scale; respective $\mathrm{FOV}=250\times 250\mu \mathrm{m}$ (scale bar, $50\mu \mathrm{m}$ ).

3.2.

Fluorescence Confocal Microscopy of Neoplastic Skin Lesions

3.2.1.

Actinic keratoses (AK)

FLSM features of AK corresponded well to the established key histopathological features of AK [Fig. 5a ]. In comparison with normal skin [Fig. 5b], FLSM was able to visualize the architectural disarray and keratinocyte pleomorphism at the level of the basal, spinous, and granular layers. [Figs. 5c and 5d]. Keratinocyte atypia are seen as cells and nuclei, varying in size and orientation. Irregular fluorescein distribution reflects the loss of clear cell-to-cell demarcation. Prominent superficial disruption associated with aberrant differentiation and hyperproliferation of epidermal keratinocytes resulted in detached corneocytes and visualization of bright superficial scale. Exocytosis and mild-to-moderate spongiosis was frequently observed, but the increased vascularity was not consistently visualized using FLSM. The absence of tumor nesting served as a distinguishing feature against BCC.

Fig. 5

FLSM morphology of AK in correlation with routine H&E and in comparison with normal skin. Panel (a) shows vertical H&E section ( $400\times$ original magnification) of AK with noted epidermal pleomorphism, parakeratosis, hyperkeratosis, and architectural disarray (black arrow indicating atypical keratinocyte). Panel (b) corresponds to en face FLSM image of normal skin showing spinous keratinocytes with homogenous appearance. Nuclei appear as uniform, bright, centrally placed structures within dark cytoplasm. Intercellular spaces appear as surrounding bright structures, indicating clear cell-to-cell demarcation. Panel (c) corresponds to en face FLSM image of AK. Image obtained at the level of the granular layer; keratinocytes showing enlarged, pleomorphic nuclei with haphazard orientation (black arrows) and noted loss of cell-to-cell demarcation. Atypical nuclei vary in size and are often placed eccentrically within dark cytoplasm. Panel (d) corresponds to en face FLSM image of AK obtained at the level of the spinous layer. Inhomogeneous staining of nuclei and irregular cell-to-cell demarcation $\left({}^{*}\right)$ illustrate the pleomorphism of atypical spinous keratinocytes (black arrows). All images resolved in gray scale; respective $\mathrm{FOV}=250\times 250\mu \mathrm{m}$ (scale bar, $50\mu \mathrm{m}$ ).

3.2.2.

Basal cell carcinoma (BCC)

FLSM was able to detect the morphologic characteristics of BCC that were in correlation with routine histology. Tumor nests corresponded to the presence of islands of atypical basal cells that are monomorphic in shape [Fig. 6c ]. Large, elongated nuclei were often oriented along the same axis, resulting in an overall polarized appearance. Areas of clefting demarcated the tumor nests against surrounding epidermal structures [Fig. 6d]. A superficial inflammatory infiltrate was visualized as multiple, round to oval structures of $8\text{to}10\mu \mathrm{m}$ in diameter [Fig. 6d]. Prominent atypia and pleomorphism of epidermal keratinocytes compared well to that observed in AK. At the level of the dermoepidermal junction [Fig. 7a ], the aberrant epidermal cells interfere with the normal honeycomb pattern of normal skin [Fig. 7b] and result in the disruption of dermal papillary architecture [Fig. 7c]. Dermal papillae exhibit abundant blood vessels with prominent tortuosity as well as increased blood vessel dilatation [Fig. 7d, 7e, 7f].

