Tissue optical clearing: new prospects in optical

imaging and therapy

Valery V. Tuchin
Research-Educational Institute of Optics and Biophotonics, Saratov State University, Russia
Laboratory of Laser Diagnostics of Technical and Living Systems,
Institute of Precise Mechanics and Control RAS, Saratov, Russia
Laboratory of Biophotonics, Tomsk State University, Russia

Abstract—Recent achievements of tissue immersion optical § π 5 a 4 n03 · 2 ª 2 º,

μ s # ρs ¨¨ ¸¸(m  1) 2 «1  2 (1)
clearing technique are demonstrated. The specific features of 2»
© λ 3
¹ ¬ ( m  1) ¼
optical clearing of tissues in the visible/NIR and terahertz ranges 0

are underlined. In vitro, ex vivo, and in vivo studies of human and where Us =fcyl/Sa2, fcyl is the surface fraction of the cylindersc
animal tissues are presented. faces, a is the cylinder radius, m=ncyl/nis is the relative index of
refraction of cylinders (scatterers) to the background (medium
Keywords—tissue; optical clearing; imaging; glucose; of the interfibrillar space, ISF).
diffusion; bound water

I. INTRODUCTION
The optical clearing (OC) technology described is based
on controlling of tissue optical properties by using immersion
technique via application of exogenous optical clearing agents
(OCAs) [1-3]. Impact of OCA and water transport in a tissue,
caused by an action of a hyperosmotic OCA and leading to
tissue reversible shrinkage and dehydration, on temporal tissue a
optical properties is discussed. The specific features of OC of
fibrous and cell-structured tissues are investigated using
different linear and nonlinear optical modalities [1-11]. In
vitro, ex vivo, and in vivo studies of human and animal tissues,
including skin, fat, eye sclera, muscle, cerebral membrane,
digestive tract tissue, cartilage, tendon, bone, blood vessels,
and blood are presented in [1-13]. The technologies of b
delivery of OCAs are also under discussion, including hidden Fig. 1. Typical tissue structure (a) and corresponding RI variation at crossing
free diffusion, enforced tissue permeability, and via blood and tissue structures; fibers (nf), ISF (nis), cytoplasm (ncp), organelle (nor), and
lymph vessel networking. Impact of different OCAs on tissue nucleus (nnc).
structure, free/bound water balance [12,13] and
microcirculation [11] are analyzed. Experimental results on Described tissue model is applicable to any fibrous soft
diffusivity of glucose and other biocompatible clearing agents tissue, including skin dermis and muscle. RI of the scatterers
in normal and pathological tissues are presented in [6,7]. is higher than for the background. To provide OC, RI of the
This paper presents fundamentals and advances of tissue OCA normally should be higher than RI of the ISF and
optical clearing (TOC) with demonstration of its recent cytoplasm. Therefore, diffusion of foreign molecules of an
achievements in a few fields of tissue optical imaging. OCA inside tissue will reduce the RI mismatch (m→1) and
correspondingly will lead to drop of the scattering coefficient
II. FUNDAMENTALS OF OPTICAL CLEARING (Ps→0). The single scattering directness, described by
scattering anisotropy factor g, is also sensitive to RI matching,
The scattering coefficient of a tissue depends on the
it increases with the better matching condition (g→1) [1]. As a
refractive index (RI) mismatch between the RIs of interstitial
fluid (ISF) nis and scatterers ns (see Fig.1). For the tissue result of these two processes, reduced scattering coefficient P's
model that can be presented as a monodisperse system of thin = Ps(1–g) becomes a very sensitive function of RI matching.
dielectric cylinders (ns ≡ ncyl) with a number of fibrils per unit Therefore, transport mean free path of a photon, which is
area Us, scattering coefficient μs has a form [1]: defined as [1]
1 , (2)
ltr
μ a  μ cs

