Optical clearing potential of immersion-based agents applied to thick mouse brain sections
Optical clearing potential of immersion-based agents applied to thick mouse brain sections
Mathew Loren 0 1 2 3
Christian Crouzet 0 1 2 3
Adrian Bahani 0 1 2 3
Vitaly Vasilevko 2 3
Bernard ChoiID 0 1 2 3
0 Department of Biomedical Engineering, University of California , Irvine, California , United States of America
1 Beckman Laser Institute and Medical Clinic, University of California, Irvine, California, United States of America, 3 Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, California, United States of America, 4 Department of Surgery, University of California, Irvine, California, United States of America, 5 Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California , Irvine, California , United States of America
2 Editor: Kristen C. Maitland, Texas A&M University , UNITED STATES
3 University of California, Irvine Undergraduate Research Opportunities Program ( , USA
We have previously demonstrated that the use of a commercially-available immersionbased optical clearing agent (OCA) enables, within 3-6 hours, three-dimensional visualization of subsurface exogenous fluorescent and absorbing markers of vascular architecture and neurodegenerative disease in thick (0.5-1.0mm) mouse brain sections. Nonetheless, the relative performance of immersion-based OCAs has remained unknown. Here, we show that immersion of brain sections in specific OCAs (FocusClear, RIMS, sRIMS, or ScaleSQ) affects both their transparency and volume; the optical clearing effect occurs over the entire visible spectrum and is reversible; and that ScaleSQ had the highest optical clearing potential and increase in imaging depth of the four evaluated OCAs, albeit with the largest change in sample volume and a concomitant decrease in apparent microvascular density of the sample. These results suggest a rational, quantitative framework for screening and characterization of the impact of optical clearing, to streamline experimental design and enable a cost-benefit assessment.
Imaging thicker tissue sections is limited due to light scattering . Recent progress in optical
clearing methods modulates optical scattering to enable volumetric imaging without the need
for sectioning. Entire organs can be cleared and imaged with minimal damage to structures
of interest and without requiring extensive post-processing of data. Several optical clearing
approaches, including variants of CLARITY [2,3], variants of Scale , FocusClear [5,6],
RIMS/sRIMS , variants of 3DISCO , and SeeDB , are reported to reduce scattering
within biological tissues and enable visualization of subsurface tissue structures, with a major
emphasis on the brain.
These approaches differ considerably in terms of sample preparation, time required for
clearing, researcher-time required during the clearing process, complexity of the clearing
analysis, decision to publish, or preparation of the
protocol, and overall cost. To maximize use of limited resources, researchers should select the
most optimal optical clearing approach to suit their individual needs. Although whole-brain
optical clearing enables detailed study of long-range connectivity , the cost in
researcherhours and materials can be relatively high. Sectioning of the brain into thick (0.5?1.0mm)
tissue sections can help reduce this cost by enabling use of immersion-based optical clearing
agents (OCAs) and parallel fluorescence labeling and clearing of the brain sections. In recent
studies, we found that the use of a commercially-available immersion-based OCA, FocusClear,
enables, within 3?6 hours, three-dimensional visualization of subsurface fluorescently-labeled
vasculature  and exogenous fluorescent and absorbing markers of fibrillar beta-amyloid and
cerebral microhemorrhages, respectively  in thick (0.5?1.0mm) mouse brain sections.
However, the performance of FocusClear versus other immersion-based OCAs remains unknown.
Here, we demonstrate immersion-based optical clearing affects both transparency and
volume of brain samples, with the temporal dynamics varying with OCA. Here, we focus on
protocols that involve a single immersion step, as opposed to multi-step immersion-based
clearing protocols (i.e., CUBIC, TDE, FRUIT, RTF, etc.) [11?15]. Moreover, the imaging
depth achieved with confocal microscopy correlates with the reduction in optical attenuation.
Together, the work presents a simple framework for quantitative comparison of OCAs.
