2.1.

Volunteers

Characterization of intrinsic and photo-aging by MPT-FLIM was conducted in accordance with approval of Princess Alexandra Hospital Research Committee Approval No. 2008001342, which is administered by the University of Queensland Human Ethics Committee. Our study involved a total of 24 healthy subjects divided into four age groups: 20 to 29, 30 to 39, 40 to 49, and 50 to 59 years old. MPT-FLIM images were taken at two sites: the dorsal (solar-exposed, photo-damaged) and volar (solar-protected) mid-forearm. Each group contained three male and three female subjects with undamaged skin and no history of cutaneous disease. Consent was obtained after informing the volunteers about the imaging details.

2.2.

MPT-FLIM Imaging

MPT of volunteer skin was carried out using the DermaInspect® system (JenLab GmbH, Jena, Germany). Source excitation light was provided by an ultra-short pulsed, mode-locked (80-MHz) femtosecond titanium sapphire laser (Mai Tai, Spectra Physics, Mount View, California), with an 85 fs pulse width and tuning range of 710 to 920 nm. All images were taken using a Plan-Neofluar high-NA oil-immersion $40\times /1.30$ objective lens (Carl Zeiss, Germany). Using a constant incident optical power of 31 mW, endogenous cellular autofluorescence was imaged at an excitation laser wavelength of 740 nm (two-photon) while collagen SHG was imaged using an excitation wavelength of 800 nm. Autofluorescence emission and SHG signals were measured with a 350 to 650 nm broad-bandpass filter (BG39, Schott glass color filter, Schott MG, Mainz, Germany). Each image was $179\times 179\mathrm{\mu m}$ wide, taken with a scan time of 4.35 s at a resolution of $512\times 512\text{pixels}$.

For FLIM measurements, a time-correlated single-photon counting (TCSPC) SPC-830 detector (Becker & Hickl GmbH, Berlin, Germany) was integrated into the MPT system. The TCSPC module constructs a photon distribution across the $x$ and $y$ coordinates of the scan area. For each photon detected, the module determines the time of arrival within the fluorescence decay. In front of the FLIM detectors, a 350 to 650 nm broad-bandpass filter (BG39, Schott glass color filter) was used to filter the emitted light. Each FLIM scan was performed using an exposure of 13.4 s at an acquisition image size of $214\times 214{\mathrm{\mu m}}^2$. For each image site on the dorsal and ventral forearm, FLIM images were taken at three depths: 5 to 10, 20 to 30 and 40 to 50 µm from the SC, corresponding to the three layers of the epidermis [stratum granulosum (SG) and stratum spinosum (SS) and stratum basale (SB)]. The correct layer was also confirmed by keratinocyte morphology.

2.3.

Autofluorescence and SHG Intensity Versus Skin Depth and Epidermal Thickness

One hundred twenty Z-stack images were taken starting from the stratum corneum (SC) surface at a spacing of 2 µm using 740 and 800 nm excitation wavelengths. Using the ImageJ application ( http://rsbweb.nih.gov/ij/), a region of interest (ROI) was drawn to encompass epidermal cells through the Z-stack and measure the ROI area and integrated density. The background fluorescence intensity (FI) was also measured from six blank regions. The normalized FI within the ROI for each Z-stack image was calculated according to:

2.4.

SAAID

The SHG to autofluorescence aging index (SAAID) was calculated as described previously.⁹ Both SHG signal (800 nm) and autofluorescence (AF; 740 nm) intensity was measured using MPT images taken from a gated area $\sim 177\times 172\mathrm{\mu m}$ at a depth of 40 μm below the epidermal-dermal junction. The following formula was used to calculate SAAID:

2.5.

FLIM Data Analysis

FLIM data was analyzed using SPCImage 3.8.9 software (Becker & Hickl GmbH, Berlin, Germany) to generate fluorescence lifetime and photon contribution histograms. This data can be considered as an array of pixels that contains many time channels distributed across the fluorescence decay, with each pixel possessing a decay curve. The decay curve is a sum of several exponentials as each pixel represents an overlay of emissions from various endogenous fluorophores (that may or may not be found in the same photo-physical state). For our study, the decay curve was fitted using a double-exponential decay model function¹:

The instrument response function (IRF) was calibrated to a sucrose crystal standard (Ajax Finechem Pty. Ltd., Sydney, NSW, Australia). For each FLIM image, the $\mathrm{IRF}(t)$ is convoluted with the model function, $f(t)$, to generate the function $F(t)$. This function corresponds to the shape of the signal if the measured optical signal was the same as the model function $f(t)$.

2.6.