978-1-4673-7926-7/15/$31.00 ©2015 IEEE

Authorized licensed use limited to: KU Leuven Libraries. Downloaded on May 22,2024 at 13:20:57 UTC from IEEE Xplore. Restrictions apply.
and is a key parameter for image contrast and probing depth, higher density of proteins and lipids in comparison with the
could be increased significantly at RI matching. ground substance and, thus, a greater index of refraction (ns =
It follows from (1), that relatively small increase in the RI 1.39–1.47). The RI of the ICF, as well as human blood
of the background matter nis will cause a few-fold decrease of plasma, is approximately 1.33–1.35, depending on the
the scattering coefficient μs. wavelength. The main scatterers in blood are red blood cells
For the soft tissues, which are well supplied by water, in (RBCs). A hemoglobin (Hb) concentration of 32 g/dl
some wavelength intervals absorption and RI are defined by represents a typical Hb concentration within a human RBC,
optical properties of water. This is especially valid for IR and and RI of the solution is approximately 1.42. For human
terahertz ranges, where strong water absorption bands exist. whole blood, depending on the wavelength, RI is
Absorption spectrum and dispersion of water in a wide approximately 1.36–1.40.
spectral range are presented in Fig. 1 [14]. There are a several main mechanisms of light scattering
reduction induced by an OCA: 1) dehydration of tissue
constituents; 2) partial replacement of the interstitial fluid by
the immersion substance; 3) structural modification (packing);
and 4) dissociation of collagen. The first and the third
mechanisms are characteristic for hyperosmotic agents. For
fibrous tissue similar to sclera, dura mater, dermis, the second
mechanism could be prevalent for many of tested chemical
agents for which molecule size is much less than the mean
cross-section of interfibrillar space. Both the first and the
second processes mostly cause matching of the RIs of the
tissue scatterers (cell constituents, collagen and elastin fibers)
and the cytoplasm and/or ISF. The RI matching is manifested
in the reduction of the scattering coefficient (μs → 0) (1) and
increase of single scattering directness (g → 1). For fibrous
tissues such as skin dermis, eye sclera, dura mater, tendon,
Fig.2. Absorption spectrum and dispersion of water in a wide spectral range decrease of reduced scattering coefficient P's can be
[14]. significant.
Structural modification is manifested as tissue shrinkage,
Refractivity of a number of tissues at 633 nm is in the it causes the near-order spatial correlation of scatterers (see
range from the lowest 1.368 for liver to the largest 1.455 for [1]) and, as a result, the increased constructive interference of
fatty tissue with other tissues between, such as 1.380 for lungs, the elementary scattered fields in the forward direction and
1.400 for blood and spleen, 1.410 for muscular tissue, and destructive interference in the perpendicular direction of the
1.418 for kidney (see [1]). There is a tendency to refraction incident light that may significantly increase tissue
decrease with the wavelength from 390 to 700 nm, in transmittance even at residual refractive index mismatch.
particular, for bovine muscle – from 1.42 to 1.39. For some tissues and for the specific pH of applied OCA,
In terahertz range, 0.5 – 2.5 THz, mean RI of water, the tissue swelling may take place that could be considered as a
main component of soft tissues, is equal to: nW # 2.2 (Fig.1). competitive process in providing TOC. In collagen-based
For the soft tissues, such as muscle and skin (dermis), n # 2.1, tissues a change in the supramolecular structure may be
for more dry epidermis n # 2.0; for a soft tissue with a less involved [4,5]. Collagen reversible solubility in sugars and
water content, such as adipose, n # 1.65. For nail which is a sugar alcohols may take place. OCA-induced destabilization
hard tissue with comparably low water content and lack of the of collagen structures may lead to an additional reduction of
mineral component, n # 1.8. For all other hard tissues with tissue scattering owing to less size of the main scatterers.
the lower content of water RI is higher than for water due to The osmotic pressure is a driving force in the generation
inclusion of mineral tissue component, for tooth dentin n # of fluid flows and controlling intensities of these flows,
2.4, for bone n # 2.5, and for tooth enamel n # 3.1 (see providing a several mechanisms of TOS; however, rather
[1]). strong osmotic pressure may destroy tissue structure. This is a
The scattering coefficient (μs) and scattering anisotropy major physicochemical mechanism of OCA toxicity.
factor (g) of a tissue are dependent on RI mismatch – relative
RI m [1] (see (1)). The RI mismatch exists between cellular III. INSTRUMENTATION
tissue components, such as cell membrane, cytoplasm, cell Optical techniques and instrumentation beneficial from
nucleus and other organelles, melanin granules, and the TOC and that were used to study TOC efficiency and
extracellular fluid (see Fig.1). For fibrous (connective) tissue, mechanisms are presented in Fig. 3. They are: diffuse
index mismatch of interstitial medium and long strands of reflectance and fluorescence; integrating sphere technique,
scleroprotein (collagen–, elastin–, or reticulin–forming fibers) used to quantify tissue optical properties at OC; collimated
is important. The scattering particles themselves (organelles, transmittance, used to clarify OC mechanisms; Laser Speckle
protein fibrils, membranes and protein globules) exhibit a

Authorized licensed use limited to: KU Leuven Libraries. Downloaded on May 22,2024 at 13:20:57 UTC from IEEE Xplore. Restrictions apply.
Contrast Imaging (LSCI); spectral OCT; photoacoustic flow IV. OPTICAL CLEARING AGENTS (OCAS)
cytometry (PAFC); and terahertz spectrometry.
The immersion TOC is based on the impregnation of a
tissue by a biocompatible chemical agent, which may have
hyperosmotic properties. The OCAs frequently used are
monosaccharides, such as glucose, dextrose, fructose;
polysaccharides made of many glucose molecules – dextrans;
sugar alcohols (polyols) – glycerol, mannitol and sorbitol;
alcohol – 1,3-butanediol; propylene glycol, polypropylene
glycol, ethylene glycol, polyethylene glycol, 1,4-butanediol
and their combinations, such as combined lipophilic
polypropylene glycol-based polymers and hydrophilic
polyethylene glycol-based polymers. Many of these OCAs are
a b
widely used as cryoprotectants for cryotherapeutics and
cryopreservation of tissue and organs for applications in
regenerative medicine [15,16].
Another class of OCAs are radiographic contrast media,
such as VerografinTM, TrazographTM, HypaqueTM,
Omnipaque , etc. [1]. For example, OmnipaqueTM is a
TM

nonionic water-soluble radiographic contrast – Iohexol with

molecular weight MW 821.14 (iodine content 46.36%) was
c d used for OC of blood and cartilage [17-19]. In aqueous
solution each triiodinated molecule remains undissociated.
The osmolalities of Iohexol solutions are close to osmolalities
of blood plasma (285 mOsm/kg water) and cerebral-spinal
fluid (CSF) (301 mOsm/kg water) (Table 1), therefore they
can be used intrathecally, intravascularly, and for oral/body
cavities with very low impact on tissue condition.