Materials and methods
We performed experiments on adult male mice (n = 5, C3H strain, 7?11 weeks old,
2530g, Charles River Laboratories, Wilmington, MA). This work was in compliance with a
protocol approved by the University of California, Irvine Institutional Animal Care and Use
Lycopersicon esulentum tomato lectin is a glycoprotein with a binding affinity to glycoprotein
moieties that are found in the vascular endothelium of rodents . In this study, we used
lectin-Dylight 650 (Vector Laboratories, Inc., Burlingame, CA) to label the vasculature (see below
for procedural details).
A 200?L dose of lectin-Dylight650 diluted 1:3 with PBS was administered via retro-orbital
injection  into the microcirculation of mice anesthetized with an intraperitoneal injection
of a cocktail of ketamine (90 mg/kg) and xylazine (10mg/kg). The lectin was allowed to
circulate for 30min prior to cardiac perfusion.
To obtain brain samples with lectin-labeled vasculature, we adapted a procedure first described
by Li et al  and used subsequently by our group [5,6,18,19]. Briefly, immediately after
euthanasia with an overdose of Sodium Pentobarbitol, we opened the chest cavity and
performed a cardiac perfusion of saline (to flush blood out of the vasculature) followed by cardiac
perfusion of buffered formalin to initiate the fixation process. We then extracted the brain and
immersed it in buffered formalin for 24 hours. We then transferred the brain to a PBS with
0.02% Sodium Azide solution to mitigate photodegradation of the fluorophore.
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Fig 1. Sample preparation protocol. (a) Brain extracted after cardiac perfusion. (b) Brain placed in mouse brain slicer matrix. (c) Two 1 mm thick
coronal sections removed from midpoint of brain. Red dashed lines in (b) denote approximate locations at which each section is removed. Each of the
two sections were bisected, resulting in four samples that were then exposed to one of the four optical clearing agents. Red solid lines denote regions of
interest probed and analyzed for attenuation measurements and confocal microscopy.
Preparation of brain sections
A brain matrix slicer (Zivic Instruments) was used to section each brain into 1-mm-thick
coronal samples. Two adjacent coronal sections were taken from the approximate middle of each
brain. In total, we studied 10 coronal sections extracted from five brains. To compare the
attenuation and physical effects of OCAs, each coronal section was cut into four samples (Fig
1) and randomly assigned to one of the four OCAs tested in this study.
Optical clearing agents (OCAs)
We studied the following four OCAs: FocusClear (CelExplorer, Taiwan), refractive index
matching solution (RIMS), sorbitol RIMS (sRIMS), and ScaleSQ. For RIMS, sRIMS, and
ScaleSQ, we followed preparation protocols described in the respective papers with reagents
purchased from Sigma Aldrich, Carlsbad, CA:
? RIMS: 88% (w/v) Histodenz (PN: S2158-100G) in 0.02 M phosphate buffer solution with
0.01% sodium azide. Bring to 7.5 pH with NaOH .
? sRIMS: 70% (w/v) sorbitol (PN: 240850) in 0.02 M phosphate buffer solution with 0.01%
sodium azide. Bring to 7.5 pH with NaOH .
? ScaleSQ: 9.1 M urea (PN: U5378), 22.5% (w/v) sorbitol solution, and 4.7% Triton X-100 (PN:
X100). Gentle heating required to fully dissolve urea crystals .
Optical attenuation spectroscopy
To estimate the attenuation coefficient of each sample, we adapted a method previously
described by d?Esposito et al . We used a custom-built optical device consisting of a
microscope light source illuminator (V-Lux-1000, Volpi USA, Auburn, NY), integrating sphere
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(Labsphere, North Sutton, NH), imaging lens (Optem, Qioptiq Photonics, Munich, Germany),
and 12-bit monochrome CCD camera equipped with an electro-optic liquid crystal tunable
filter (LCTF) (Nuance, PerkinElmer). The light from the halogen bulb was coupled to the input
port (25mm) of the sphere. The light exited the 6mm output port of the sphere positioned at
90 degrees with respect to the input port. The sample holder (described below) was positioned
in direct contact with the output port. The transmitted light was spectrally resolved with the
LCTF set at a wavelength in the range of 420 to 720nm and imaged with the camera.
