1.1.

Tissue Optical Clearing

The response of tissue to chemical agents such as glycerol and dimethyl sulfoxide is a reduction in light scattering and corresponding increase in optical clarity. “Tissue optical clearing” permits delivery of near-collimated light deeper into tissue, potentially improving the capabilities of various optical diagnostic and therapeutic techniques. Numerous technical publications discuss methods and applications of tissue optical clearing using chemical agents, ^{1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11} yet understanding of the mechanisms of clearing remains incomplete.

Three hypothesized mechanisms of light scattering reduction induced by chemical agents include (1) dehydration of tissue constituents, (2) replacement of interstitial or intracellular water with an agent that better matches the higher refractive index $\left(n\right)$ of the proteinaceous structures, and (3) structural modification or dissociation of collagen. Although the refractive index matching mechanism (2) requires dehydration (1), this mechanism is differentiated by an additional feature: replacement of water with a chemical agent. Mechanisms 1 and 2 were first proposed by Tuchin, ² and mechanism 3 was first proposed by Yeh.⁷ These and possibly other unspecified dynamic mechanisms may be working synergistically or antagonistically with different relative contributions dependent on tissue type and vitality, chemical agent, and delivery method.

Dehydration and refractive index matching of intrinsic structures may be coupled. Other researchers have suggested that dehydration can reduce scattering in soft tissue by displacing water from the space between collagen fibrils, increasing protein and sugar concentrations, and decreasing refractive index mismatch. ^{12, 13, 14, 15, 16} Such refractive index matching is due to native species and is a passive process, and we explicitly differentiate it from index matching due to exogenous chemical agents intentionally introduced into the tissue.

Motivation for this paper is twofold: (1) provide evidence that dehydration is an important mechanism of optical clearing in tissues exposed to chemical agents and (2) present an analysis of how dehydration can contribute to reduced light scattering. By comparing two methods of tissue optical clearing: air-immersion and agent-immersion, we are able to isolate and investigate the dehydration mechanism of optical clearing by chemical agents in relation to other proposed mechanisms. Optical scattering changes between native and dehydrated tissue states are measured using transmission-reflection photography and optical coherence tomography (OCT). Examining changes in tissue ultrastructure using transmission electron microscopy (TEM) images and predicting modification of reduced scattering coefficient using a heuristic particle-interaction model provides a better understanding of how dehydration contributes to reduce light scattering.

1.2.

Light Scattering in Tissue

Scattering results from the interaction of light with a material of nonisotropic refractive index. The spatial refractive index distribution of a material specifies the magnitude and direction of light scattered elastically at a given wavelength. Biological tissue, such as skin, is highly scattering to visible and near infrared light because it contains high-refractive index submicron scattering particles such as collagen fibrils $(n\sim 1.47)$ and cellular organelles ( $n\sim 1.39$ to $1.42$ ) surrounded by low-refractive index fluid $(n\sim 1.35)$ .^{17, 18} Light scattering theories such as Mie and Rayleigh-Gans and computational methods such as the finite difference time domain method further support that submicron scattering particles are primarily responsible for large-angle scattering.^{19, 20, 21}

Light scattering theories such as Mie and Rayleigh-Gans predict a reduced scattering cross section $\left({\sigma }_s^{\prime }{\mathrm{cm}}^2\right)$ for a single particle as a function of the scattering particle radius and refractive index, the surrounding medium refractive index, and the wavelength of light in vacuum.^{1, 22} For a dense distribution of scattering particles, the reduced scattering coefficient $\left({\mu }_s^{\prime }{\mathrm{cm}}^{-1}\right)$ is related to the reduced scattering cross section by a simple heuristic particle-interaction model:^{1, 23}

Fig. 1

Relationship between reduced scattering coefficient, ${\mu }_s^{\prime }$ , and scattering particle volume fraction, $\varphi$ , provided $r$ , $n_{in}$ , $n_{ex}$ , and $\lambda$ remain constant.

Equation 1 is useful for examining the mechanisms of optical clearing. Chemical agents may alter the scattering properties of tissue by changing scattering particle size, shape, packing density, refractive index, and surrounding media refractive index. These parameters experience a controlled transient excursion dependent on water and agent transport kinetics. The ${\mu }_s^{\prime }$ parabolic dependence on $\varphi$ is useful for understanding dehydration effects that modify the scattering particle packing density. Refractive index matching and shape or structural changes to individual scattering particles affect the reduced scattering cross section $\left({\sigma }_s^{\prime }\right)$ .

2.1.

