2.1.

Patients and Treatment

The study involved four healthy Caucasian volunteers (two women and two men, age range 23 to 43) who had not used topical or systemic corticosteroids for at least two months before the study. Before enrolment, each volunteer received information about the investigation procedure with the DermaInspect^® medical device, was fully informed about the nature of the test product, and signed an informed consent. The experimental protocol was approved by the Saint Louis Hospital ethics committee, and we followed the Declaration of Helsinki protocols.

Clobetasol propionate (0.05% cream, Dermoval^®, GlaxoSmithKline, Marly-le-Roi, France) was applied under overnight occlusion for three weeks on the dorsal side of the forearm. The application zone was limited to an area of approximately $0.8{\mathrm{cm}}^2$, and Tegaderm sheets (3 M Health Care, St. Paul, Minnesota) were used for the occlusion. The treated regions were identified by tracing natural anatomic marks, such as skin folds, beauty spots, and neavus on transparent plastic sheets (Monaderm, Monaco), which allowed us to investigate the same region of the skin at D0 (before the treatment), D7, D15, D22 (end of the treatment), and D60 (38 days after the end of the treatment).

2.2.

Multiphoton Imaging

Multiphoton imaging was performed with DermaInspect^® (JenLab GmbH, Jena, Germany), a CE-marked medical system integrating two time-correlated single photon-counting detectors for fluorescence lifetime imaging (FLIM) (SPC-830, Becker & Hickl, Berlin, Germany).^14,15 SHG and 2PEF were simultaneously excited by a femtosecond titanium-sapphire laser adjusted to 760 nm (MaiTai Spectra-Physics, Mountain View, California).

The treated regions were identified using marked transparent plastic sheets, and the human skin was imaged by use of a $40\times$, 1.3-NA oil immersion objective lens (Zeiss, Jena, Germany). A cover glass was placed at the surface of the skin, and a small drop of water was used for the optical contact between the skin and the cover glass. The excitation power was exponentially increased from 12 mW at the surface of the skin up to 47 mW at a depth of 75 µm and kept constant for imaging depths greater than 75 µm. 2PEF and SHG signals were dispatched to two time-correlated single photon-counting detectors using a dichroic mirror and appropriate spectral filters.

For each volunteer, we recorded two 2PEF/SHG $z$-stacks of FLIM data (see Fig. 1). Each $130\times 130\times 162{\mathrm{\mu m}}^3$ $z$-stack was acquired with 2.346 µm $z$-step and 0.255 µm pixel size, and for each pixel the multiphoton signals were integrated using only four temporal channels with an integration time of $2\mathrm{ns}/\text{channel}$. The total acquisition time was 7.4 s per image ($511\times 511\text{pixel}$) and $9.4\min /z$-stack.

Fig. 1

Montage of a $z$-substack of combined 2PEF/SHG in vivo images of normal human skin.2PEF signal (cyan hot color) reveals the endogenous fluorophores distribution inside the epidermis and dermis (elastic fibers, fibroblasts) whereas the SHG signal (red color) reveals the collagen fibers distribution in the dermis. This figure shows images 6 to 61 extracted from a stack of 70 images; the image number is indicated on the top of each image. Images 6 to 9 correspond to stratum corneum disjunctum, and images 9 to 14 to stratum corneum compactum (the numbers are indicated on top of each image). The keratinocytes in the stratum granulosum appear in image 14, and melanin distribution in the basal layers of the epidermis can be seen in images 22 to 32. The black arrow in image 22 indicates a melanized keratinocyte with high melanin 2PEF intensity signal highlighted in white. Image 28 was acquired at the epidermis–dermis junction level as indicated by the onset of elastic and collagen fibers of the dermis. A blood capillary can be followed from images 35 to 61 (see yellow arrows). A typical 3-D region of interest (ROI, $130\times 130\times 21{\mathrm{\mu m}}^3$) used for the quantification of melanin volume density in the basal layers of the epidermis is depicted as a white rectangle. Acquisition time: $7.4\mathrm{s}/\mathrm{image}$ of $511\times 511$ pixel and $8.5\min /\mathrm{stack}$ of $130\times 130{\mathrm{\mu m}}^2\times 70$ images acquired every 2.346 µm.

Fig. 2

Qualitative scoring of melanin density in the basal layers of the epidermis. We arbitrarily defined three score levels: (0) corresponds to no or very low melanin density images, ($+$) to moderate, and ($++$) to high melanin density images. The same imaging conditions applied as in Fig. 1.