Table 1. Physical properties of OmnipaqueTM: Iohexol,N,N´-Bis(2,3-

dihydroxypropyl)-5-[N-(2,3-dihydroxypropyl)-acetamido]-2,4,6-
triiodoisophthalamide.
Concentration Osmolality Absolute viscosity (cp)
(mgI/mL) (mOsm/kg water) 20°C 37°C
e
140 322 2.3 1.5
180 408 3.1 2.0
240 520 5.8 3.4
300 672 11.8 6.3
350 844 20.4 10.4

V. COLLIMATED TRANSMITTANCE, FREE AND BOUND

WATER
Using the experimental assembly presented in Fig. 3c, the
f collimated transmittance of rat muscle tissue samples of
thickness l=0.5 mm at treatment by glucose-water solutions of
different concentration were measured in the wavelength
range between 400 and 1000 nm (Fig. 4) [12]. The treatment
with the lowest concentration of glucose (20%) provides a fast
diffusion and significant kinetic curve saturation with decay
(Fig. 4 (a)). This means that the immersion solution contains a
great amount of water (80%) compared with the tissue water
content. Therefore, because of the strong involvement of small
g
water molecules in the diffusion process, the total time
Fig.3. Optical techniques and instrumentation beneficial from TOC: Diffuse response τ of the diffusion process is short (fast diffusion).
reflectance/fluorescence (a), Integrating sphere technique (b), Collimated With time, after the saturation is reached, the tissue needs to
transmittance measurement (c), Spectral OCT (d), Photoacoustic flow compensate the partial dehydration caused by the impact of
cytometry (PAFC) (e), Terahertz spectrometry (f), Laser Speckle Contrast
Imaging (LSCI) (g).
glucose, and so extra water from the solution is coming to

Authorized licensed use limited to: KU Leuven Libraries. Downloaded on May 22,2024 at 13:20:57 UTC from IEEE Xplore. Restrictions apply.
 create a balance and tissue swelling may be created with some For a particular glucose concentration, the mean and
decay of the collimated transmittance for longer times. standard deviation of τ, measured at 11 wavelengths, was
As the glucose concentration increases (Fig. 4(b) and (c)), calculated and presented in Fig.5. Dependence has an
the water content in the solution becomes similar to the free extrapolated maximum for a concentration of 40.5%. This
water content in the tissue and the water flux decreases, maximum indicates that an aqueous solution with that
becoming close to zero at a glucose concentration around 40% concentration of glucose would have the same amount of
(Fig. 4(b)). That means that only glucose diffuses freely in the water as the free water in the tissue, thus no reason for water
system, and the diffusivity parameters depend only on the flux to exist. Only glucose diffuses into the tissue.
diffusion parameters of the larger glucose molecules, thus we From the plot in Fig. 5 for glucose τG = 302.9 s is
see a maximal value of the measured diffusion time τ (Fig. 5). obtained. This value represents the true diffusion time of
However, at higher glucose concentration τ decrease (Fig. glucose in the rat breast muscle. Using this value the diffusion
4(c)), because water is once again involved in the diffusion coefficient of glucose in muscle tissue can be calculated [1]:
process, with the main flux directed from the tissue to the l2
surrounding solution due to glucose hyperosmolarity. DOCA , (4)
Selecting the data sets observed for wavelengths between π2τ
600 and 800 nm (Fig.4) and using equation for time-dependent where l is the sample thickness, l=0.5 mm. Thus, for glucose
collimated transmittance Tc(λ,t), which can be presented as a diffusion in rat breast muscle tissue:
ratio of OCA concentration in the sample at time moment t
0
COCA t to concentration of applied OCA COCA , [1] DG 8.36 u 10 7 cm 2 /s . (5)

COCA t ª § t ·º As the equilibrium glucose concentration for this tissue is

Tc λ, t 0
# «1  exp¨  ¸» , (3)
40.5%, then the volume fractions of bound and free water in
COCA ¬ © τ ¹¼
the tissue should satisfy to the following relation with the
values of the diffusion time τ can be calculated. volume fraction of the total water of 0.756 [12]:

f water natural fbound water  f free water 0.161  0.595 0.756 . (6)

The similar measurements and estimations for another

OCA - ethylene glycol (EG), give τG = 446 s (Fig. 5) and

DEG 5.68 u10 7 cm 2 /s . (7)

Fig. 5. Diffusion time τ of glucose (solid line) and ethylene glycol (EG)
(dashed line) molecules as a function of OCA concentration in water solution
at OC of rat muscle [12, 13].

