2.1.

Near-Infrared Fluorescence Imaging System

The NIR fluorescence imaging system has been described in detail previously.¹² Briefly, it is composed of two wavelength-isolated excitation sources, one generating $0.5\mathrm{mW}∕{\mathrm{cm}}^2$ of $400\text{to}650\text{-}\mathrm{nm}$ white light, and the other generating $5\mathrm{mW}∕{\mathrm{cm}}^2$ of $725\text{to}775\text{-}\mathrm{nm}$ NIR excitation light, all over a $15\text{-}\mathrm{cm}$ -diameter field of view (FOV). Emitted photons are collected by custom optics that separate the white light and NIR fluorescence $(>795\mathrm{nm})$ channels. Through computer-controlled image acquisition via LabVIEW software (LabVIEW, National Instruments, Austin, Texas), anatomic (white light) and functional (NIR fluorescence light) images can be displayed separately and merged. NIR fluorescence images were acquired on a $12\text{-}\text{bit}$ Orca-AG camera (Hamamatsu, Bridgewater, New Jersey). Color video images were acquired on an Imitech IMC-80F (Seoul, Korea) camera. All images were refreshed up to 15 times per second. The entire apparatus is suspended on an articulated arm over the surgical field at a working distance $18\mathrm{in.}$ , thus permitting noninvasive and nonintrusive imaging.

2.2.

Animals

Animals were used in accordance with an approved institutional protocol. Female Yorkshire pigs (E. M. Parsons and Sons, Hadley, Massachusetts) weighing $35\mathrm{kg}$ (age $11\text{weeks}$ ) were induced with $4.4\text{-}\mathrm{mg}∕\mathrm{kg}$ intramuscular Telazol (Fort Dodge Labs, Fort Dodge, Iowa), intubated, and maintained with 1.5 to 2% isoflurane (Baxter Healthcare Corporation, Deerfield, Illinois). It should be noted that each animal had only one procedure or experiment; multiple studies were not performed on a single animal.

2.3.

Perforator Vessel Near-Infrared Fluorescence Angiography

The imaging system was positioned above the ventral surface of $n=3$ animals. $10\mathrm{ml}$ of a $0.25\text{-}\mathrm{mg}∕\mathrm{ml}$ ( $2.5\mathrm{mg}$ total; $0.092\mu \mathrm{mol}∕\mathrm{kg}$ ) solution of indocyanine green (ICG, Sigma-Aldrich, Saint Louis, Missouri) in saline was injected via a central venous catheter as a rapid bolus. Skin perforator vessels were imaged preinjection and for $1\text{-}\min$ postinjection at a frame rate of $500\mathrm{msec}$ and a camera exposure time of $150\mathrm{msec}$ . 100% glycerol was topically applied on a $15\times 15\text{-}\mathrm{cm}$ area that covered the entire FOV. The glycerol was allowed to soak into the skin for $30\min$ . Once the fluorescence intensity in the tissue dropped to the preinjection level, another ICG injection was given, and the perforator vessels were imaged in the same manner described before. The animals were anesthetized and secured for the entire procedure to ensure a consistent sampling region between measurements. For quantitation, two images corresponding to identical vascular phases were chosen. The fluorescence intensity was quantified over two straight lines drawn over the same part of each image. The same procedure was then performed using 50% glycerol in water and polypropylene glycol:polyethylene glycol (PPG:PEG).¹⁴

2.4.

Near-Infrared Fluorescence Sentinel Lymph Node Mapping

ICG was preadsorbed to Cohn Fraction V bovine serum albumin (BSA, Sigma-Aldrich) at a 1:1 molar ratio,¹⁵ and the complex (ICG:BSA) was diluted to a final concentration of $10\mu \mathrm{M}$ in saline. 22 groin SLN mappings were performed on $n=16$ animals by subcutaneously injecting $200\mu \mathrm{L}$ of ICG:BSA. The procedure was performed bilaterally in six animals. SLNs were successfully identified within $1\min$ in all animals. 100% glycerol, 50% glycerol in water, and PPG:PEG were topically applied $10\min$ after the fluorophore injection on $15\times 15\text{-}\mathrm{cm}$ areas, which covered the entire FOV. Images were obtained preinjection and 2- and $5\text{-}\min$ postinjection, then every $5\min$ for $60\min$ after injection. In treated groups, the images were also acquired immediately following the application. Fluorescence-guided SLN resection was performed after the experiment to confirm the depth of the SLN from the skin surface. For quantitation, a circular region of interest (ROI) was drawn over the SLN and nearby background (BG) tissue. The contrast-to-background ratio (CBR) was calculated as $\mathrm{CBR}=\left(\mathrm{SLN}\text{intensity}\text{-}\mathrm{BG}\text{intensity}\right)∕\mathrm{BG}$ intensity. The percent (%) change of CBR with optimal exposure time was compared in treated and nontreated groups, with CBR set to 100% at the time of glycerol application $(T=10\min )$ . The animals were anesthetized and secured for the entire procedure to ensure a consistent sampling region between measurements.