The two $z$-stacks of images were acquired within a small region of the skin (less than 1 mm distance between the $z$-stacks) situated in the central part of the $0.8{\mathrm{cm}}^2$ area, identified using the marked transparent plastic sheets. We did not exactly follow the same region of $130\times 130\times 162{\mathrm{\mu m}}^3$ over time, but we approximately investigated the same central part of the $0.8{\mathrm{cm}}^2$ treated region.

2.3.

Image Processing

The 2PEF/SHG images of a $z$-stack of FLIM data are individually saved in an sdt file format. In order to have fast access to the data, the sdt image files were opened under ImageJ (Rasband, National Institute of Health) using an appropriate plugin (LOCI) and further processed using a macro developed with ImageJ. This macro converts the sdt image files to a more accessible file format—bmp, for example—that provides direct access to the photon numbers per pixels and allows for a rapid quantitative analysis of the whole stack of images. Two-dimensional (2-D) images were combined using ImageJ, and 3-D reconstructions were performed with Imaris (Bitplane, Zurich, Switzerland).

2.4.

Analysis of Epidermis Morphology and Qualitative Evaluation of Melanin Density

2PEF/SHG images were combined using ImageJ to obtain montages of multimodal $z$-stacks and to qualitatively and quantitatively analyze the different constituents of the epidermis (see Fig. 1). We estimated the thickness of stratum corneum compactum by the number of images in which at least 50% of the image pixels are occupied by this layer. The thickness of the epidermis was estimated by the number of images between the skin surface and the top of the dermal papillae. In Fig. 1 the stratum corneum and the compactum layer correspond to images 6 to 14 and 9 to 14, respectively.

In stratum granulosum, we counted the keratinocytes in the region of interest defined by the first three images below the compactum layer, which allows for an estimation of the keratinocytes size, as there is an inverse correlation between their number and their sizes.

We also characterized the global aspect and the morphology of the keratinocytes in stratum granulosum and the melanin density. The global aspect and the morphology of the cells were qualified as normal before the corticosteroid therapy and as modified if any modifications were visible during or following the corticosteroid therapy. A qualitative evaluation of melanin density was performed using a visual assessment of the extent of white pixels that correspond to melanin in the basal layers of the epidermis. We arbitrarily defined three score levels: (0) for no or very low melanin density, ($+$) for moderate melanin density, and ($++$) for high melanin density (see Fig. 2).

2.5.

Quantitative Analysis of Melanin Density in the Basal Layers of the Epidermis

2PEF images were processed to assess the melanin density in the basal layers of the epidermis using a macro developed under ImageJ. For each $z$-stack, 3-D regions of interest (ROI) of $130\times 130\times 21{\mathrm{\mu m}}^3$ were defined inside the epidermis and just above the epidermal–dermal junction (in Fig. 1 the 3-D ROI corresponds to images 18 to 27). These ROIs were further processed to obtain the volume of the pixels occupied by the melanin 2PEF signal. For that purpose, we used a similar procedure to the ones developed for fibrosis scoring and for the assessment of 3-D collagen matrix remodeling.^18–20 We performed a Gaussian blur with 1 pixel sigma radius and applied a threshold equal to the maximum 2PEF signal intensity of nonmelanized keratinocytes to obtain a mask corresponding to melanin distribution [see Fig. 3(d)]. In order to choose the threshold value, we performed histograms of our images and found that in our experimental conditions the maximum 2PEF signal intensity in nonmelanized keratinocytes corresponds to 40 photons whereas in melanized keratinocytes it reaches 120 photons. The threshold value was fixed to 45 photons. We then expressed the melanin density as the ratio of the number of voxels occupied by melanin and the total number of voxels in the 3-D ROI.

Fig. 3

Assignment of highly intense 2PEF endogenous signals in the living epidermis to melanin and image processing for quantitative analysis of melanin density. (a) Transmitted light image and (b) corresponding 2PEF image of a fixed histological section of normal human skin stained with Fontana-Masson, showing the epidermis and the upper part of the dermis (image size is 210 µm). In the living epidermis the melanin regions colored in black in the conventional histological image (black arrow) clearly colocalize with the highly intense 2PEF signal in the multiphoton image (white arrow). The biopsy was taken from a 25-year-old female volunteer included in another clinical study. (c) In vivo 2PEF raw image with melanized keratinocytes in the basal layers of the epidermis; (d) 2PEF mask obtained after application of a threshold. The volume of the pixels occupied by the melanin 2PEF signal was obtained from the 2PEF mask and is expressed as the ratio of the number of voxels occupied by melanin and the total number of voxels in the 3-D ROI.