VI. OCT, CARTILAGE OPTICAL CLEARING

Optical Coherence Tomography (OCT) is often used in

conjunction with TOC [1-3]. Recently it was applied for
c assessment of deep cartilage layers and cartilage-bone
Fig. 4. Time dependences for collimated transmittance of the rat muscle at
interface (Figs. 6 and 7) [19]. A commercially available OCT
treatment with glucose-water solutios of different concentrations 20% system from Thorlabs Inc. (930 nm) used in this study is
(a); 40% (b), and 50% (c), measured forjj different wavelengths from presented schematically in Fig. 3d. The solution of Iohexol
400 to 1000 nm [12]. (OmnipaqueTM) (see Table 1) in water has been used as an

Authorized licensed use limited to: KU Leuven Libraries. Downloaded on May 22,2024 at 13:20:57 UTC from IEEE Xplore. Restrictions apply.
OCA. The cartilage-bone boundary becomes visible after 15 result in loss of PAFC sensitivity and resolution. The rapid
min of OC that enabling non-invasive estimation of its and efficient OC of skin to minimize light scattering and thus,
roughness: Sa = 10±1μm. to increase optical resolution and sensitivity of PAFC was
The results show that for 0.9 mm thick cartilage optical recently demonstrated (Figs. 8 and 9) [21]. PAFC
clearing is significant and saturated after 50 min. Current experimental assembly is shown in Fig. 3e. OC effect was
approach enables more reliable detection of arthroscopically achieved in 20 min by advanced skin cleaning,
inaccessible regions, including cartilage/bone boundary and microdermabrasion, and then glycerol application as the OCA
subchondral bone, and potentially improves accuracy of the which delivery into skin was enhanced by massage and
osteoarthritis diagnosis [19]. sonophoresis. Using ~0.8 mm mouse skin layer over a blood
vessel as an in vitro phantom, 1.6-fold decrease in laser spot
blurring accompanied by 1.6-fold increase in PA signal
amplitude against blood background were found. As a result,
peak rate for B16F10 melanoma cell counting in blood flow
increased 1.7-fold. By using OC, the feasibility of PA contrast
improvement for human hand veins was also demonstrated.

Fig. 6. The osteochondral samples: natural appearance of the sample before

(a) and after OC by Iohexol (OmnipaqueTM) solution has been completed (b)
[19].

Fig. 8. OC improvement of PAFC detection sensitivity for B16F10 melanoma

cells demonstrated for in vitro phantom (~0.8 mm mouse skin layer over a
blood vessel). Typical PAFC traces for melanoma cells in flow before (a) and
after OC (b) of skin with glycerol. Dependence of peak rate of melanoma cell
counting (cells/min) on the laser pulse energy before and after OC (c) [21].

Fig. 7. 3D OCT image of the cartilage after 15 min of OC by Iohexol

(OmnipaqueTM) solution. The extracted from OCT data average roughness of
the cartilage-bone interface Sa = 10 μm.
The first use of phase-stabilized swept source optical
coherence elastography (PhS-SSOCE) to monitor
quantitatively the change in elasticity of hyaline cartilage
during the OC process noninvasively was recently
demonstrated [20]. The results showed that PhS-SSOCE was
able to assess the increase in cartilage stiffness during the OC
process over time and with different concentrations of glucose.
In addition, the results demonstrated that the elasticity of the
cartilage was reversed once the clearing agent was replaced
with saline.
Fig. 9. OC of human skin: visual contrast of the vein (a); typical changes in
Therefore, the proposed optical coherence elastography can PA signal waveform before/after OC (b); PAFC traces for a vein in human
dynamically assess the effects of OC and associated changes hand before/after OC (c); US-imaging of the selected vein (d) [21].
in tissue biomechanical properties noninvasively and
nondestructively. This technique may be potentially useful in
orthopedic studies such as early detection and monitoring of VIII. TISSUE CLEARING IN TERAHERZ RANGE
osteoarthritic diseases. The terahertz frequencies belong to far infrared optical range
and are adjacent to the microwave frequency range. The
VII. PHOTOACOUSTIC MICROSCOPY AND FLOW broadband sources of THz radiation based on femtosecond lasers
CYTOMETRY have demonstrated their high potentialities in biomedical
Clinical applications of photoacoustic (PA) flow applications, including medicine [22]. The experimental laser
cytometry (PAFC) for detection of circulating tumor cells in THz time-domain system is presented in Fig. 3f. The main
deep blood vessels are hindered by laser beam scattering, that advantages of terahertz range for the medical and biological
studies are the small frequency dispersion of tissues and blood in

