2.1.

System Setup

Angio-OCT is home-built based on a typical SDOCT configuration, as shown in Fig. 1. Briefly, the light source is a broadband superluminescent diode (SLD; Superlum, Carrigtwohill, Ireland, Broadlighters D855-HP2) with a central wavelength of 850 nm and a full-width-half-maximum bandwidth of 100 nm, theoretically offering a high axial resolution of $\sim 3.2\mu \mathrm{m}$ in air. An objective lens with a focal length of 40 mm was used to focus the probing light beam on the region of interest, yielding a measured lateral-resolution of $\sim 15\mu \mathrm{m}$. The OCT detection unit in our system was a high-speed spectrometer, equipped with a fast line-scan CMOS camera (Basler, Ahrensburg, Germany, Sprint spL4096-140k) providing a 120-kHz line-scan rate and 2048 active pixels. The spectrometer had a designed spectral resolution of 0.062 nm, providing an imaging range of $\sim 2.9\mathrm{mm}$ on each side of the zero-delay line in air. The system sensitivity was measured at $\sim 100\mathrm{dB}$ at the depth position of 0.5 mm, with an incident optical power of 2 mW upon the sample surface.

Fig. 1

Schematic of the imaging system used to collect the 3-D spectral interferogram cube for OCT angiography. SLD, super luminescent diode; PC, polarization controller; CL, collimating lens; FL, focusing lens; OL, objective lens.

2.2.

Chemical Agents

FPT was used in this study for optical clearing. As described in our previous study,¹⁶ FPT was a mixture of saturated fructose (Sinopharm Chemical Reagent Co., Ltd.) solution (78%, w/w) with PEG-400 (Kemiou Reagent Development Corporation) and thiazone (Heming Trading Company Limited) at a volume ratio of $11:8:1$. FPT has a refractive index of 1.4801 and a pH value of 6.0. Norepinephrine (NE), purchased from Affiliated Hospital of Huazhong University of Science and Technology (Wuhan, PRC), was used for the dynamic study of cutaneous blood flow.

2.3.

Animal Preparations

Male Balb/c mice ($N=15$, 8 weeks old) were obtained from Shanghai Animal Experimental Center (Shanghai, PRC) and fed under specific pathogen-free conditions. Mice were anesthetized by intraperitoneal injection of a mixture of 10% urethane and 2% $\alpha$-chloralose with a dosage of $0.8\mathrm{ml}/100\mathrm{g}$. The dorsal hair was shaved and the residual hair was thoroughly removed with depilatory cream. Then mice were placed on the experimental platform for OCT imaging. All procedures in the experiments were carried out with the guidelines of the Institutional Animal Care and Use Committee of Zhejiang University, PRC. To evaluate the clearing efficacy of the FPT agent in vivo, the dorsal skin of 15 prepared mice was imaged with Angio-OCT before and after topical application of OCAs. Then six of these mice were injected with $100\mu \mathrm{l}$ of NE ($0.2\mu \mathrm{g}/\mu \mathrm{l}$) after skin became transparent, and the dynamic response of blood flow to the vasoactive drug was monitored for 25 min with a time interval of 1 min.

2.4.

Quantification of Imaging Performance and Skin Optical Properties

OCT provides depth-resolved images of tissue. Due to the optical scattering and absorption of tissues, the OCT probe light is attenuated as the beam penetrates deeper into the tissue. The OCT depth profile reveals the depth-dependent attenuation coefficient in the tissue. According to Lambert–Beer’s law, the OCT depth profile is related to the attenuation coefficient ${\mu }_{\mathrm{t}}$ of tissues as^21,33

The scattering coefficient of tissue is closely dependent on the refractive-index mismatching extent between extracellular fluid ($n_{\mathrm{ECF}}$) and tissue components (e.g., collagen, fibers, and cell) ($n_{\mathrm{s}}$). In a simple model of scattering dielectric sphere, ${\mu }_{\mathrm{s}}$ can be approximated as³⁵

In addition, the permeability rate ($P$) is an important parameter for the evaluation of efficiency of OCAs, which can be estimated by dividing the thickness $z_{\text{region}}$ of the region that OCAs reach by the time $t_{\text{region}}$ of molecular permeation in the monitored region²¹

The correlation between the relative changes in refractive-index mismatching ($m-1$) and Angio-OCT signal strength $I$ can be calculated as

3.1.