Topical Staining of Solar-Exposed and Solar-Protected In Vivo Skin

For topical staining of the skin, we applied 20 μl of a 10 μM solution of acriflavine ($52\mathrm{ng}/{\mathrm{cm}}^2$; Sigma-Aldrich Pty. Ltd, Castle Hill, NSW, Australia) and/or 6-carboxyfluorescein (6-CF; $75\mathrm{ng}/{\mathrm{cm}}^2$; Sigma-Aldrich Pty. Ltd.) in PBS to a $1{\mathrm{cm}}^2$ area on the skin surface of the dorsal (solar-exposed) and volar (solar-protected) forearms of volunteers aged 20 to 29 ($n=3$) and 50 to 59 years old ($n=3$). After 1 h, the staining area on the skin was wiped with a lint-free tissue paper and imaged by MPT-FLIM at an excitation wavelength of 740 nm (2-photon) within the SG ($\sim 5$ to 10 μm beneath the SC and morphologically validated). Fluorescence emission was collected and analyzed by a third TCSPC FLIM detector through a broad bandpass filter (515 to 620 nm) to measure changes in the average ${\tau }_m$ fluorescence lifetime of the viable epidermis due to the presence of acriflavine and/or 6-CF. The pixel intensity (pixel frequency multiplied by the photon count) was normalized to the area of the region of interest, gated using SPCImage. FLIM images of the SG were pseudo-coloured according to the average weighted lifetime (${\tau }_m$; 0 to 5000 ps).

2.7.

Statistical Analysis

Data is expressed as $\text{mean}\pm \text{standard}$ error of the mean (SEM) ($n=6$). Statistical comparison of the average lifetime, epidermal thickness or SAAID between solar-exposed and protected forearms, within each age group and epidermal layer, was made using Wilcoxon matched-pairs signed rank test. Statistical differences within an epidermal layer, of either the solar-exposed or protected forearm, across the age groups was determined using a one-way ANOVA with a post-hoc Tukey’s test. To assess differences in the average lifetime, epidermal thickness or SAAID due to intrinsic aging and photo-damage simultaneously, a repeated measures two-way ANOVA was performed with a Bonferroni’s post-test. All the statistical analysis was done using GraphPad Prism v5.03 (GraphPad Software Inc., La Jolla, California). A $p$ value of less than 0.05 was considered to be statistically significant.

3.1.

Change in Epidermal AF and Dermal Collagen SHG Intensity with Skin Depth

Figure 1 shows representative AF and SHG images within the stratum corneum (SC), granulosum (SG), spinosum (SS), basale (SB) and upper dermis of volar and dorsal forearm from a subject of the 30 through 39 years old group. The images show a marked reduction of collagen observed within the dermis in the dorsal (solar-exposed) forearm compared to the volar (solar-protected) side.

Fig. 1

Representative multiphoton images of the dorsal (solar-exposed) and volar (solar-protected) forearm skin (male subject aged 20 to 29 years old). Images are an overlay of the fluorescence emission (350 to 450 nm) at an excitation of 740 (green) and 800 nm (blue) to capture endogenous AF and collagen SHG, respectively. The yellow scale bar indicates a length of 40 μm.

The AF intensity was comparable between volar and dorsal forearms for individuals of all age categories [Fig. 2(a)]. For all age groups there was a tendency for reduced collagen SHG within the dermis of the dorsal forearm [Fig. 2(a)]. At a depth of 60 μm from the SG, the collagen SHG signal in the solar-protected forearm was significantly ($p<0.05$) higher than the solar-exposed forearm.

Fig. 2

Autofluorescence and SHG intensity curve of the viable epidermis and dermis. (a) In each group (20 to 29, 30 to 39, 40 to 49 and 50 to 59 years old), the normalized fluorescence intensity was measured after excitation at 740 and 800 nm (2-photon) in solar-exposed (grey) and solar-protected (black) forearm skin. (b) The autofluorescence and SHG intensity curves of 20 to 29 (black), 30 to 39 (blue), 40 to 49 (orange) and 50 to 59 (red) year old subjects are compared within solar-exposed or solar-protected groups. Error bars represent the SEM of the average normalized fluorescence intensity ($n=5$ to 6/forearm side/age group). Significant differences are indicated with an asterisk (* for $p<0.05$).

Figure 2(b) demonstrates that for either the solar-exposed or protected forearm, the AF and SHG signal tended to be higher for subjects aged between 50 and 59 years old than for other age groups. However, these differences were not statistically significant.

3.2.