For three-dimensional imaging of Dylight650 fluorescence from brain samples, we utilized a
commercial confocal microscope (Fluoview FV3000, Olympus, Tokyo, Japan). We used
640nm laser excitation and a 10x objective (Plan-Apo 10x/0.4 WD 3.1mm).
Clearing and attenuation analysis
Each sample was immersed in a specific OCA for a total of 24 hours. During immersion, the
samples were placed in an incubator (37?C). At time points of 0, 3, 6, and 24 hours, optical
transmittance data were collected using the optical attenuation spectroscopy device described
above. To study reversal of the optical clearing effect, each OCA-treated sample subsequently
was immersed in the PBS-sodium azide solution for 24 hours and attenuation data collected.
For the attenuation measurements, each sample was removed from the OCA and placed in a
custom holder consisting of two glass slides of known thickness. The sample thickness was
estimated using a digital micrometer . The holder was placed at the exit port of the
integrating sphere and a portion of the transmitted light was collected by the lens attached to the
camera. We paired each sample measurement with a maximum transmittance measurement
collected using the sample holder containing saline. Using the LCTF, we collected
transmittance data at center wavelengths of 420 to 720 nm, with a step size of 10 nm. We used
customwritten MATLAB code
(R2017b, The MathWorks, Natick, MA)
to estimate a spectrum of
optical attenuation coefficients [?(?)] within a selected region of interest, using Beer?s law:
where I(?) and Io(?) are the transmitted spectral intensities measured through the holder
containing the brain sample and saline, respectively; and L the measured sample thickness. To
calculate normalized attenuation coefficients, we calculated for each sample and each time point,
the quotient of ?(?,t) and ?(?,t = 0). Similar to our previous work , we define the optical
clearing potential (OCP) as the quotient of ?(?,t = 0) and ?(?,t). In addition, measurements of
the lateral dimensions of each sample were collected at each time point with a ruler visible in
each frame for calibration of pixel size and binarized images in MATLAB used to estimate
Confocal fluorescence microscopic imaging
Samples were imaged using an excitation wavelength of 640 nm and emission wavelength
band of 650-750nm. To achieve consistency among samples, imaging was performed within
the midbrain/thalamus region of each section, with z-stacks collected starting at the surface of
the sample proximal to the objective. For each z-stack, 101 images (512x512 pixel resolution,
2.5?m lateral resolution) were collected with a step size of 10?m, resulting in a total imaging
depth of 1mm. Care was taken to control the starting position of the depth scan for each
sample, to facilitate comparison of the data collected from each sample. To quantify vascular
density, which is a measure of total vessel length per area, we used an algorithm described in our
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Fig 2. Representative photos of brain samples taken during immersion in each OCA. Time points of 0,3,6, and 24h
are shown as well as 24h after re-immersion of samples in PBS-sodium azide.
previous publication . To account for the difference in fluorescence emission intensity at
each depth, the intensity and size thresholds were changed for each depth but applied equally
for images collected with each OCA.
To test various hypotheses (described in the Results section), we used either repeated measures
or one-way ANOVA with Holm-Sidak?s multiple comparison tests. All analyses were
performed using Prism (Version 7, GraphPad Software, La Jolla, CA).
Optical clearing affects both transparency and volume of brain samples
To investigate the relative effects of immersion-based OCAs on brain samples, we performed
qualitative and quantitative inspection of each sample at different time points. Immersion
within each OCA increased its transparency (Fig 2). The reversal of clearing effects can be
observed after samples were returned to PBS-sodium azide. Samples immersed in FocusClear,
RIMS, or sRIMS retained some hue innate to the samples, whereas samples immersed in
ScaleSQ became nearly colorless and more translucent than the other samples. Samples immersed
in ScaleSQ increased in volume, while samples immersed in each of the other three OCAs
decreased in volume during the initial 6h (Table 1). For RIMS and sRIMS, the volume at 24h
was greater than the volume at 6h. A comparison of changes in thickness and lateral dimensions
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Volume percentage normalized to initial volume of respective sample before optical clearing. Thickness percentage represents the thickness normalized to initial
thickness of respective sample before optical clearing. Values represent medians of data set with minimum and maximum shown in parenthesis.
suggest that sample expansion and contraction occur in an anisotropic manner, as the data do
not follow a quadratic trend (S1 Fig). Immersion in each of the OCAs led to diminished sample
integrity, as each sample post immersion required more delicate handling to avoid tearing.
