1.

Introduction

Researchers commonly use fluorescence microscopy to investigate and study disease progression in biological tissues. Prior to imaging, researchers may incubate the tissue with exogenous biomarkers conjugated with fluorescent probes that bind to specific sites of interest, enabling precise localization and visualization of fine structural features. Since many biological tissues strongly scatter visible wavelengths, fluorescence microscopy applications often remain limited to superficial ($<200\mu \mathrm{m}$ in depth) regions.

Recently, several research groups have developed protocols involving the use of chemical agents to reduce the degree of optical scattering in turbid tissues.^1,2 These reports describe the use of various optical clearing agents, including glycerol,³ dimethylsulfoxide (DMSO),⁴ benzyl alcohol benzyl benzoate,⁵ tetrahydrafuran,⁶ FocusClear,⁷ Scale,⁸ CLARITY,⁹ and 3DISCO.¹⁰ In addition to chemical optical clearing, both the CLARITY and 3DISCO protocols involve complex tissue processing; CLARITY requires electrophoretic optical clearing to remove highly scattering lipids, while 3DISCO requires several tissue dehydration steps with toxic and flammable chemicals, which necessitate special chemical safety considerations. The result is that both techniques are more challenging to implement than simply immersing the tissue in an optical clearing agent. We recently described a method that we call optical histology,^11,12 which involves applying optical clearing using FocusClear to thick ($\sim 1\mathrm{mm}$) tissue sections and subsequent imaging with confocal fluorescence microscopy. With optical histology, we demonstrated high-resolution visualization of fluorescent structures up to depths of 500 to $700\mu \mathrm{m}$ within each section.

The efficacy of optical clearing depends on multiple factors including tissue type and selected chemical agent. In general, researchers desire a substantial increase in tissue transparency, which is associated with a decrease in optical scattering. Previous studies related to optical clearing^2,13 describe the use of measurements of optical transmittance of native and cleared tissues to assess changes in tissue optical properties after optical clearing. However, since transmittance depends on optical scattering, tissue thickness, and (to a lesser extent) optical absorption, transmittance alone does not enable quantification of optical scattering. Hence, additional measurements are required to fully characterize the optical clearing process.

Optical properties in ex vivo thick tissue can be quantified using the integrating sphere technique coupled with an appropriate model of light propagation.¹⁴ Measurements can involve either a single integrating sphere, which enables sequential measurement of diffuse reflectance and total transmittance, or a double integrating sphere,¹⁵ which consists of two adjoined integrating spheres that enable simultaneous measurement of diffuse reflectance and total transmittance. From measurements of total transmittance, diffuse reflectance, and sample thickness one can estimate the optical scattering and absorption of the tissue using a model based approach,¹⁶ such as diffusion theory,¹⁷ Monte Carlo simulation,¹⁸ or the inverse adding doubling (IAD) algorithm.¹⁹ We previously used an integrating sphere and the IAD algorithm to evaluate the efficacy of various optical clearing agents¹⁹ and introduced the metric of optical clearing potential (OCP). OCP is a ratio of the reduced optical scattering coefficient before optical clearing to the value after clearing, and quantitatively describes the change in optical scattering that occurs due to optical clearing. In this paper, we describe the use of integrating sphere-mediated optical spectroscopy and the IAD algorithm to quantify the reduced optical scattering coefficient, and to determine the time-resolved change in optical scattering associated with FocusClear-mediated optical clearing of ex vivo mouse brain samples.

2.

Materials/Methods

2.2.

Tissue Holder

We placed each slice into a tissue holder consisting of a 30-mm SM1 threaded optic holder (CP08, Thor Labs, New Jersey), two 25-mm circular cover glasses (Fisherbrand Cover Glass No. 1, Fisher Scientific, Waltham, Massachusetts), and two 30-mm SM1 threaded retaining rings (SM30RR, Thor Labs, New Jersey; Fig. 1). We inserted the slice between the circular cover glasses and used the retaining rings to hold it in place. With this apparatus, the tissue remained fixed in position and its thickness was maintained during the optical measurements. We measured the sample thickness more precisely by measuring the thickness of the entire apparatus (with slice in place) and subtracting the measured thickness of the apparatus alone (i.e., without the slice).

Fig. 1

Components of tissue holder, which is used to immobilize the tissue and keep the tissue thickness uniform during imaging. The tissue holder consists of a 30-mm threaded optic holder, two 25-mm circular glass slides, and two 30-mm threaded retaining rings used to evenly compress and sandwich the glass slides together against the tissue.

2.3.

Optical Spectroscopy

We used an integrating sphere setup to acquire optical transmittance and reflectance data from the brain slices both before and after optical clearing (Fig. 2). The setup consisted of an integrating sphere (Labsphere Inc., North Sutton, New Hampshire), a broadband halogen light source whose output was collimated to a diameter of 4 mm, and spectrometer (Prime-X, B&W Tek, Newark, Delaware). The range of the spectrometer was 400 to 1000 nm, with a spectral resolution of 4.8 nm (FWHM). We used a custom-written LabVIEW program (National Instruments, Austin, Texas) to communicate with the spectrometer, and a 99% reflectance standard (Spectralon, LabSphere Inc.) to calibrate the reflectance measurements.