SAAID and Epidermal Thickness

The SAAID parameters for solar-exposed skin—at a depth of 40 μm below the epidermal-dermal junction—for the 20 to 29, 30 to 39, 40 to 49, and 50 to 59 year old age groups were $0.07\pm 0.04\mathrm{au}$, $0.03\pm 0.03\mathrm{au}$, $0.17\pm 0.06\mathrm{au}$ and $0.06\pm 0.08\mathrm{au}$, respectively [Fig. 3(a)]. For solar-protected skin, the SAAID values were $0.23\pm 0.07\mathrm{au}$, $0.20\pm 0.11\mathrm{au}$, $0.23\pm 0.09\mathrm{au}$ and $0.26\pm 0.08\mathrm{au}$, respectively. Figure 3(a) shows that the SAAID index was significantly lower in solar-exposed forearm of 20 to 29 year old subjects than solar-protected skin. For each respective age group, the SAAID index in solar-exposed skin trended lower than solar-protected skin, but significant differences were only seen for 20 to 29 year old subjects. The tendency for the SAAID intensity to be lower in the forearms of solar-exposed volunteers, across all age groups, was statistically significant ($p<0.05$). The SAAID index for either solar-exposed or protected skin did not appear to change with age [Fig. 3(a)].

Fig. 3

SAAID (a) aging index and epidermal thickness (b) values of solar-exposed (gray) and solar-protected (white) forearm skin of each indicated age group. Error bars represent the SEM of the average SAAID or epidermal thickness ($n=5$ to 6/forearm side/age group). Significant differences are marked by an asterisk (* for $p<0.05$).

Figure 3(b) indicates that there was no significant difference in the epidermal thickness of solar-exposed versus protected skin for volunteers aged 20 through 29 ($35\pm 2.91$ versus $31\pm 1.34\mathrm{mm}$), 30 through 39 ($37.67\pm 4.27$ versus $36.67\pm 3.17\mathrm{mm}$), 40 through 49 ($27.2\pm 4.45$ versus $39.6\pm 4.4\mathrm{mm}$) and 50 through 59 ($40\pm 4.07$ versus $31.7\pm 5.15\mathrm{mm}$) years old, respectively. Similarly, there was no significant change in the epidermal thickness with age.

3.3.

Redox Imaging of Sun-Protected and Exposed In Vivo Skin

Figure 4 shows representative redox images from solar-protected and exposed forearm skin of each age category. Figure 5(a) and 5(b) chart the average weighted lifetime histograms from all the volunteers, comparing solar-protected and exposed forearm skin for each skin stratum and age group.

Fig. 4

Representative MPT-FLIM images of the viable epidermis (SG, SS and SB) within solar-exposed and solar-protected forearm skin of each indicated age group. FLIM images were measured within a spectral range of 350 to 450 and 450 to 515 nm to capture NAD(P)H and NAD(P)H/FAD, respectively. Redox images were pseudo-colored according to the average-weighted fluorescence lifetime (${\tau }_m$) within a range of 0 to 2000 ps (blue-to-red). Each image is $214\times 214\times 1{\mathrm{\mu m}}^3$. Scale bars (yellow) indicate a length of 40 μm.

Fig. 5

${\tau }_m$ lifetime histograms of NAD(P)H (a) and NAD(P)H/FAD (c) within the viable epidermis (SG, SS and SB) of solar-exposed and solar-protected forearm skin of each indicated age group. The histogram represents the average normalized pixel frequencies for each ${\tau }_m$ lifetime within the gated ROI, encompassing viable epidermal cells. The corresponding average ${\tau }_m$ lifetime of NAD(P)H (b) and NAD(P)H/FAD (d), for solar-exposed and solar-protected skin, is charted for each age group. Error bars representing the SEM ($n=6/\text{forearm side}/\text{age group}$), with significant differences marked by an asterisk (* $p<0.05$, ** $p<0.01$).

We observed a significant increase ($p<0.05$) in the average ${\tau }_m$ lifetime of NAD(P)H in the SG of the solar-protected forearm ($832.1\pm 94.7\mathrm{ps}$) compared to the solar-exposed forearm ($682\pm 96.4\mathrm{ps}$) of 20 to 29 year old subjects [Fig. 5(a)]. Within the SB, the average lifetime was significantly higher in the solar-protected forearm versus solar-exposed skin of 40 to 49 ($1005\pm 81.7$ versus $521.7\pm 72.9\mathrm{ps}$ respectively) and 50 to 59 ($656.6\pm 42.9$ versus $596.9\pm 57.2\mathrm{ps}$ respectively) year old subjects. Similarly, the average lifetime of NAD(P)H/FAD was significantly higher in solar-protected skin ($1257\pm 70.7\mathrm{ps}$) than solar-exposed skin ($826.5\pm 98.8\mathrm{ps}$) for 40 to 49 year old subjects.