Optical clearing achieves a reduction in attenuation of each sample over
the visible spectrum
To investigate quantitative effects of optical clearing, we estimated the attenuation of each
sample using transmittance imaging. Due to an error during data collection, one sample of
ScaleSQ was omitted from analysis. The median attenuation of light through each sample
gradually decreased at each measured wavelength (Fig 3, Table 2).
Dynamics of optical clearing varied among OCAs
We hypothesized that immersion of mouse brain sections in OCAs would decrease light
attenuation through the samples. Additionally, prolonged immersion would decrease attenuation
further. Fig 4 depicts results from repeated measures ANOVA and Holm-Sidak?s multiple
comparison tests were performed. Immersion in each OCA led to a significant decrease in
attenuation through the samples when compared to attenuation at t = 0 (p<0.001 in all cases).
However, the significance of attenuation reduction among 3, 6, and 24h vary for each agent. A
significant decrease in attenuation was observed for ScaleSQ and RIMS comparisons of 3h and
6h to 24h.
ScaleSQ and RIMS had a significantly greater optical clearing potential
than FocusClear and sRIMS
We then hypothesized that the magnitude of attenuation reduction would vary across OCAs.
Ordinary one-way ANOVA and Holm-Sidak?s multiple comparison tests were performed for
evaluation. As depicted in Fig 5, significant differences during 3h and 6h were only found
between ScaleSQ and the other OCAs. However, by 24h differences in optical clearing
potential were found throughout all comparisons (p<0.01 for FocusClear/sRIMS and p<0.001 for
Optical clearing effects of OCAs are reversible
After a total 24 hours of immersion in an OCA, each sample was re-immersed in the initial
storage condition of PBS-sodium azide. After 24 hours of re-immersion in PBS-sodium azide,
the sample attenuation coefficient increased (p<0.001 for each OCA). The 0h attenuation
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Fig 3. Normalized attenuation coefficients measured for each OCA over the 420?720 nm wavelength range. Data points represent median values of
measurements. Error bars representing spread are omitted for clarity.
Normalized to attenuation values at t = 0. Values represent medians of data set with minimum and maximum shown in parenthesis.
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Fig 4. Attenuation reduction significance between time points. Immersion in each optical clearing agent led to a significant reduction in
attenuation coefficient. and denote p<0.05 and p<0.001, respectively. PBS-SA = solution of PBS and sodium azide.
coefficients of samples that then were immersed in FocusClear, were equivalent to the values
after re-immersion in PBS-sodium azide (p = 0.25). For the other OCAs, the 0h attenuation
coefficients and the values after re-immersion were slightly different (p 0.02).
ScaleSQ enabled deeper microscopic imaging of the cerebral
microvasculature, albeit with an apparent decrease in microvascular
Imaging depth after 24 hours of clearing was consistently greater than after 3 hours of clearing
(Fig 6). ScaleSQ showed the greatest achievable imaging depth at both 3h and 24h, with some
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Fig 5. Attenuation reduction significance between OCAs. Of the four optical clearing agents, ScaleSQ had the
highest and sRIMS the lowest optical clearing potential at each evaluated time point (3, 6, and 24h). , , and
denote p<0.05, p<0.01, and p<0.001, respectively.
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Fig 6. (A) Representative confocal fluorescence microscope scans and (B) vascular density at specific immersion
time points and at three imaging depths. Note that the colorbar differs for each row of images, to facilitate
comparison of the visibility of microvasculature among clearing agents. Only ScaleSQ enables visualization of
microvessels at 800?m depth.
vasculature evident at 800?m depth. With ScaleSQ immersion, the microvascular density was
lower at 100?m, as compared with samples treated with other OCAs.