Authorized licensed use limited to: KU Leuven Libraries. Downloaded on May 22,2024 at 13:20:57 UTC from IEEE Xplore. Restrictions apply.
this range and the low scattering of radiation by inhomogeneities and propylene glycol. As an example, Fig. 10 shows spectral
smaller than 10 μm (cell size). The use of ultrashort pulses allows dependences of the absorption coefficient and refractive index
for investigation of a wide range of frequencies in one of the bovine muscle tissue treated with glycerol [23]. A
measurement; one can also attain high temporal resolution and significant reduction of absorption coefficient during 6-8 min
extract information on the phase and, therefore, the refractive and the stability of the effect during 2 hrs are well seen. A
index. corresponding decay of RI is also important for better THz
The main disadvantage of application of THz waves in radiation transport in layered tissues, because of lesser Fresnel
biomedicine is their small probing depth caused by strong water refraction on the interfaces of tissue layers.
absorption (see Fig. 1). Therefore, tissue dehydration methods To obtain a direct evidence for skin dehydration under
can be used to enhance THz penetration depth. However, in exposure to agents and their further use in THz measurements,
vivo protocols require the appropriate changes in the tissues to clinical studies on eight volunteers were performed. A
be temporary and reversible. In [22-24] to perform reversible Callegari Soft Plus system designed for dermatological studies
tissue dehydration there was proposed to apply hyperosmotic was used to measure skin hydration. Figure 11 shows the time
agents, the use of which is based on their ability to form a dependences of relative skin hydration, averaged over all
flow of free water directed outside from the tissue, as well as volunteers. Before averaging, the data were normalized for
to penetrate into the tissue and temporarily substitute the free each individual to take into account the personal features of
water. The promising agents are glycerol, polyethylene glycol, skin. The hydration values obtained in 3 min after the agent
and glucose solutions [1]. These agents are not toxic and have deposition were used for normalization.
no strong absorption bands in THz range. Besides, they It can clearly be seen that the degree of human skin
possess reasonably high diffusion rate in tissues and can be hydration sharply decreases 10−17 min after applying the
used as OCA to provide in parallel an enhanced optical agent and then sharply increases to 20−22 min (rehydration
imaging. with higher moistening of the upper skin layers occurs).
Specifically, this is the manifestation of agent assignment to
provide the moistening effect which is based on the formation
of a water flux from the water-saturated deep layers of skin.
However, this is happened only in 20 min after agent
application due to skin physiological reaction. Concerning the
practical application of THz radiation in diagnostics, one must
take into account that the skin transparency window exists
from 10 to 22 min after agent application.

Fig.11. Time dependences of the average normalized hydration (moistening)

(TEWL) for the skin of volunteers (N =8) affected by hyperosmotic agents
[24].
b
Fig. 10. Spectral dependences of the absorption coefficient (a) and refractive
index (b) of the bovine muscle tissue treated with glycerol [23].
IX. OC OF BLOOD
The laser terahertz spectrometer (0.25 to 2.5 THz) was Laser speckle contrast imaging (LSCI) shows a great
used to study the influence of dehydrating agents on the potential for monitoring of blood flow in tissues and organs,
efficiency of propagation and reflection of THz radiation in however the spatial resolution suffers from the strong
tissues [23, 24]. The object of study was the muscle tissue, scattering in tissues. Using the experimental LSCI assembly
chosen as a model of fibrous soft tissue with high content of presented in Fig. 3g the capability of a combination method of
free water. Broadband terahertz absorption and reflection LSCI and TOC was demonstrated to characterize the dynamic
spectra of the bovine skeletal muscle tissue were obtained response of cutaneous vasculature to vasoactive noradrenaline
under the action of glycerol, polyethylene glycol (PEG-600), (NA) injection in mice [11]. The superior resolution, contrast

Authorized licensed use limited to: KU Leuven Libraries. Downloaded on May 22,2024 at 13:20:57 UTC from IEEE Xplore. Restrictions apply.
and sensitivity due to TOC made it possible to reconstruct nm, detection wavelength, 800 nm) and glycerol as an optical
arteries-veins location and quantitatively assess the blood flow clearing agent to enhance imaging from under 6.2-mm-thick
velocity and vascular diameter at single artery or vein level. porcine muscle tissue samples was demonstrated (Fig. 14)
Figure 12 presents monitoring of mouse cutaneous blood [26]. The improvement of image of UCNP luminescent label
flow dynamics after NA tail-vein injection accompanied with is caused by transformation of the diffuse light emitted by the
OCA-treatment [11]. label into the direct component due to less scattering of the
luminescent light (800 nm). More efficient excitation (at 980
nm) of the label due to bigger probing depth at TOC is also
give input in the excited intensity of UCNPs, thus also
promotes a brighter image. These results in 2-fold increase in
visibility (contrast) of the label and 20-fold increase in
maximal signal intensity thus making the combination of the
UCNPs and TOC promising for precise detection of tissue-
embedded labeled abnormalities (Fig. 14).

Fig.12. Monitoring of cutaneous blood flow dynamics after NA injection

accompanied with OCA-treatment: white-light images, blood flow velocity
maps; profiles of the flow velocity along the horizontal white line. Bar = 500
μm. OCA is a mixture of two biocompatible agents, PEG-400 and thiazone at
a volume ratio of 9:1 [11].
Thus, combination of skin OC and LSCI provides a
valuable technology to monitor and diagnosis of some
vascular diseases, such as diabetes, cancer, arteriosclerosis and
a
venous leg ulceration. 140
6
120
195 min
100 5

Maximum, norm.
Intensity, a.u.