2.5.

Preparation and Handling of Porcine Skin for Ex-Vivo Quantitation

In 22 pigs, porcine skin was harvested from the lower part of the abdomen $4\text{to}8\mathrm{h}$ prior to the experiment. Subcutaneous fat was surgically removed and the skin was cut into two $9\times 9\text{-}\mathrm{cm}$ pieces. One piece was stored in saline at $4°\mathrm{C}$ . The other was exposed to air at both dermal and epidermal sides for between 4 and $8\mathrm{h}$ prior to the experiment. Four skin samples each from hydrated and nonhydrated harvested skin were trimmed into $2\times 1.5\text{-}\mathrm{cm}$ sections, and the thickness of each sample measured with precision calipers three times: at the sample preparation, before each measurement, and after measurement. The thicknesses of the hydrated samples were divided into three groups including 0.5, 1.0, and $1.5\mathrm{mm}$ . Eight samples were harvested from one animal. If samples deviated by more than $0.1\mathrm{mm}$ in either direction, then the samples were excluded. All nonhydrated skin samples $(n=40)$ were $0.2\mathrm{mm}$ thick and were acquired from 10 animals. Hydrated skin samples with thicknesses of $0.5\mathrm{mm}$ $(n=20)$ , $1.0\mathrm{mm}$ $(n=30)$ , and $1.5\mathrm{mm}$ $(n=32)$ originated from 5, 7, and 6 animals, respectively. The standard deviation of 0.2, 0.5, 1.0, and $1.5\text{-}\mathrm{mm}$ -thick samples at the time of sample preparation was 0.038, 0.059, 0.053, and 0.058, respectively. 60 samples were treated with glycerol, while 62 with no treatment served as controls. The glycerol-treated group consisted of 20 nonhydrated and 40 hydrated samples, while the control group included 20 nonhydrated and 42 hydrated samples.

2.6.

Preparation of Near-Infrared Fluorescent Targets and Image Acquisition

NIR standards were prepared by mixing ICG:BSA with 25% gelatin at a ratio of 1:4 to produce a final ICG concentration of $10\mu \mathrm{M}$ . $10\mu \mathrm{L}$ of each NIR fluorescent standard was pipetted into equally spaced chambers in a plastic tray and allowed to harden. Excitation fluence rate was measured, and a NIR fluorescence image of the standards within the linear range of the camera was acquired. Skin samples were then sutured to wires placed at a distance of either 3 or $5\mathrm{mm}$ above the NIR standards with an air gap to recapitulate the conditions of the study by He and Wang, epidermal side up.¹⁶ 29 of the treated and 31 of the nontreated samples were placed $3\mathrm{mm}$ over a NIR standard, while the other half of the samples, including 31 of the nonhydrated and hydrated, were placed $5\mathrm{mm}$ over a NIR standard (Table 1 ). Baseline images were acquired, followed immediately by application of 100% glycerol to half the samples. Samples were imaged every $10\min$ for $60\min$ after glycerol application. Skin samples were removed from the device, and the NIR standards were reimaged.

Table 1

Sample number in ex-vivo evaluation.

2.7.

Quantitation of Fluorescence Emission Through Skin

All acquired images of NIR fluorescence emission were within the linear range of the $12\text{-}\text{bit}$ camera. Images were corrected for variations in excitation fluence rate and evaporation of water over the course of the experiment as follows. Obtained fluorescence intensity through each skin sample $(n=122)$ was divided by the corresponding fluence rate (range $2.6–5.8\mathrm{mW}∕{\mathrm{cm}}^2$ , mean $4.2\mathrm{mW}∕{\mathrm{cm}}^2$ ). Due to water evaporation in the NIR fluorescent standards, the change of fluorescence intensity in NIR standards pre- and postexperiment ranged between 94 and 245% (mean 145%). Since room temperature $(25\text{to}28°\mathrm{C})$ , air flow velocity $(\approx 0\mathrm{m}∕\mathrm{s})$ , and humidity (50 to 60 %) were stable over the course of the experiment, evaporation rate $\left(Wq\right)$ was considered constant.¹⁷ The dye concentration at time $x$ (min) $\left(Cx\right)$ is relative to NIR standard volume $\left(V\right)$ :

2.8.

