Non-Invasive Skin Imaging Assessment of Human Stress During Head-Down Bed Rest Using a Portable Handheld Two-Photon Microscope

Abstract

Spaceflight presents a series of physiological and pathological challenges to astronauts resulting from ionizing radiation, microgravity, isolation, and other spaceflight hazards. These risks cause a series of aging-related diseases associated with increased oxidative stress and mitochondria dysfunction. The skin contains many autofluorescent substances, such as nicotinamide adenine dinucleotide phosphate (NAD(P)H), keratin, melanin, elastin, and collagen, which reflect physiological and pathological changes in vivo. In this study, we used a portable handheld two-photon microscope to conduct high-resolution in vivo skin imaging on volunteers during 15 days of head-down bed rest. The two-photon microscope, equipped with a flexible handheld scanning head, was used to measure two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) images of the left forearm, left front chest, and forehead of volunteers. Changes in TPEF, SHG, and the extended SHG-to-AF(TPEF) aging index of the dermis (SAAID) were measured. It was found that TPEF intensity increased during bed rest and was restored to normal levels after recovery. Meanwhile, SHG increased slightly during bed rest, and the skin aging index increased. Moreover, we found the skin TPEF signals of the left forearm were significantly negatively associated with the oxidative stress marker malondialdehyde (MDA) and DNA damage marker 8-hydroxy-2'-desoxyguanosine (8-OHdG) values of subjects during head-down bed rest. Meanwhile, the SHG signals were also significantly negatively correlated with MDA and 8-OHDG. A significant negative correlation between the extended SAAID of the left chest and serum antioxidant superoxide dismutase (SOD) levels was also found. These results demonstrate that skin autofluorescence signals can reflect changes in human oxidant status. This study provides evidence for in-orbit monitoring of changes in human stress using a portable handheld two-photon microscope for skin imaging.

Keywords: SHG; TPEF; head-down bed rest; portable handheld two-photon microscope; skin.

FIGURE 1

Optical biopsy of in situ human skin during 15 days of head-down bed rest. (A) Corresponding two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) fitted images of the skin obtained from different depths below the skin surface, as shown in the images. (B) Measurement schedule. The indicated measurements were performed at each time point.

FIGURE 2

Portable handheld two-photon microscope imaging of a skin structure. (A,B) Image of the clinical device portable two-photon microscope to perform non-invasive, label-free, rapid in vivo histology. Photograph of indicated parts of the body coupled to the probe of portable two-photon microscope imaging system during imaging. TPEF (green) and SHG (red) images of skin obtained from different depths below the skin surface. SC, stratum corneum; SG, stratum granulosum; SS, stratum spinosum; SB, stratum basale.

FIGURE 3

Qualitative comparison of skin TPEF during 15 days of head-down bed rest. (A) Typical images of TPEF intensity in the subcutaneous basale cell layer in different parts of the skin at each time point. (B–D) Changes of TPEF intensity in the skin basale cell layer of left forearm, left chest and forehead during 15 days of head-down bed rest. Data represent mean ± SEM. Significant values are determined as *p < 0.05, **p < 0.01 (Tukey’s multiple comparisons paired test).

FIGURE 4

Qualitative comparison of skin SHG during 15 days of head-down bed rest. (A) Typical images of SHG intensity in the subcutaneous basals cell layer in different parts of the skin as a function of time in bed. (B–D) Changes of SHG intensity in the skin basale cell layer of left forearm, left chest, and forehead during 15 days of head-down bed rest. Data represent mean ± SEM. Significant values are determined as *p < 0.05 (Tukey’s multiple comparisons paired test).

FIGURE 5

Qualitative comparison of skin SAAID during 15 days of head-down bed rest. (A–C) Changes in SAAID index calculated from TPEF and SHG values of left forearm, left anterior chest, and forehead during 15-day bed rest with head down. Data represent mean ± SEM. Significant values are determined as *p < 0.05 (Tukey’s multiple comparisons paired test).

FIGURE 6

Quantitative comparison of serum biochemical indicators during 15 days of head-down bed rest. (A) Changes of serum superoxide dismutase content during 15 days of head-down bed rest. Data represent mean ± SEM. Significant values are determined as *p < 0.05 (Tukey’s multiple comparisons paired test). (B) Changes of serum 8-hydroxydeoxyguanosine during 15 days of head-down bed rest. Data represent mean ± SEM. (Tukey’s multiple comparisons paired test). (C) Changes of serum malondialdehyde content during 15 days of head-down bed rest. Data represent mean ± SEM. (Tukey’s multiple comparisons paired test).

FIGURE 7

Pearson correlation coefficients of two-photon detection indexes and serum biochemical indicators. (A,B) Pearson correlation coefficients of serum MDA, 8-OHDG and gray value of left forearm skin TPEF. There was a significant negative correlation. (C,D) Pearson correlation coefficients of serum MDA, 8-OHDG, and gray value of left forearm skin SHG. The left forearm SHG gray value was significantly negatively correlated with serum MDA and 8-OHDG levels. (E) The SAAID index calculated by TPEF and SHG intensity of left anterior chest were significantly negatively correlated with serum SOD.