Fig. 6

Correlation between vertical H&E and FLSM morphology of BCC, superficial type. Panel (a) corresponds to vertical H&E showing nuclear polarization and epidermal pleomorphism. Arrow denoting atypical keratinocytes and aberrant proliferation of basal cells. Individual nuclei appear dark on H&E stains. Panel (b) corresponds to representative en face FLSM morphology of normal skin. Individual nuclei appear as bright, centrally placed structures within the dark appearing cytoplasm; each spinous keratinocyte is surrounded by a bright rim due to intercellular and cytoplasmatic accumulation of fluorescein, and cells appear morphologically homogenous. Panel (c) corresponds to en face FLSM morphology of BCC, atypically large cells with noted heterogeneity of fluorescein uptake and aberrant cell morphology (white arrowheads). The presence of inflammatory cells is shown as a cluster of small, round to oval, bright cells of $8\text{to}10\mu \mathrm{m}$ in diameter (white arrow). Image obtained at the level of the granular layer. Panel (d) illustrates en face FLSM morphology of lobular tumor nests (white ${}^{*}$ ). Lateral tumor edges (white arrowheads) appear surrounded by dark areas of clefting (white arrows), demarcating tumor nests; images were obtained at the level of the spinous layer. All images resolved in gray scale; respective $\mathrm{FOV}=250\times 250\mu \mathrm{m}$ (scale bar, $50\mu \mathrm{m}$ ).

Fig. 7

FLSM morphology of BCC in correlation with routine H&E and in comparison with normal skin. Panel (a) represents a vertical H&E section of BCC illustrating the aberrant morphology of basal cells at the level of the dermoepidermal junction with widening of the intercellular spaces (arrow). Panel (b) corresponds to representative FLSM morphology of normal skin with homogenous appearance of spinous keratinocytes; selected papillary structures indicated by (PS). Panel (c) corresponds to FLSM morphology of BCC, showing architectural disruption of the epidermis with pleomorphism of basal keratinocytes, decreased cell-to-cell demarcation, and widening of the intercellular spaces (white arrows); no regular papillary structures can be identified. Panels (d) to (f) illustrate the aberrant morphology of tumor vasculature. Panel (d) corresponds to vertical H&E histology $(40\times )$ of BCC with nesting of atypical basal cells in the upper dermis (black ${}^{*}$ ) and dilated blood vessels in the superficial dermis (black arrows). Panel (e) corresponds to FLSM of normal skin at the level of the dermoepidermal interface with small vessel diameter (black arrowhead). Blood vessels are shown as dark canalicular structures within the tip of the characteristic bright appearance of dermal papillae. Panel (f) corresponds to FLSM of BCC at the level of the dermoepidermal interface; The white arrow denotes the increased vessel diameter and elongated appearance of atypical vasculature. All images resolved in gray scale; respective $\mathrm{FOV}=250\times 250\mu \mathrm{m}$ (scale bar, $50\mu \mathrm{m}$ ).

3.3.

FLSM for Monitoring of Therapy

Upon initial FLSM examination, BCC sites showed characteristic atypia of basal keratinocytes with loss of cell-to-cell demarcation, tumor nesting, and nuclear pleomorphism [Fig. 8a ]. Similar evaluations following localized Imiquimod application showed spongiosis and an inflammatory infiltrate seen as multiple, round to oval structures with bright appearance. At three weeks into treatment, FLSM illustrated an inhomogenous appearance of pleomorphic basal cells and atypical keratinocytes remained. Residual atypia, nuclear pleomorphism, and a scarce inflammatory infiltrate can be observed. [Fig. 8b]. At 8 and $12\text{weeks}$ after initiation of therapy, a normalization of epidermal architecture can be observed by FLSM in selected treatment sites, and residual atypia and nuclear pleomorphism was still detectable in others [Fig. 8c].

Fig. 8

Example of FLSM for noninvasive monitoring of treatment response in BCC. Panel (a) corresponds to FLSM image of superficial BCC obtained at the level of the spinous layer prior to treatment with Imiquimod. Increased brightness is associated with architectural disruption, loss of clear cell-to-cell demarcation, and irregular staining of nuclei; fluorescein accumulation indicates nuclear atypia and cellular polymorphism (black arrows). Brightness in lower-right corner relates to the image artifact. Panel (b) corresponds to FLSM image obtained three weeks after the start of treatment. Inhomogeneous appearance of pleomorphic spinous keratinocytes (white arrows) remains. Panel (c) was imaged eight weeks posttreatment. The characteristic honeycombed pattern of the epidermis corresponds to increased normalization of epidermal architecture and homogeneous staining relates to improved cell-to-cell demarcation. Images resolved in gray scale; $\mathrm{FOV}=250\times 250\mu \mathrm{m}$ . (scale bar, $50\mu \mathrm{m}$ ).