Our results reveal that immersion of brain sections in specific OCAs affects both their
transparency and volume; the optical clearing effect occurs over the entire visible spectrum and is
reversible; and that ScaleSQ had the highest optical clearing potential and increase in imaging
depth of the four evaluated OCAs, albeit with the largest change in sample volume and a
concomitant decrease in apparent microvascular density of the sample.
An increase in sample transparency is an obvious feature of optical clearing. Quantitation
of optical clearing effects is necessary to arrive at a true comparison of different approaches
[21,23,24]. To maximize comparison of data from the four OCAs evaluated in this study, we
divided coronal brain sections into four smaller sections and randomly selected a different
OCA to apply to each of these smaller sections. Due to the small sample sizes, we adopted a
simpler approach to estimate an optical attenuation coefficient than the dual integrating
sphere setup we previously used to study FocusClear . Our current data show a decrease in
optical attenuation coefficient with wavelength, which is similar to the scattering trend we and
others have shown previously [19,20]. It is important to note that our attenuation estimates are
not a true optical attenuation coefficient, which is equal to the sum of the absorption and
scattering coefficients of the sample.
We focused our study on comparison of FocusClear with three OCAs (RIMS, sRIMS, and
ScaleSQ) that also can be used with simple immersion of the sample. Our data at 640nm
(selected as a representative wavelength) demonstrate that the OCAs have different temporal
clearing effects. The OCP of FocusClear and sRIMS reached a plateau at 3h, while the OCP of
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ScaleSQ and RIMS continued to decrease over the 24h clearing period. Our FocusClear OCP
observations differed from our previous study , in which we observed a significant increase
in OCP from 3h to 6h. A potential explanation is the difference in ambient temperature during
immersion in the OCA. In our previous work, the temperature was ~25?C, while we
performed experiments at 37?C in the current study. The higher temperature may have increased
the rate of mass diffusion of FocusClear into the sample and thus led to a faster saturation of
the optical clearing effect.
At 24h, the OCP associated with ScaleSQ and RIMS were significantly greater than those of
FocusClear and sRIMS, and the OCP of ScaleSQ was nearly twice as high as the OCP of RIMS.
Hence, our results suggest that ScaleSQ is the best OCA of the four, in terms of tissue
transparency. Our microscopy data are in agreement with the OCP measurements, as the imaging
depth achieved with optical clearing trends with increased OCP. It is important to note that,
with these OCAs, changes in sample volume also were observed. FocusClear, RIMS, and
sRIMS each induced a reduction in volume, while ScaleSQ induced an increase in volume.
Reports of volume changes associated with optical clearing in general exist in the literature
. Out of the four immersion-based OCAs used in this study, quantitative data exists only
for ScaleSQ; Wan et al  also observed an increase in sample volume. These volume changes
should be considered during interpretation of imaging data. For example, with ScaleSQ, we
observed both an increase in imaging depth with immersion time and a decrease in
microvascular density. The increase in volume most likely decreases the density of structures that can
scatter light, as well as the microvasculature. Such changes may alter quantitative
interpretation of the data (i.e., quantification of cerebral microbleed distance from nearby blood vessels
In previous work on optical clearing of unfixed skin samples, we demonstrated that optical
clearing potential high for sorbitol due primarily to its high collagen solubility , and that
chemical fixation of skin negates the ability of hyperosmotic OCAs to achieve clearing .
Design of OCA formulations for clearing of fixed tissues involves two primary constituents: 1)
an index-matching agent and 2) a detergent that removes lipids. The increased effectiveness of
ScaleSQ over the other OCAs tested is likely due to the combination of sorbitol, urea, and
Triton X-100. Compared to sRIMS, which uses a high concentration of only sorbitol
(indexmatching agent), ScaleSQ combines sorbitol with a small quantity of Triton X-100 to remove
lipids. The composition of ScaleSQ also suggests the reasons for volume expansion. Although
sorbitol is a hyperosmotic agent that would remove tissue shrinkage, the high concentration of
urea induces hydration and hence tissue expansion that apparently counteracts the sorbitol
Over the 420-720nm wavelength range interrogated in this study, the optical clearing effect
in general is more pronounced at longer wavelengths. The shortest typical excitation
wavelength used for vessel painting is 488nm, for excitation of fluorescein isothiocyanate (FITC).