Optical Coherence Tomography Structural and Flow Imaging of Mouse Dorsal Skin

Figure 2 shows the representative OCT structural and angiographic images of mouse dorsal skin in vivo. Figure 2(a) is a cross-sectional schematic of the layered skin. Figures 2(b1) and 2(c1) show the representative OCT structural cross-sections before and after the topical application of the FPT agent, respectively. The layered structures of skin are quite vague and difficult to distinguish in Fig. 2(b1). In contrast, the imaging contrast and depth are improved greatly in Fig. 2(c1), and the layered structures and features are clearly identified and manually depicted, including the epidermis (ED), dermis (D), hypodermis (HD), and muscles (M), according to cutaneous anatomical features and the obvious boundaries in the Angio-OCT structural cross-section. The ED-D and HD-M boundaries are visualized clearly, and the dermis exhibits a decreased OCT scattering intensity. The hypodermis appears to be an interwoven structure that is correlated to the adipose cells in the layer. The muscle has a sandwich structure. After the topical application of the FPT agent, the thickness of the epidermis and dermis decreases obviously due to agent-induced dehydration. Figures 2(b2) and 2(c2) are the corresponding OCT angiograms before and after FPT treatment, respectively. The blood vessels are isolated from the tissue bed. The big vessels mainly appear at the HD layer, as indicated by the bold arrows, and the small vessels above the HD layer come from the dermis, as indicated by the thin arrows. As shown in Fig. 2(c2), most of the dermal small vessels disappear after the FPT treatment, and the HD blood vessels present an improved visibility, most likely due to the decreased scattering attenuation in the dermis, which can be also observed in the projection view of the 3-D angiography in Figs. 2(b3) and 2(c3). The dermal vasculature can be observed before the FPT treatment in Fig. 2(b3), while the HD big vessels exhibit an improved contrast after the FPT treatment in Fig. 2(c3).

Fig. 2

Representative OCT structural and angiographic images of mouse dorsal skin in vivo, before (column b) and after (column c) FPT treatment, respectively. (a) Cross-sectional schematic of the layered skin. (b1) and (c1) Structural cross-sections. (b2) and (c2) Cross-sectional angiograms. (b3) and (c3) Projection view of 3-D angiography. ED, epidermis; D, dermis; HD, hypodermis; M, muscles. Thin arrows indicate small vessels. Bold arrows indicate big vessels.

3.2.

Quantifications of Skin Optical Properties

The skin surface can be identified in the OCT structural cross-sections. The outermost surface was flatted to align all the depth profiles. Then, the OCT depth profile was averaged along the lateral direction so as to reduce effects of the heterogenetic distribution of the cutaneous absorbers and scatters as much as possible. As shown in Fig. 3(a), the solid blue and dashed red curves correspond to the depth profiles before and after the FPT treatment, respectively. After the FPT treatment, OCT intensity strength exhibits a decrease of $\sim 25.6\%$ in the D and HD layers, with an increase of $\sim 42.5\%$ in the M layer. The attenuation coefficient image was calculated according to Eq. (2); thereafter, the outermost surface was flattened and the depth-resolved attenuation coefficient was calculated by averaging the attenuation coefficient image along the lateral direction, as shown in Fig. 3(b). After the FPT treatment, the attenuation coefficients were lower: $\sim 40.5\%$ for the ED, D, and HD layers, and $\sim 11.6\%$ for the M layer.

Fig. 3

(a) Normalized OCT depth profiles and (b) depth-resolved attenuation coefficients, before (solid blue curves) and after (dashed red curves) FPT treatment, respectively. (c) Relative changes in refractive-index mismatching extent (solid blue curve) and signal intensity (dashed red curve). The vertical dashed lines roughly indicate the skin layers.

As mentioned before, the relative changes in the refractive-index mismatching extent ($m$) between tissue components and extracellular fluid could be quantified using Eq. (3). As shown in Fig. 3(c), the solid blue line represents the relative changes in ($m-1$) after FPT treatment, indicating an average decline of $\sim 25.3\%$ in the ED, D, and HD layers, and $\sim 6.1\%$ in the muscle layer, respectively. The dashed red line is the relative changes in Angio-OCT signal strength. Thereafter, we calculated the correlation between the relative changes in refractive-index mismatching ($m-1$) and Angio-OCT signal strength using Eq. (5), and the results demonstrate that the correlation between the relative changes in refractive-index mismatching ($m-1$) and Angio-OCT signal strength are about 90% and $-55\%$ for the superficial (ED + D + HD) layers and the deeper (M) layer, respectively. In addition, the permeability rate $P$ is $\sim 5.83\times {10}^{-5}\mathrm{cm}/\mathrm{s}$ using Eq. (4).

3.3.

Blood Flow Dynamic Responses

With a combination of optical clearing methods, Angio-OCT exhibits enhanced performance in imaging skin blood flow, allowing cutaneous hemodynamic analysis with high spatiotemporal resolution and contrast. The vasoactive drug-induced changes of vascular diameter and flow flux are estimated over time. The flow flux is measured based on the signal strength of OCT angiograms and normalized with the baseline.

Figure 4(a) shows the M-mode Angio-OCT image of the time-lapse blood flow dynamic responses to vasoactive NE after FPT treatment, and the dynamic changes in vessel diameter, which were fitted with red curves, are obvious to see. Figure 4(b) reports the quantifications of the dynamic responses of vessel diameter and normalized blood flow. The baseline of vessel diameter is $64\mu \mathrm{m}$. After NE injection, the diameter exhibits a gradual decrease of up to 32.8% at the time instant $\sim 14\min$, then returns to $\sim 96\%$ of the baseline in 25 min. Along with the dynamic change in diameter, the flow flux exhibits a gradual increase of up to $\sim 30\%$ at the time instant $\sim 15\min$, and then returns to $\sim 105\%$ of the baseline in 25 min.

Fig. 4

Blood flow dynamic responses to vasoactive drug monitored by Angio-OCT. (a) The M-mode Angio-OCT image of the time-lapse blood flow dynamic responses (the fitted red curves enhance the visualization of response changes) and (b) the dynamic changes in vessel diameter and normalized blood flow flux.