Photographic Imaging

Dorsal sections of full-thickness skin (approximately $4{\mathrm{cm}}^2$ each) are placed in separate glass vials containing either anhydrous glycerol or DMSO. An adjacent tissue specimen is allowed to dehydrate in air. At nine time points over $100\mathrm{h}$ samples are weighed. Concurrently at these times a photographic image is recorded of each tissue sample. Each sample is positioned so that half is overlying transparent glass and half is overlying an opaque ruler that functions as a visible light transmission mask. Samples are epi- and transilluminated simultaneously with visible light and photographed using an Olympus C-3040 digital camera. Lighter regions of tissue over glass and darker regions of tissue over the opaque ruler indicate increased transmission and decreased reflectance, respectively. The samples are otherwise kept undisturbed at room temperature.

Individual tail tendon fascicles are imaged with phase contrast microscopy ( $10\times$ , Ph1 objective) in native, glycerol-immersed, and air-immersed states. The native sample is immersed in isotonic saline solution to prevent dehydration. The sample immersed in glycerol is imaged after $10\min$ of exposure, and the sample immersed in air is imaged after $30\min$ of exposure.

2.2.

OCT Imaging

Individual tendon fascicles are imaged with an 820-nm wavelength OCT system during glycerol and air-immersion. The fascicle immersed in glycerol is imaged for $16\min$ , and the fascicle exposed to ambient air is imaged for $2.5\mathrm{h}$ . OCT images allow measurement of two dynamic parameters: (1) macroscopic cross-sectional diameter of the tendon fascicle and (2) depth-resolved backscattered light intensity.

2.3.

TEM Imaging of Tissue Ultrastructure

The second aim of this paper is to detect and investigate the dehydration mechanism of optical clearing. Lateral resolution of most light-based imaging techniques are limited by diffraction and unable to reveal structural information of nanometer-sized particles responsible for light scattering. TEM, with $5\text{-}\mathrm{\AA }$ resolution, is capable of imaging ultrastructural changes in tissue to help understand the scattering reduction mechanisms of optical clearing. TEM is used to inspect the ultrastructure of native, glycerol-immersed and air-immersed tissue.

Specimens include rat-tail tendon fascicles and rat hepatocytes. Tendon fascicles are composed of Type I collagen, the primary constituent of human dermis. The utility of tendon as an experimental specimen is that the collagen fibrils are arranged parallel along the fascicle axis, unlike the more random fibril orientation in skin. The orderly fibril arrangement facilitates specimen orientation prior to TEM embedding and permits ultrasectioning perpendicular to the fibril axis. Hepatocytes are used in lieu of more clinically relevant epidermal keratinocytes because of the difficulty in separating them from the dermis. In addition, the liver is a homogeneous cellular structure whereas epidermis is an inhomogeneous structure composed of differentiated keratinocyte layers.

Tail tendon fascicles are cut into 2-cm-long sections and liver is cut into $1\text{-}{\mathrm{mm}}^3$ cubes. Both specimens are immediately submerged into anhydrous glycerol. Agent-immersed samples are extracted from respective solutions after $20\min$ and the excess agent is removed from the surface by placing the tissue specimens onto filter paper. The air-immersed specimens are exposed to ambient atmosphere for $2\mathrm{h}$ . All specimens, including a control, are then fixed in 5% gluteraldehyde in Hepes buffer at pH 7.2 for $1\mathrm{h}$ and later embedded in resin according to a standard protocol for TEM ultramicrotomy.²⁸

Stained tissue sections are imaged with an AMT Advantage HR digital camera at magnifications of up to $28000\times$ on an FEI EM208 TEM at $80\mathrm{kV}$ . For consistency, images are recorded near the center of each fascicle. Cross-sectional area (and volume) fraction of the fibrils, defined as the ratio of cross-sectional area occupied by fibrils to the total image area, is quantified using a conventional thresholding image processing technique.

3.1.

Photographic Imaging

Photographic imaging assesses optical clearing efficacy of air, glycerol, and DMSO-immersed in vitro rat skin. Half of each tissue specimen is placed overlying glass while the other half is placed overlying a black opaque ruler. The entire tissue specimen is simultaneously epi- and transilluminated (Fig. 2 ). Lighter regions of tissue overlying glass and darker regions of tissue overlying the opaque ruler indicate increased transmission and decreased reflectance, respectively. Tissue transmittance was calculated by subtracting the reflectance intensity [upper portion of Figs. 2a, 2b, 2c] from the corresponding total intensity [lower portion of Figs. 2a, 2b, 2c]. Reflectance from the black (highly absorbing) ruler is negligible. The ratio of transmittance to reflectance (T/R) is an indicator of scattering strength assuming absorption remains constant. A high ratio corresponds to weak scattering, while a low ratio corresponds to strong scattering.

Fig. 2

Photographic images of rat skin immersed in DMSO (a), glycerol (b), and air (c). Corresponding immersion times are listed for each image.

