In vivo imaging with a fast large-area multiphoton exoscope (FLAME) captures the melanin distribution heterogeneity in human skin

Abstract

Melanin plays a significant role in the regulation of epidermal homeostasis and photoprotection of human skin. The assessment of its epidermal distribution and overall content is of great interest due to its involvement in a wide range of physiological and pathological skin processes. Among several spectroscopic and optical imaging methods that have been reported for non-invasive quantification of melanin in human skin, the approach based on the detection of two-photon excited fluorescence lifetime distinguishes itself by enabling selective detection of melanin with sub-cellular resolution, thus facilitating its quantification while also resolving its depth-profile. A key limitation of prior studies on the melanin assessment based on this approach is their inability to account for the skin heterogeneity due to the reduced field of view of the images, which results in high dispersion of the measurement values. Pigmentation in both normal and pathological human skin is highly heterogeneous and its macroscopic quantification is critical for reliable measurements of the epidermal melanin distribution and for capturing melanin-related sensitive dynamic changes as a response to treatment. In this work, we employ a fast large-area multiphoton exoscope (FLAME), recently developed by our group for clinical skin imaging, that has the ability to evaluate the 3D distribution of epidermal melanin content in vivo macroscopically (millimeter scale) with microscopic resolution (sub-micron) and rapid acquisition rates (minutes). We demonstrate significant enhancement in the reliability of the melanin density and distribution measurements across Fitzpatrick skin types I to V by capturing the intra-subject pigmentation heterogeneity enabled by the large volumetric sampling. We also demonstrate the potential of this approach to provide consistent measurement results when imaging the same skin area at different times. These advances are critical for clinical and research applications related to monitoring pigment modulation as a response to therapies against pigmentary skin disorders, skin aging, as well as skin cancers.

Figure 1

3D melanin density for Fitzpatrick skin types I–V. (a) The z-projection of MVF for representative subjects with skin types I-V, from the volar and dorsal forearm. Scale bar = 1 mm. (b) 2D-melanin density as a function of epidermal depth from the basal layer (0) to stratum granulosum (1) for all the skin types. The data and error bars represent the average and standard deviation of the melanin fraction in each layer, respectively. The position across the y-axis is normalized against the epidermal thickness. (c) The global MVF values for all the skin types from both dorsal and volar forearms. (*, #) indicate a significant difference (P < 0.01) among the average MVF values in the dorsal and volar forearm, respectively, except for skin types sharing letter ‘a’. (°) indicate a significant difference (P < 0.01) between dorsal and volar forearm within a skin type. (N = 32 z-stacks, the 16 stacks from each subject (total of 2) were separately considered).

Figure 2

Comparison of 3D melanin density for skin types with different levels of heterogeneity. (a, c) z-projection of the MVF values from the dorsal forearm of representative subjects with skin types I and V, respectively. (b, d) 2D melanin density as a function of epidermal depth from the basal layer (0) to stratum granulosum (1) for skin types I and V, respectively. The position across the y-axis is normalized against the epidermal thickness. The plots in (b) and (d) correspond to the ROIs of 0.25 × 0.25 mm² marked in (a) and (c) with the same color and number.

Figure 3

The effect of field of view on the accuracy and precision of the MVF measurements. (a, b) The MVF for skin types I to V for the volar and dorsal forearm, respectively. The data points and error bars correspond to the average MVF and their standard deviation (S.D.) (c, d) The S.D. of the MVF values as a function of imaging area for skin types I to V for the volar and dorsal forearm, respectively. (0.25 × 0.25 mm², N = 338 z-stacks, 1.6 × 1.6 mm, N = 8 stacks. Complete list in “Methods”).

Figure 4

Comparison of the MVF measured by selectively sampling a small-area (0.25 × 0.25 mm²) versus sampling a large-area (1.1 × 1.1 mm²). (a, b) Schematic diagrams for Methods A and B, respectively. (c–e) The resulting MVF values using the sampling methods described in (a) and (b) for skin types I, III, and V, respectively (N = 9 z-stacks). (***) Indicate significant difference between the MVF values obtained using Method A and B using a two-sample unpaired t-test with resulting P-values indicated in the figure.

Figure 5

Repeatability of the MVF measurements. MVF values were calculated from three different measurements by imaging the dorsal forearm of a skin type IV subject. (N = 9 stacks, FOV: 1.1 × 1.1 mm² for each measurement). All measurements were performed based on images acquired from approximately the same area using skin landmarks as guidance.