Fig. 2

Schematic of integrating sphere setup for (a) transmittance measurements and (b) reflectance measurements.

3.

Results

Figure 3 is a time course set of images of different brain slices optically cleared with FocusClear for different optical clearing incubation times. Under visual inspection, the tissues appeared to become more transparent with longer optical clearing time, which was an expected result. Table 1 summarizes the baseline ${\mu }_{\mathrm{s}}^{\prime }$ values at the interrogated wavelengths, which are averaged over all samples and all time points. We observed an expected monotonic decrease in ${\mu }_{\mathrm{s}}^{\prime }$ with an increase in wavelength. Tissue slice thickness before optical clearing ranged from 0.7 to 1.4 mm for the different optical clearing time points. Figure 4 is a plot of OCP versus wavelength for each of the different optical clearing times. At each wavelength, OCP increased with increasing optical clearing time, though we observed that the amount of OCP increase was lower between 6 and 24 h of optical clearing time than between 3 and 6 h optical clearing times. However, for a given optical clearing time, the OCP spectrum is relatively flat over the visible-NIR wavelength range.

Table 1

Baseline reduced optical scattering coefficient values of the mouse brain slices (n=19) used in this study. SD = standard deviation.

Fig. 3

Color images of representative brain slices (a) before and (b–e) after optical clearing with FocusClear for (b) 1 h, (c) 3 h, (d) 6 h, and (e) 24 h. $\text{Scale bar}=3\mathrm{mm}$.

Fig. 4

Line plot of average optical clearing potential versus wavelength for all time points (1, 3, 6, and 24 h).

4.

Discussion

We immersed samples in FocusClear for different periods of time. We observed that an increase in immersion time induced an increase in sample transparency (Fig. 3). We quantified this increased transparency as an increase in mean OCP at all wavelengths between 400 and 1000 nm (Fig. 4).

To illustrate the effects of different levels of OCP achieved, we used methodology described previously,¹¹ to image four 1-mm thick coronal slices of mouse brain with microvasculature labeled with DiI (Fig. 5). We optically cleared brain slices with the same four optical clearing times used in the integrating sphere measurements. An immersion time $>3\mathrm{h}$ did not further increase the depth that vessels could be visualized, as we visualized vessels at similar tissue depths in the 3, 6, and 24 h time points. However, there was a clear effect on the microvascular density that could be visualized at all depths, as the brain slices cleared for 6 and 24 h showed a higher density of vasculature as compared to the brain slices cleared for 1 and 3 h. This is consistent with the trends seen in OCP (Fig. 4), as OCP increased between 1 and 3 h of optical clearing, increased further between 3 and 6 h, and increased slightly between 6 and 24 h of optical clearing. Interestingly, the mean OCP did not exhibit a dependence on wavelength (Fig. 4). The generic shape of an optical scattering spectrum is²³

Fig. 5

Maximum intensity projection (MIP) maps of tissue microvasculature in 1-mm brain slices optically cleared for (a) 1 h, (b) 3 h, (c) 6 h, and (d) 24 h showing the increased density of vasculature that can be visualized with increasing optical clearing time. MIP images are constructed by determining the maximum intensity pixel at each pixel location from all of the image slices in a confocal microscopy image stack and projecting these pixels onto a two-dimensional (2-D) image. Each pixel is assigned a color corresponding to the originating slice in the image stack resulting in a depth-encoded MIP map that gives three-dimensional information in a 2-D image.

Furthermore, since FocusClear is associated with an OCP that is relatively constant with wavelength, we propose that optical histology with FocusClear is conducive to optical imaging of a wide range of fluorescent molecular probes that emit over the visible and near-infrared wavelength range. Hence, with optical histology using FocusClear, researchers have flexibility in selection of an exogenous probe that best matches their specific application.

The exact mechanism by which FocusClear achieves enhanced optical transparency of brain tissue is unclear. In optical clearing of skin with glycerol, a factor contributing to reduced scattering is the matching of refractive index between collagen ($n=\sim 1.44$) and glycerol ($n=1.47$).²⁵ Interestingly, the refractive index of FocusClear ($n=1.46$) is relatively high compared with native brain tissues ($n=1.36$ to 1.40).²⁶ Hence, alternate factors may play a contributing or major role in the optical clearing properties of FocusClear on mouse brain—further study is warranted, as a complete understanding of the mechanism of action of clearing will lead to more rational optimization of protocols based on FocusClear.

In summary, our measurements suggest that optical clearing of fixed brain slices with FocusClear achieves a substantial increase in OCP at immersion times as short as 6 h. Furthermore, because the wavelength dependence of the OCP is flat over the range from 400 to 1000 nm, these results suggest that this optical histology approach can improve ex vivo visualization of a wide range of fluorescent molecular probes.