Statistical Analysis and Histological Staining

Corrected intensity and percentage change from baseline were statistically analyzed at all data points in four categories: distance between sample and NIR standard, hydration, thickness of the sample, and glycerol treatment. A Mann-Whitney test was performed to evaluate differences in distance, hydration, and glycerol treatment with two-sided 95% confidence intervals. A Kruskal-Wallis test was used to investigate the impact of skin thickness. Multivariate analysis was subsequently performed to assess the difference by condition within each group, including distance between sample and NIR standard, hydration, and thickness of the sample. Samples were divided by each condition and the impact of glycerol on corrected intensity; the percent change from baseline was compared between treated and nontreated samples. A Mann-Whitney test was used for statistical evaluation with two-sided 95% confidence intervals. All statistical evaluations were made using Prism Version 4.0 (GraphPad Software, La Jolla, California) Skin samples were histologically evaluated with hematoxylin and eosin (HE) staining after the experiment to determine if glycerol had any impact on sample morphology.

3.1.

Magnitude of the Optical Clearing Effect In Vivo

The perforator arteries and draining veins (perforator vessels) were within a few millimeters of the surface of the skin, and thus provided an ideal model system for assessing the magnitude of the optical clearing effect. Based on previously published studies, we expected a dramatic effect of glycerol on perforator vessel intensity and/or deblurring. However, as shown in Fig. 1a, there was no qualitative difference in perforator vessel imaging using the intravenously administered NIR fluorophore ICG, even after a $30\text{-}\min$ pretreatment with 100% glycerol, nor was there a noteworthy difference in perforator vessel intensity [Fig. 1b] with such treatment. The same results were obtained using 50% glycerol and PPG:PEG-treated animals [Fig. 1c].

Fig. 1

The impact of optical clearing on perforator vessel NIR fluorescence angiography. (a) Intravenous injection of $0.092\mu \mathrm{mol}∕\mathrm{kg}$ of ICG either pretreatment (top row) or $30\text{-}\min$ post- treatment (bottom row) of porcine skin with 100% glycerol. Shown are color video (left column) and NIR fluorescence emission (right column). NIR camera exposure time was $150\mathrm{msec}$ and normalization was identical in both sets of images. $\mathrm{N}=\text{nipple}$ . (b) Quantitation of fluorescence intensity, preglycerol application (black curve) and postglycerol application (gray curve), using lines (1 and 2) drawn across different vascular regions. The $12\text{-}\text{bit}$ intensity at each point (line plots) is shown at the bottom. (c) Quantitation of $12\text{-}\text{bit}$ fluorescence intensity, preapplication (black curve) and postapplication (gray curve) of PPG:PEG (left) and 50% glycerol (right).

Surprised by this result, we next measured the optical clearing effect during SLN mapping. As shown in Fig. 2a, although the SLN could be identified using NIR fluorescence in all animals, application of 100% glycerol to the skin surface over the course of an hour resulted in no substantial improvement in image quality $(n=16)$ . The percent change of CBR of the SLN showed no significant difference in animals that were treated or not treated with 100% glycerol $(p=0.3263)$ , 50% glycerol $(p=0.1333)$ , and PPG:PEG $(p=0.1191)$ [Fig. 2b]. Of note, the mean and standard deviation of the measured depth from the skin surface to the identified SLNs was $5.2\pm 0.075\mathrm{mm}$ .

Fig. 2

Sentinel lymph node (SLN) mapping of the groin with and without glycerol treatment. (a) ICG:BSA was injected at $T=-10\min$ to identify the SLN. At $T=0$ , 100% glycerol was applied topically, and NIR fluorescence emission followed over the next $50\min$ . NIR camera exposure time was $500\mathrm{msec}$ and normalization was identical in all images. $\mathrm{N}=\text{nipple}$ . (b) Percent change of the CBR measured at the SLN over time in nontreated animals $(n=10)$ and animals treated with 100% glycerol $(n=4)$ , 50% glycerol $(n=4)$ , and PPG:PEG $(n=4)$ . Point and error bar represent mean and SEM. The Mann-Whitney test showed no statistical significance between nontreated and treated groups.

3.2.

Ex-Vivo Evaluation of the Optical Clearing Effect

To understand the in-vivo results, and to reconcile previously published studies on optical clearing, we systematically evaluated the effect of skin thickness, skin hydration, distance from the skin to a fluorescent target, and glycerol treatment on NIR fluorescent target intensity. A typical experiment is shown in Fig. 3 . It should be noted that all measured values were within the linear range of the $12\text{-}\text{bit}$ camera. However, to see all targets well in Fig. 3, contrast was enhanced such that the brightest standards appear saturated.