2.6.

Statistical Analysis

We performed a descriptive statistic followed by a normality test and a two-sample t-test. The analyses were performed using OriginPro software (OriginLab Corporation, Northampton, Massachusetts). The quantitative parameters extracted from the multiphoton images are expressed as mean $\pm 95\%$ confidence intervals of the mean.

3.1.

Normal Human Skin

Typical in vivo multiphoton images of normal human skin are presented in Fig. 1. The images acquired from the surface of the skin up to a depth of 10 µm reveal the organization of the stratum corneum disjunctum. This superficial layer contains polygonal-shaped keratinized cells, the corneocytes, which create a high-intensity fluorescence signal that mainly arises from keratin.¹¹ The next 10 µm below the stratum corneum disjuctum show a decreased intensity signal and no distinguishable cell structure. This region corresponds to the stratum corneum compactum (see Fig. 1).

About 20 µm below the surface of the skin, the transition to the stratum granulosum is detected by the occurrence of living cells that present large homogenous fluorescent cytoplasms and nonfluorescent membranes and nuclei. Deeper inside, the keratinocytes of the spinous layer show decreasing cell diameter and a decreasing ratio between the cytoplasmic and nuclear volumes. In this layer the cytoplasm fluorescence is mainly due to mitochondrial NAD(P)H.⁹

At depths between 50 and 80 µm, basal keratinocytes appear as small and polygonal cells. Some of them contain melanin, which is found in different concentrations within the cells (see Fig. 1). Under fs excitation, the highly concentrated melanin regions create a high-intensity fluorescence signal stronger than the one created by the other cellular fluorophores.^{10,12,15,21–23}

The epidermis–dermis junction is revealed in our images by the appearance of the dermal papillae. These structures, as well as the deeper parts of the dermis, are mainly characterized by a 2PEF signal created by the elastic fibers and an SHG signal created by the collagen fibers. Blood capillaries and blood cells are often seen in the papillary dermis (see yellow arrows in Fig. 1).

3.2.

Skin Modifications Following Topical Corticosteroids Occlusive Treatment

Several modifications were evidenced by in vivo multiphoton microscopy in the treated areas.

We regrouped the results into:

Qualitative observations of known corticosteroid-induced skin modifications: The stratum corneum and, more precisely, the compactum layer showed a decreased thickness for all the volunteers from D7 until the end of the treatment. Most of the time, very strong modifications could be observed, as evidenced in Fig. 4(b), in which the compactum layer seems to have disappeared completely.

Fig. 4

Multiphoton images of (a) normal human skin and (b) skin after three weeks of topical overnight occlusive treatment with clobetasol propionate in the treated regions, of the stratum corneum compactum seem to have completely disappeared, and the morphology of the cells in the granulosum layer has changed. Substack of $130\times 130{\mathrm{\mu m}}^2\times 12$ images acquired every 2.346 µm; the same imaging conditions applied as in Fig. 1.

A decrease in the global epidermis thickness could also be observed during the treatment at D15 [see Fig. 5(b)]. The thickness of the epidermis, measured from the skin surface to the top of the dermal papillae, varied from $48\pm 4\mathrm{\mu m}$ at D0 to $46\pm 6\mathrm{\mu m}$ at D7, $37\pm 2\mathrm{\mu m}$ at D15, $55\pm 10\mathrm{\mu m}$ at D22, and $56\pm 6\mathrm{\mu m}$ at D60. To check whether the decrease in the epidermis thickness observed at D15 is significant, we performed a descriptive statistic followed by a normality test and a two-sample t-test. This statistical analysis confirmed that there is a significant difference between D15 and D0, D7, D22, or D60 ($p\le 0.008$). Moreover, we observed a significant increase in the epidermis thickness at D60 as compared to D0 or D7 ($p\le 0.023$). This increase, appearing at 38 days after the end of the treatment, could be due to an increase in the proliferation rate of the epidermal cells.