In our previous work , we estimated baseline mouse brain reduced scattering coefficients
of 2.62mm-1 at 485nm and 1.93mm-1 at 642nm. In the present study, at 24h of optical clearing,
the median difference in normalized attenuation coefficient between 490 and 640nm
wavelengths was ~25%, suggesting that the vascular imaging depth at ~640nm would be nearly
twice that at ~488nm. However, it is important to note that imaging depth is not the only
consideration for selection of a vessel painting agent, as the quality of vessel labeling differs for
different fluorescent dyes [27,28]. Future work should focus on study of both vessel painting
approaches and optical clearing approach.
Another relevant factor, specifically for experiments requiring large quantities of OCA, is
the cost per volume of OCA used in a given experiment. With the thick sections used in this
study, clearing a single sample ranged from approximately $0.06 (ScaleSQ) to upwards of $20
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(FocusClear). RIMS and sRIMS also boast lower costs (~$3 and $0.20 respectively) with
comparable OCP to FocusClear. Researchers may wish to consider the ratio of price per sample
cleared when designing procedures and selecting an OCA that is best suited for their
Our study has limitations. Here we assess only four OCAs paired with a single fluorescent
labeling method. As many options exist for both optical clearing approaches and vascular
fluorescence labeling, care must be taken in extrapolating our findings to other combinations of
OCAs and labeling. Here, we focused on brain sections instead of intact brains, so differences
in trends may exist if the latter were used as samples . Optimal clearing time often varies
among OCAs, ranging from one hour to multiple days, so a more complete timeline of the
clearing processes may reveal additional details regarding peak effectiveness.
In summary, our results indicate that immersion of mouse brain sections in FocusClear,
RIMS, sRIMS, or ScaleSQ lead to a decrease in optical attenuation coefficient over the visible
spectrum, demonstrate the spatiotemporal dynamics associated with sample immersion, and
show that confocal microscopy imaging depth trends with optical clearing potential. We
present a rational, quantitative framework for screening and characterization of the impact of
optical clearing. Selection of an OCA tailored to the requirements of a given set of experiments
should help streamline experimental design and enable a cost-benefit assessment.
S1 Fig. Sample expansion and contraction occur in an anisotropic manner. For each sample
and each timepoint, the lateral change in area is plotted versus the measured change in sample
thickness. If expansion and contraction occurred in an isotropic manner, the data should fall
on a parabola. However, a best-fit quadratic model is associated with a R2 value of 0.24,
suggesting that volume changes do not occur in an isotropic manner.
We would like to thank Allia Fawaz, Paul Nguyen, Natalie Mai, Malcom Su (University of
California, Irvine) for their intellectual contributions to this project.
Conceptualization: Christian Crouzet, Vitaly Vasilevko, Bernard Choi.
Data curation: Mathew Loren, Bernard Choi.
Formal analysis: Mathew Loren, Christian Crouzet, Adrian Bahani, Bernard Choi.
Funding acquisition: Bernard Choi.
Investigation: Mathew Loren, Christian Crouzet, Adrian Bahani.
Methodology: Mathew Loren, Christian Crouzet, Bernard Choi.
Project administration: Bernard Choi.
Resources: Bernard Choi.
Software: Mathew Loren, Christian Crouzet, Bernard Choi.
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Supervision: Bernard Choi.
Visualization: Bernard Choi.
Writing ? original draft: Mathew Loren, Bernard Choi.
Writing ? review & editing: Mathew Loren, Christian Crouzet, Adrian Bahani, Vitaly
Vasilevko, Bernard Choi.
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