Fig. 3

Experimental setup for ex-vivo quantitation of the optical clearing effect in glycerol-treated porcine skin. The experimental setup described in detail in Sec. 2 is shown at top. Also shown are images of the NIR fluorescent targets before application of skin samples (NIR standards), then over time. All NIR fluorescence images were acquired with a $60\text{-}\mathrm{msec}$ exposure time and were flat field corrected.

Quantitation of results measured for the effect of distance between skin and NIR fluorescent target, hydration, skin thickness at the time of pretreatment, and glycerol treatment (at $T=0$ , 30, and $60\text{-}\min$ postapplication) are shown in Fig. 4a . For all variables tested, hydration status of the skin and skin thickness consistently resulted in a statistical improvement in intensity. Distance failed to show a significant impact on corrected fluorescence. None of these three demonstrated statistical differences in terms of percentage change from baseline (Table 2 ). Glycerol treatment did not show statistical improvement in either corrected intensity or percentage change from baseline over time (Table 3 ). When samples were stratified based on glycerol treatment [Figs. 4b and 4c], glycerol showed a statistically significant improvement in nonhydrated specimens in terms of percentage change from baseline $(p=0.0006)$ as well as corrected intensity $(p=0.0030)$ .

Fig. 4

Statistical evaluation of optical clearing in ex-vivo specimens. (a) The impact of skin to target distance, sample hydration, skin thickness, and glycerol treatment on the corrected intensity of NIR fluorescent targets imaged through porcine skin. Asterisks indicate statistical significance. Three box plots shown in the top row represent pretreatment status. (b) Multivariate analysis by hydration status of samples. Corrected intensity and percentage change from baseline of NIR fluorescent targets imaged through hydrated (top row) or nonhydrated (bottom row) skin, with or without treatment with 100% glycerol. Point and error bar indicates mean and SEM. Asterisks indicate statistical significance. (c) Multivariate analysis by distance between sample and NIR standard (top row) and thickness of the sample (bottom row). Corrected intensity and percentage change from baseline of NIR fluorescent targets imaged with or without treatment with 100% glycerol were shown. Point and error bar indicates mean and SEM. Asterisks indicate statistical significance. Note that the corrected intensity of nontreated groups was higher than treated groups when the sample thickness was more than $0.5\mathrm{mm}$ (bottom, left).

Table 2

Impact of distance, hydration, and thickness ex-vivo. Significant* (glycerol treated> nontreated ).

Table 3

Statistical evaluation of optical clearing effects of glycerol ex-vivo.

On the other hand, in the hydrated specimen, corrected intensity appeared to be better without glycerol treatment $(p=0.0011)$ . No statistically significant difference was seen in hydrated group in terms of percentage change from baseline [Fig. 4b, Table 4 ). Subgroup analysis of thickness revealed that glycerol treatment only had a significant impact on skin that was $0.2\mathrm{mm}$ thick, the thinnest samples of skin both in terms of intensity and change from baseline ( $p=0.0030$ and 0.0006, respectively) [Fig. 4c]. Surprisingly, glycerol treatment showed an adverse effect in terms of intensity in the other samples with $p$ values of 0.0030, 0.0020, and 0.0019 for 0.5, 1.0, and $1.5\text{-}\mathrm{mm}$ -thick samples. There was no statistical difference in change from baseline in those samples. According to the distance between skin and NIR fluorescent target, we found glycerol treatment improved the intensity in the distance of the $3\text{-}\mathrm{mm}$ group $(p=0.0070)$ , while no improvement was detected in the distance of the $5\text{-}\mathrm{mm}$ group $(p=0.0830)$ . It should be mentioned that the change from baseline of the glycerol-treated group was statistically higher than that of the nontreated group in both the distances over time ( $p=0.006$ and 0.0041 for 3 and $5\mathrm{mm}$ distance) (Table 4).

Table 4

Multivariate analysis of clearing impact of the glycerol ex-vivo. Significant* (glycerol treated> nontreated ). Significant** (glycerol treated<nontreated ).

3.3.

Histological Analysis of Ex-Vivo Skin Samples

A distinctive morphological difference was observed in the SC of glycerol-treated skin samples. In hydrated skin, glycerol treatment resulted in smoothing of the SC, although no notable consistent changes were noted in the dermis or other layers of the epidermis (Fig. 5 ). In nonhydrated skin, glycerol treatment appeared to reduce the air gaps within the SC, and may have helped re-expand an otherwise collapsed epidermis (Fig. 5).

Fig. 5

Ex-vivo histological analysis of skin samples. HE staining of hydrated (top row) and nonhydrated (bottom row) skin samples either not treated (left column) or treated (right column) with 100% glycerol. $\mathrm{SC}=\text{stratum}$ corneum.