80 4
60
3
40
0 min
2
20

0 1

250 300 350 400 450 500 550 600 650 0 25 50 75 100 125 150 175 200 225
X pixel no. Clearing time, min
b c

d e
Fig.13. Enhancement of transmittance for various OCAs mixed with blood. Fig.14. Enhanced UCNPs luminescence imaging from tissue depth: outline
Data were obtained using the OCT system working at 930 nm. Polyethylene of the imaging system (a); image of a star-shaped upconversion
glycol (PEG), polypropylene glycol (PPG), and propylene glycol (PG) were luminescence label at 800 nm before (0 min) and after (195 min) of
used at 100%. glycerol OC through the 6.2-mm-thick porcine muscle tissue in vitro (b);
the corresponding kinetic clearing curve (c); images observed in mice in
The improvement of light penetration depth in blood after vivo before (d) and after TOC (e) [26].
application of such OCAs, as polyethylene glycol,
polypropylene glycol, propylene glycol, and hemoglobin B. Optical Diagnosis of Rheumatoid Arthritis at Skin OC
solutions was quantified using an OCT system (see Fig. 3d) A new method of enhanced optical imaging of finger
[25]. The influence of OCAs on light transport in tissues and proximal interphalangeal (PIP) joints in humans based on skin
blood was also investigated at an OCA injection into mouse OC was recently proposed [27]. A set of illuminating laser
tail-vein and surrounding tissues [25]. diodes with the wavelengths 670, 820, and 904 nm were used
as a light source (Fig.15a). The laser diodes, monochromatic
X. APPLICATIONS digital CCD camera and specific software allowed for
detection of the PIP joint image in a transillumination mode.
A. Enhanced Luminescence Imaging from Tissue Depth
The experiments were carried out in vivo with human fingers.
The deep-tissue imaging using upconversion nanoparticles
(UCNPs) β-NaYF4:Yb3+:Tm3+ (excitation wavelength, 980

Authorized licensed use limited to: KU Leuven Libraries. Downloaded on May 22,2024 at 13:20:57 UTC from IEEE Xplore. Restrictions apply.
 Dehydrated glycerol and hand cream with urea (5%) were
used as OCAs. The contrast of the obtained images was
analyzed to determine the effect of the OCA. The results have
shown that glycerol and the hand cream with 5% urea allow
for obtaining of more distinct image of finger joint in the NIR
(Fig 15b and Fig. 15c). It was found that glycerol application
to the human skin during 60 min caused the increase of
contrast in 1.5 and 1.7 fold for 820 nm and 904 nm,
respectively. At the same time, the hand cream application to
the human skin during 60 min caused the increase of contrast a
in 1.3 and 1.1 fold for 820 nm and 904 nm, respectively.
Obtained data can be used for development of optical
diagnostic methods of rheumatoid arthritis.

b
a
b c
Fig.15. The exterior of the experiment and the transillumination system with 3
laser diodes for human finger joint imaging (a); images of finger joint with
pseudo color imaging in HSV color space within ROI region under the action
of glycerol: before agent application (b) and 60 min after agent application (c)
[27].

C. Electro-Kinetic Tissue Response: dcOCT and TOC

The influence of a low-frequency electric field applied to
soft tissues ex vivo at normal conditions and upon the topical
application of OCAs has been studied by the double
correlation OCT (dcOCT) (Fig. 16) [28]. This novel technique
utilizes consistent application of an adaptive Wiener filtering c
and Fourier-domain correlation algorithm (Fig. 16b). The
electro-kinetic response of tissues has been observed and
quantified by dcOCT.
Schematics of the experimental setup is presented in Fig.
16a. The OCT probe (commercially available swept source
OCT working on 1300 nm (OCM1300SS, Thorlabs Inc.)) is
placed above the surface of tissue sample to acquire OCT
images from the area between two electrodes embedded into
the tissue at a0.5 mm depth and separated by a 5 to 6 mm
from each other. Figure 16b presents the double correlation
OCT (dcOCT) approach: step 1 corresponds to I(x, z) image d
acquisition by OCT, step 2 shows the Wiener filtering of the Fig.16. Study of electro-kinetic tissue response with dcOCT and TOC:
OCT probe and tissue holder with electrodes (a); schematic representation
obtained images, step 3 represents the cross-correlation of the dcOCT approach (b); the 2-D dcOCT images of fresh chicken breast
procedure between the Wiener filtered images Iw(x, z) ex vivo (alternating current 10 V and 1 Hz) at 0, 1, 4, 60, 120, 200, 450,
obtained at t and t + 1 time intervals and the generation of and 600 s after topical application of 50%-glycerol–water solution, images
correlation map. from (a) to (h), respectively, scale bar 250 μm; results of resistance
measurements across the tissue sample as a function of time corresponding
to tissue heating induced by the applied electric current, triangles and
circles are, respectively, tissue with and without exposure to an OCA, the
solid and dashed lines show the results of best fittings [28].