Overall, we observed a statistically significant ($p<0.05$) tendency for average ${\tau }_m$ lifetime of NAD(P)H [Fig. 5(a)] and NAD(P)H/FAD [Fig. 5(b)] to be elevated within each epidermal stratum for solar protected skin versus solar-exposed skin, in each age group. This indicates photo-damage may be responsible for a redox difference observed between solar-exposed and solar-protected skin.

We observed NAD(P)H lifetime changes due to intrinsic aging within the solar-protected forearm in all of the epidermal layers [Fig. 5(a)]. In the SG, the average NAD(P)H lifetime for solar-exposed skin from 20 to 29 year old subjects ($682\pm 96.4\mathrm{ps}$) was significantly lower than for 50 to 59 year old subjects ($994.6\pm 71.5\mathrm{ps}$). In the SS, the average NAD(P)H lifetime of the solar-protected forearm skin from 20 to 29 year old volunteers ($789.3\pm 57.9\mathrm{ps}$) was significantly lower ($p<0.05$) than subjects aged 40 to 49 ($1114\pm 91.1\mathrm{ps}$).

For the SB, the average NAD(P)H lifetime of solar-protected forearm skin from 20 to 29 year old subjects ($576.5\pm 78.8\mathrm{ps}$) was significantly lower ($p<0.01$) than the average NAD(P)H lifetime 40 to 49 year olds ($1005\pm 81.7\mathrm{ps}$). However, the average NAD(P)H lifetime of solar-protected forearm skin from 40 to 49 year olds was significantly ($p<0.05$) higher than 50 to 59 year olds ($654.4\pm 52.5\mathrm{ps}$).

In the solar-exposed forearm, the average ${\tau }_m$ NAD(P)H lifetime within the SG was significantly lower ($p<0.05$) in 20 to 29 year olds ($536.6\pm 54\mathrm{ps}$) compared to 30 to 39 year old subjects ($808.7\pm 115\mathrm{ps}$), which was also significantly higher ($p<0.05$) than the age-related change in the average ${\tau }_m$ NAD(P)H lifetime of 40 to 49 year olds ($521.7\pm 72.9\mathrm{ps}$). The only statistically significant age-related difference in the ${\tau }_m$ NAD(P)H/FAD lifetime was observed in the SG of solar-exposed forearm skin where the average lifetime for 20 to 29 year olds ($991.1\pm 98.5\mathrm{ps}$) was lower than that of 30 to 39 year old subjects ($1263\pm 65.9\mathrm{ps}$) [Fig. 5(b)].

Collectively, these data demonstrate an increase in the average weighted lifetime of solar-protected skin compared to solar-exposed skin, suggesting a redox change associated with solar exposure and photo-damage. We also observed an increase in the average lifetime of NAD(P)H associated with intrinsic aging, predominantly within solar-protected skin.

In Fig. 6, we further examined the lifetime (${\tau }_1$ and ${\tau }_2$) and amplitude coefficient (${\alpha }_1$) differences between the solar-exposed and solar-protected forearm of a representative subject aged 40 through 49 years old. Figure 6(a) shows FLIM images of the SG and SB, of the solar-exposed and solar-protected forearm, pseudo-colored for the short (${\tau }_1$) and long (${\tau }_2$) NAD(P)H lifetimes and ${\tau }_1$ amplitude coefficient (${\alpha }_1$). The FLIM images of the solar-protected side of the forearm, of both the SG and SB, appeared brighter than the solar-exposed side [Fig. 6(a)].

Fig. 6

Representative ${\tau }_1$, ${\tau }_2$ and ${\alpha }_1$ FLIM images (a) and histograms (b) of solar-exposed (gray) and solar-protected (red) forearm skin (SG and SB) of a subject aged 40 to 49 years old. (a) FLIM images were pseudo-colored (blue-to-red) according to ${\tau }_1$ (0 to 2000 ps), ${\tau }_2$ (0 to 2000 ps) and ${\alpha }_1$ (50 to 100%) parameters. (b) Histograms display the pixel frequencies for each FLIM parameter within the gated ROI, encompassing viable epidermal cells.