Fig. 5

Impact of clobetasol propionate treatment on the size of granular keratinocytes and epidermis thickness. (a) The number of granular keratinocytes in the chosen region of interest was dramatically modified from D7 until D22 for all the volunteers. The results show an increased cell $\mathrm{number}/\mathrm{image}$, indicating a decreased cell size during the treatment. (b) The thickness of the epidermis measured from the skin surface to the top of the dermal papillae shows a significant decrease at D15 (p-value $<0.008$). Error bars correspond to 95% confidence intervals of the mean.

The keratinocytes in the granulosum layer were also dramatically modified from D7 until D22 for all the volunteers. The number of the granular keratinocytes in the chosen region of interest varied from $20\pm 2$ at D0, $38\pm 7$ at D7, $37\pm 3$ at D15, $35\pm 3$ at D22, and $22\pm 3$ at D60, indicating a decreased cell size during the treatment [see Fig. 5(a)]. This modification is significantly different as compared to D0 and D60 ($p<0.002$).

New qualitative observations: the keratinocytes in the granulosum layer were also characterized by irregular shape and an inhomogeneity in the fluorescence signal intensity of their cytoplasm at D22. Highly intense 2PEF signals were observed close to the nucleus. These signals were also irregularly distributed all over the cytoplasm [see Fig. 4(b)] whereas in normal skin [see Fig. 4(a)] the intensity of the cytoplasm fluorescence was rather homogenous. The increase of the 2PEF signal intensity at D22 indicates an increased concentration of the intracellular fluorophores. Furthermore, as the intracellular fluorescence is dominated by mitochondrial NAD(P)H emission upon excitation at 760 nm,²⁴ the increased 2PEF signal around nuclei is probably due to increased NAD(P)H concentration and thus reflects a change of the metabolic state of the cells.

Epidermal modifications also include whitening. In order to characterize this effect, we first performed a qualitative analysis of the melanin content based on a visual examination of multiphoton images (see Fig. 2). Our results show that the high-intensity fluorescence signals observed in normal skin that correspond to melanin almost completely disappeared after three weeks of corticosteroids treatment, which induced a bleaching of the treated region that was visually observed at D22 (see the multiphoton images at D22 in Fig. 6).

Fig. 6

Example of time-dependent evolution of melanin density with clobetasol propionate treatment in the basal layers of the epidermis for one volunteer. (left) 2PEF images used for qualitative scoring of melanin density; (right) 3-D melanin distribution before (D0), during (D7, D15, D22), and after (D60) clobetasol propionate treatment; (bottom right) mean values of melanin volume density calculated in 3-D ROIs. All the volunteers show a significant decrease in the melanin volume density at D22. Error bars correspond to 95% confidence intervals of the mean. The same imaging conditions applied as in Fig. 1.

New quantitative observations: in the living epidermis the highly concentrated melanin regions create a high-intensity fluorescence signal stronger than the one created by the other cellular fluorophores.^{10,12,15,21,23} We verified this point in a previous unpublished clinical study using conventional histological techniques, as displayed in Fig. 3. We imaged a fixed histological section of normal human skin stained with Fontana-Masson, using transmitted-light microscopy [see Fig. 3(a)] and 2PEF microscopy [see Fig. 3(b)]. Both images reveal the same structures, namely the melanin regions colored in black in the conventional histological image (black arrow) clearly colocalize with the highly intense 2PEF signal in the multiphoton image (white arrow). It validates the attribution of the highly intense 2PEF signal in the living epidermis to melanin. Based on this criterion, a more precise evaluation of the melanin changes upon corticosteroids treatment can be assessed. For that, we developed a melanin quantification procedure. We selected 3-D regions of interest (ROI) inside the basal layers of the epidermis (see Fig. 1), extracted melanin masks after image processing (see Fig. 3), then calculated a melanin volume density as the ratio of the number of voxels occupied by melanin to the total number of voxels in the 3-D ROI (see Fig. 6).

The 2PEF images and the 3-D reconstructions in Fig. 6 show an example of time-dependent evolution of melanin distribution with corticosteroids treatment for one volunteer. The melanin volume density decreased at D22 as shown in the 3-D reconstruction in Fig. 6. This result was also observed for all the volunteers. The graph in Fig. 6 shows a decrease in the mean melanin volume density after three weeks of topical occlusive corticosteroid treatment. We confirmed this result by statistical analysis of the data ($p\le 0.03$ between D22 and D0, D7, D15, or D60).

Two months after the end of the treatment, stratum compactum and epidermis thickness, cell morphology, and pigmentation had all almost reached their baseline values (see the results obtained at D60 in Figs. 5 and 6).