Authorized licensed use limited to: KU Leuven Libraries. Downloaded on May 22,2024 at 13:20:57 UTC from IEEE Xplore. Restrictions apply.
 The 2-D dcOCT images of fresh chicken breast ex vivo REFERENCES
obtained during exposure with 10 V and 1 Hz alternating [1] V. V. Tuchin, Tissue Optics: Light Scattering Methods and Instruments
current at 0, 1, 4, 60, 120, 200, 450, and 600 s after topical for Medical Diagnosis, SPIE Press, PM 254, 3rd ed., SPIE Press, 2015.
application of 50%-glycerol–water solution are shown in Fig. [2] D. Zhu, K.V. Larin, Q. Luo, and V.V. Tuchin, “Recent progress in tissue
16c. Results of resistance measurements across the tissue optical clearing,” Laser Photonics Rev. 7(5), 732–757, 2013.
sample as a function of time corresponding to tissue heating [3] E.A. Genina, A.N. Bashkatov, Yu.P. Sinichkin, I.Yu. Yanina, V.V.
Tuchin, Optical clearing of biological tissues: prospects of application in
induced by the applied electric current are presented in Fig. medical diagnostics and phototherapy [Review], J. of Biomedical
16d. Photonics & Eng. 1(1), 22-58, 2015.
It was demonstrate that in comparison to impedance [4] J. M. Hirshburg, K. M. Ravikumar, W. Hwang, A. T. Yeh, “Molecular
measurements and the mapping of the temperature profile at basis for optical clearing of collagenous tissues,” J. Biomed. Opt. 15(5),
the surface of the tissue samples, the dcOCT is much more 055002, 2010.
sensitive to the changes associated with the tissues’ electro- [5] J. Wang, N. Ma, R. Shi, Y. Zhang, T. Yu, and D. Zhu, “Sugar-induced
skin optical clearing: from molecular dynamics simulation to
kinetic response. The topical application of the OCA reduces experimental demonstration,” IEEE J. Select. Tops. Quant. Electron. 20
the tissues’ electro-kinetic response (Fig. 16c) and provides (2), 7101007-1–7, 2014.
tissue cooling (temperature induced by the electric current [6] Z. Zhu, G. Wu, H. Wei, H. Yang, Y. He, S. Xie, Q. Zhao, and X. Guo,
reduces by a few degrees). That phenomenon can be “Investigation of the permeability and optical clearing ability of different
analytes in human normal and cancerous breast tissues by spectral
associated with tissue dehydration (Fig. 16d). It was domain OCT,” J. Biophoton. 5(7), 538–545, 2012.
anticipated that dcOCT can find a new application in [7] D.K. Tuchina, R. Shi, A.N. Bashkatov, E.A. Genina, D. Zhu, Q. Luo,
bioelectrical impedance analysis and monitoring of the electric and V.V. Tuchin, “Ex vivo optical measurements of glucose diffusion
properties of tissues, including the resistivity of high water kinetics in native and diabetic mouse skin,” J. Biophotonics, 8(4), 332–
content tissues and its variations at OC. 346, 2015.
[8] O. Nadiarnykh and P.J. Campagnola, “SHG and optical clearing,” in
XI. SUMMARY Second Harmonic Generation Imaging, F.S. Pavone and P.J.
Campagnola (eds.), CRC Press, Taylor & Francis Group, Boca Raton,
Tissue optical clearing method based on interaction of London, NY, 2014, pp. 169−189.
exogenous biocompatible agents with tissues components [9] V. Hovhannisyan, P.-S. Hu, S.-J. Chen, C.-S. Kim, C.-Y. Dong,
allows for significant improvement of optical and terahertz “Elucidation of the mechanisms of optical clearing in collagen tissue
with multiphoton imaging,” J. Biomed. Opt. 18 (4), 046004-1–8, 2013.
techniques at application to tissue imaging, spectroscopy and
[10] G. Vargas, J. K. Barton, A. J. Welch, “Use of hyperosmotic chemical
control of laser treatment. agent to improve the laser treatment of cutaneous vascular lesions,” J.
It may have great benefits at monitoring of pathology Biomed. Opt. 13(2), 021114, 2008.
development as well as at use of the least invasive laser [11] R. Shi, M. Chen, V.V. Tuchin, D. Zhu, “Accessing to arteriovenous
therapy and surgery. blood flow dynamics response using combined laser speckle contrast
imaging and skin optical clearing,” Biomedical Optics Express 6 (6),
TOC is an emerging technique for dynamically modifying 1977–1989, 2015.
tissue optical properties to increase imaging depth, which is [12] L.Oliveira, M.I. Carvalho, E. Nogueira, V. V. Tuchin, “The
useful in applications such as imaging and functional characteristic time of glucose diffusion measured for muscle tissue at
diagnostics of many diseases. optical clearing,” Laser Phys. 23, 075606-1–7, 2013.
[13] L.Oliveira, M.I. Carvalho, E. Nogueira, V. V. Tuchin, “Diffusion
ACKNOWLEDGMENTS characteristics of ethylene glycol in skeletal muscle,” J. Biomed. Opt.
20(5), 051019-1–10, 2015.
Author is thankful to his colleagues and PhD students from
[14] U. Møller, D.G. Cooke, K. Tanaka, and P.U. Jepsen, “Terahertz
SSU, A.N. Bashkatov, E.A. Genina, I.Yu. Yanina, Yu.P. reflection spectroscopy of Debye relaxation in polar liquids [invited],” J.
Sinichkin, V.I. Kochubey, E. Kolesnikova, D. Tuchina, P. Opt. Soc. Am. B 26 (9), A113–A125, 2009.
Timoshina, O. Zhernovaya, A. Kolesnikov, and many others. [15] K.G. Brockbank, M.J. Taylor, “Tissue preservation.” Advances in
Author also do appreciate collaboration with many Biopreservation, Baust J.G., Baust J.M., eds., Boca Raton: CRC Press,
157–196, 2007.
research groups all over the world, Q. Luo, D. Zhu, R. Shi,
[16] L.M. Alvarez, Compendium of organ & tissue banking concepts, 2015;
HUST University; K.V. Larin, University of Houston; I. http://science.dodlive.mil/files/2015/01/Organ-and-Tissue-Banking-
Meglinski, A. Bykov, M. Kinnunen, A. Popov, University of Compendium-2015-Jan.pdf
Oulu; M. Leahy, National University of Ireland, Galway; O. [17] Y. Ozaki, H. Kitabata, H. Tsujioka, S. Hosokawa, M. Kashiwagi, K.
Minet, Charite University Clinic, Berlin; L. Oliveira, Ishibashi, K. Komukai, T. Tanimoto, Y. Ino, S. Takarada, T. Kubo, K.
University of Porto; V.P. Zharov, E.I. Galanzha, Y.A. Kimura, A. Tanaka, K. Hirata, M. Mizukoshi, T. Imanishi, T. Akasaka,
“Comparison of contrast media and low-molecular-weight dextran for
Menyaev, D.A. Nedosekin, University of Arkansas Medical frequency-domain optical coherence tomography, Circulation J. 76(4),
Science; A.P. Shkurinov, Moscow State University; M. 922–927 (2012).
Nazarov, Institute on Laser and Information Technologies, [18] J. Li, H. Minami, E. Steward, T. Ma, D. Mohar, C. Robertson, K. Shung,
Russian Academy of Sciences. Q. Zhou, P. Patel, Z, Chen, “Optimal flushing agents for integrated
The work was supported by Russian Presidential grant optical and acoustic imaging systems,” J. Biomed. Opt. 20(5), 056005,
2015.
NSh-703.2014.2, the Government of the Russian Federation
[19] A. Bykov, T. Hautala, M. Kinnunen, A. Popov, S. Karhula, S.
grant 14.Z50.31.0004, and The Tomsk State University Saarakkala, M.T. Nieminen, V. Tuchin, I. Meglinski, “Imaging of
Academic D.I. Mendeleev Fund Program. subchondral bone by optical coherence tomography upon optical
clearing of articular cartilage,” J. Biophotonics (in press).