Air-immersion past $50\mathrm{h}$ and glycerol-immersion past $24\mathrm{h}$ induce similar increases in optical clarity. Equilibrium T/R is approximately $1.5\mathrm{\times }$ greater for glycerol and air-immersion than DMSO-immersion. Both chemical agents increase T/R much faster than air-immersion [Fig. 3a ]. DMSO, glycerol, and air-immersion induce 15, 40, and 55% decreases in skin mass, respectively. The decrease in skin mass is fastest for DMSO and slowest for air-immersion [Fig. 3b]. By nondimensionalizing with respect to time, T/R increases for increasing mass loss as shown in Fig. 3c.

Fig. 3

(a) Normalized dynamic transmissivity/reflectivity ratio. (b) normalized dynamic skin mass. (c) normalized transmissivity/reflectivity ratio versus normalized skin mass. All plots normalize to initial values.

Tail tendon fascicle immersion in glycerol and air causes similar appearances when observed using phase contrast microscopy. Although the tendon fascicle immersed in glycerol optically clears more rapidly than the air-immersed specimen, at equilibrium the two tissue specimens appear similarly transparent. See Fig. 4 .

Fig. 4

Phase contrast images of tail tendon fascicle. (a) Native state. (b) 10-min glycerol-immersion. (c) $30\min$ air-immersion.

3.2.

OCT Imaging

OCT M-scan images $(\lambda =850\mathrm{nm})$ of tendon fascicle immersed in glycerol and air are shown in Figs. 5a and 5b , respectively. A M-scan is a time sequence of depth scans into tissue at a single lateral position. The $x$ axis of each image represents time of image acquisition, and $y$ axis represents scan depth. Backscattered light intensity (in decibels) is plotted as a function of time and depth. Initially after exposure to glycerol or air, backscattered light from the fascicle is large because the specimen is highly turbid. At early times, effect of multiply scattered light is evident from M-scan images. Multiply scattered photons undergo a longer optical path length than single-scattered photons and artifactually appear as backscattering from beneath the fascicle.

Fig. 5

OCT M-scan images (in decibels) of tail tendon fascicles immersed in glycerol (a) and air (b). Arrows indicate multiple light scattering. Normalized back-reflected light intensity integrated over fascicle diameter during immersion in glycerol (c) and air (d). Normalized fascicle optical thickness (diameter) during immersion in glycerol (e) and air (f).

Glycerol- and air-immersion induce similar responses in fascicle size and scattering, although the response due to glycerol is approximately an order of magnitude faster. The time constants for fascicle shrinkage and scattering reduction due to glycerol and air exposure are approximately $2\min$ and $20\min$ , respectively. Tendon fascicle scattering changes were indicated by the percentage of backscattered light intensity integrated over the fascicle thickness. Interestingly, with either glycerol- or air-immersion, scattering first increases slightly and then exponentially decays to near 5% of its original value [Figs. 5c and 5d].

Fascicle optical thickness exponentially drops 25% of its original value in response to both glycerol and air exposure [Figs. 5e and 5f]. In the case of both glycerol- and air-immersion, the average refractive index is expected to increase by a small percentage. For example, assuming complete replacement of all water within the tendon fascicle $(\sim 50\%)$ with either glycerol or proteinaceous fibrils $(n=1.47)$ , the average fascicle refractive index would increase 4%. The measured decrease in fascicle optical thickness of 25% may result from either a 25% decrease in physical diameter assuming no change in refractive index or approximately 29% decrease in physical thickness in combination with a 4% increase in refractive index.

3.3.

TEM Imaging of Tissue Ultrastructure

TEM images of collagen fibril distribution are shown in Fig. 6 corresponding to (a) the native state, (b) 5-min anhydrous glycerol-immersion, and (c) 2-h air-immersion. The largest (approximately 200-nm diameter) fibrils account for the majority of total fibril area fraction. Fibril diameter and shape do not appear significantly altered due to air-immersion; however, degraded image contrast of the glycerol-immersed specimen may be attributed to changes in fibril size and shape.

Fig. 6

TEM images of tail tendon fibrils. (a) native state. (b) 20-min glycerol-immersion. (c) 2-h air-immersion.

Images reveal higher fibril packing density in the glycerol- and air-immersed state versus the native state. Volume fraction of tendon fibrils increased from approximately 0.65 to approximately 0.90 due to glycerol- and air-immersion. Measured increase in scattering particle volume fraction ( $\varphi$ ) corresponds to an approximately 60% decrease in reduced scattering coefficient assuming no change in refractive index ratio between fibrils and surrounding fluid or change in scattering particle size or shape [Eq. 1, Fig. 1].

TEM images of rat hepatocytes are shown in Fig. 7 corresponding to (a) the native state, (b) 10-min glycerol-immersion, and (c) 2-h air-immersion. Images reveal higher intracellular organelle volume fraction in glycerol- and air-immersed states versus the native state. Organelle area fraction is not quantitatively analyzed.

Fig. 7

TEM images of rat hepatocyte organelles. (a) native state. (b) 20-min glycerol-immersion. (c) 2-h air-immersion.