The ${\tau }_1$ (287.6 ps) and ${\tau }_2$ (2269.9 ps) lifetimes of the solar-exposed SG displayed a lower lifetime shift relative to the solar-protected SG (481.6 and 2644.6 ps, respectively) (Fig. 6). Similarly, the solar-exposed SB ${\tau }_1$ (107.5 ps) and ${\tau }_2$ (1847.5 ps) lifetimes were lower than the solar-protected SB (605.8 and 2810.1 ps, respectively). In contrast, the solar-exposed ${\alpha }_1$ values of the SG (75%) and SB (93.5%) displayed a red shift relative to the solar-protected skin (67.7% and 71.6%, respectively) [Fig. 6(b)]. This data suggests that the lower average lifetime of solar-exposed skin (Fig. 5) is due to both shorter ${\tau }_1$ and ${\tau }_2$ lifetimes as well as an increase in the ${\tau }_1$ component compared to solar-protected skin.

3.4.

Fluorescent Staining of Intrinsically Aged and Photo-Aged Skin

Figure 7(a) shows representative ${\tau }_m$ FLIM images (${\lambda }_{\mathrm{Em}}$: 515 to 620 nm) of the solar-protected forearm skin (SG) of a subject aged 20 to 29 years old after the topical application of acriflavine ($52\mathrm{ng}/{\mathrm{cm}}^2$) and/or 6-CF ($75\mathrm{ng}/{\mathrm{cm}}^2$). The ${\tau }_m$ histograms of the untreated and vehicle controls, within the viable epidermal cells and furrows (Fig. 7(a), i though iv), display ${\tau }_m$ lifetimes ranging between 0 and 1500 ps. After topical application, both acriflavine and 6-CF were predominantly localized within the furrow, with ${\tau }_m$ lifetimes between $\sim 2000$ to 3000 and $\sim 3000$ to 3500 ps, respectively. Acriflavine, in combination with 6-CF, also localized within the furrow, showing a ${\tau }_m$ lifetime between $\sim 3000$ to 4000 ps [Fig. 7(a)].

Fig. 7

Representative ${\tau }_m$ lifetime FLIM images (a) of solar-protected skin (SG) after staining with acriflavine and/or 6-CF, along with negative and vehicle controls. The cellular and furrow ROI are indicated by the purple and red boxes, respectively. Normalized pixel intensities of the ${\tau }_m$ lifetimes are plotted for either the cellular or furrow ROI of each treatment: untreated control (black; cells [i]: continuous line, furrow [ii]: hashed line), PBS (vehicle control; gray; cells [iii]: continuous line, furrow [iv]: hashed line), acriflavine (orange [v]), 6-CF (green [vi]) and acriflavine + 6-CF (red [vii]). (b) Representative ${\tau }_m$ lifetime FLIM images of solar-exposed and solar-protected forearm skin (SG), 1 h following the topical application of acriflavine ($52\mathrm{ng}/{\mathrm{cm}}^2$) and/or 6-CF ($75\mathrm{ng}/{\mathrm{cm}}^2$) along with negative and vehicle (PBS) controls. FLIM data were measured within a spectral range of 515 to 620 nm and pseudo-colored (blue-to-red) according to a ${\tau }_m$ lifetime range of 0 to 5000 ps. Viable epidermal cells can be observed within the corresponding grayscaled images (${\lambda }_{\mathrm{Em}}$: 350 to 450 nm) displayed in the bottom right corner of each FLIM image. (c) The average ${\tau }_m$ lifetime histograms of the solar-exposed and solar-protected forearm skin (SG) from subjects aged 20 to 29 and 50 to 59 years old. Normalized pixel intensities of the ${\tau }_m$ lifetimes are plotted for the cellular ROI of each treatment: untreated control (black), PBS (vehicle control; gray), acriflavine (orange), 6-CF (green) and acriflavine + 6-CF (red). The error bars indicate the SEM ($n=3$/forearm side/age group).

Figure 7(b) shows representative FLIM ${\tau }_m$ images (${\lambda }_{\mathrm{Em}}$: 515 to 620 nm) of solar-exposed and solar-protected forearm skin (SG), from volunteers aged 20 to 29 ($n=3$) and 50 to 59 years old ($n=3$), stained with acriflavine and/or 6-CF. The data demonstrates that there were no apparent differences in the staining of viable epidermal cells, within the SG, between intrinsically aged or photo-damaged skin after the topical application of these vital dyes [Fig. 7(b)]. Figure 7(c) demonstrates that the ${\tau }_m$ lifetime histograms of the viable epidermis cells largely overlap with the negative and vehicle controls ($\sim 0$ to 1500 ps). Despite the observation of some lifetimes $>2000\mathrm{ps}$ in solar-exposed skin for both age groups, the lifetime histograms do not correspond with acriflavine and/or 6-CF lifetimes measured within the furrow [Fig. 7(a)], suggesting minimal penetration of the dyes into the SG. This data indicates that the topical application of acriflavine and 6-CF dyes was unable to reveal any impaired barrier protection associated with photo-aging.