In vivo multiphoton NADH fluorescence reveals depth-dependent keratinocyte metabolism in human skin

Abstract

We employ a clinical multiphoton microscope to monitor in vivo and noninvasively the changes in reduced nicotinamide adenine dinucleotide (NADH) fluorescence of human epidermal cells during arterial occlusion. We correlate these results with measurements of tissue oxy- and deoxyhemoglobin concentration during oxygen deprivation using spatial frequency domain imaging. During arterial occlusion, a decrease in oxyhemoglobin corresponds to an increase in NADH fluorescence in the basal epidermal cells, implying a reduction in basal cell oxidative phosphorylation. The ischemia-induced oxygen deprivation is associated with a strong increase in NADH fluorescence of keratinocytes in layers close to the stratum basale, whereas keratinocytes from epidermal layers closer to the skin surface are not affected. Spatial frequency domain imaging optical property measurements, combined with a multilayer Monte Carlo-based radiative transport model of multiphoton microscopy signal collection in skin, establish that localized tissue optical property changes during occlusion do not impact the observed NADH signal increase. This outcome supports the hypothesis that the vascular contribution to the basal layer oxygen supply is significant and these cells engage in oxidative metabolism. Keratinocytes in the more superficial stratum granulosum are either supplied by atmospheric oxygen or are functionally anaerobic. Based on combined hemodynamic and two-photon excited fluorescence data, the oxygen consumption rate in the stratum basale is estimated to be ∼0.035 μmoles/10(6) cells/h.

Figure 1

MPM-based clinical tomograph (MPTflex) during the occlusion experiment.

Figure 2

(a) Reflectance map at 850 nm including the ROI selected for the SFDI analysis. SFDI absorption (b) and scattering (c) maps corresponding to 850 nm. SFDI quantitative oxyhemoglobin (d) and deoxyhemoglobin (e) concentration maps.

Figure 3

Schematic 7-layer Monte Carlo model of skin with dermis subdivided into four layers with different blood volume fractions. The NADH fluorescence source was placed at a location in the epidermis 35 μm below the tissue surface, and a 1 mm diameter detector was used corresponding to the size of the collection area of the objective (see text).

Figure 4

Representative TPEF in vivo images of human keratinocytes from time series corresponding to (a) t = 2 min (b) t = 4 min (c) t = 6 min. Scale bar is 20 μm. The graph represents the average of normalized mean intensities of TPEF images as a function of time. Error bars represent standard deviation.

Figure 5

Normalized mean intensity of TPEF images as a function of time before, during, and after arterial occlusion for human keratinocytes in stratum granulosum (a) and in a layer close to stratum basale (b). Representative TPEF in vivo images recorded before (a1, b1), during (a2, b2), and after (a3, b3) occlusion corresponding to human keratinocytes in stratum granulosum (a1, a2, a3) and in a layer close to stratum basale (b1, b2, b3). Scale bar is 20 μm. (c) Hemodynamic response to arterial occlusion; oxyhemoglobin concentration ctO₂Hb (square), deoxyhemoglobin concentration ctHHb (diamond). The dashed lines represent the start and the release points of the occlusion. Error bars represent standard deviation.

Figure 6

Relationship between the change in deoxyhemoglobin concentration ctHHb (a), hemoglobin oxygen saturation stO₂ (b), total oxyhemoglobin concentration ctHbT (c), and the change in the NADH fluorescence intensity (Int), during arterial occlusion. Error bars represent standard deviation.

Figure 7

Absorption (a) and scattering (b) coefficients at 658 nm obtained from SFDI measurements. The dashed lines represent the start and the release points of the occlusion. Error bars represent SFDI measurement standard deviation (39).

Figure 8

Interrogation maps showing the skin layers sampled by photons emitted by the fluorescent source corresponding to 450 nm (a), 500 nm (b), and 550 nm (c). The z axis represents the skin tissue depth (the origin is on the tissue surface). The white horizontal lines mark the separation between the skin layers considered in the Monte Carlo simulation. The colorbar is on a log scale with a spectrum ranging from 1 to 10⁻⁵.