Authorized licensed use limited to: KU Leuven Libraries. Downloaded on May 22,2024 at 13:20:57 UTC from IEEE Xplore. Restrictions apply.
[20] C.-H. Liu, M. Singh, J. Li, Z. Han, C. Wu, S. Wang, R. Idugboe, R. biological tissues affected by hyperosmotic agents,” Phys. Wave
Raghunathan, E. N. Sobol, V. V. Tuchin, M. D. Twa, and K. V. Larin, Phenomena 22(3),169–176, 2014.
"Quantitative assessment of hyaline cartilage elasticity during optical [25] O. Zhernovaya,V.V. Tuchin, M.J. Leahy, “Blood optical clearing
clearing using optical coherence elastography," Modern Technologies in studied by optical coherence tomography,” J. Biomed. Opt. 18(2),
Medicine 7, 44–51, 2015. 026014-1–8, 2013.
[21] Y.A. Menyaev, D.A. Nedosekin, M. Sarimollaoglu, M.A. Juratli, E.I. [26] A.P. Popov, E.V. Khaydukov, A.V. Bykov, V.A. Semchishen, V.V.
Galanzha, V.V. Tuchin, and V.P. Zharov, “Skin optical clearing for in Tuchin, “Enhancement of upconversion deep-tissue imaging using
vivo photoacoustic flow cytometry,” Biomed. Opt. Express 4 (12), 3030- optical clearing,” Proc. SPIE 9540 (in press).
3041, 2013.
[27] E.A. Kolesnikova, A.S. Kolesnikov, U. Zabarylo, O. Minet, E.A.
[22] M. Nazarov, A. Shkurinov, V. Tuchin, X.-C.Zhang, “Terahertz tissue Genina, A.N. Bashkatov, V.V. Tuchin, “Optical clearing of human skin
spectroscopy and imaging” in Handbook of photonics for biomedical for the enhancement of optical imaging of proximal interphalangeal
science, CRC Press, Taylor and Francis Group, 2010, pp. 591−617. joints,” Proc. SPIE 9031, 90310C, 2014.
[23] A.S. Kolesnikov, E.A. Kolesnikova, A.P. Popov, M.M. Nazarov, A.P. [28] A.F. Peña, A. Doronin, V.V. Tuchin, I. Meglinski, “Monitoring of
Shkurinov, V.V. Tuchin, “In vitro terahertz monitoring of muscle tissue interaction of low-frequency electric field with biological tissues upon
dehydration under the action of hyperosmotic agents,” Quant. Electr. 44 optical clearing with optical coherence tomography,” J. Biomed. Opt.
(7), 633–640, 2014. 19(8), 086002-1–6, 2014.
[24] A. S. Kolesnikov, E. A. Kolesnikova, K. N. Kolesnikova, D. K. Tuchina,
A. P. Popov, A. A. Skaptsov, M. M. Nazarov, A. P. Shkurinov, A. G.
Terentyuk, and V. V. Tuchin, “THz Monitoring of the dehydration of

Authorized licensed use limited to: KU Leuven Libraries. Downloaded on May 22,2024 at 13:20:57 UTC from IEEE Xplore. Restrictions apply.