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Non-invasively Imaging Subcutaneous Tumor Xenograft by Handheld Raman Detector with Assistance of Optical Clearing Agent Yunfei Zhang, Haoran Liu, Jiali Tang, Zhuoyun Li, Xingyu Zhou, Ren Zhang, Liang Chen, Ying Mao, and Cong Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 05 May 2017 Downloaded from http://pubs.acs.org on May 11, 2017

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Non-invasively Imaging Subcutaneous Tumor Xenograft by Handheld Raman Detector with Assistance of Optical Clearing Agent Yunfei Zhanga, Haoran Liua, Jiali Tangb, Zhuoyun Lia, Xingyu Zhoua, Ren Zhangc, Liang Chend, Ying Maod* and Cong Lia* a

Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai 201203, China b

Department of Pharmaceutical Engineering, School of Engineering, China Pharmaceutical University, Nanjing 210009, China c

d

Center of Analysis and Measurement, Fudan University, Shanghai 200433, China

Department of Neurosurgery, Huashan Hospital, Fudan University, 12 Wulumuqi Middle Road, Shanghai 200040, China

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Abstract: Handheld Raman detector with operational convenience, high portability and rapid acquisition rate has been applied in clinic for diagnostic purposes. However, the inherent weakness of Raman scattering and strong scattering of the turbid tissue restrict its utilization to superficial locations. To extend the applications of handheld Raman detector to deep tissues, a gold nanostar-based surface-enhanced Raman scattering (SERS) nanoprobe with robust colloidal stability, a fingerprint-like spectrum and extremely high sensitivity (5.0 fM) was developed. With assistance of FPT, a multi-component optical clearing agent (OCA) efficiently suppressing light scattering from the turbid dermal tissues, the handheld Raman detector noninvasively visualized the subcutaneous tumor xenograft with a high target-to-background ratio after intravenous injection of the gold nanostar-based SERS nanoprobe. To the best of our knowledge, this work is the first example to introduce the optical clearing technique in assisting SERS imaging in vivo. The combination of optical clearing technology and SERS is a promising strategy for the extension of the clinical applications of the handheld Raman detector from superficial tissues to subcutaneous or even deeper lesions that are usually “concealed” by the turbid dermal tissue.

KEYWORDS: Surface-enhanced Raman scattering (SERS), Optical clearing, Gold nanostar, Non-invasive diagnosing, Handheld Raman detector

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1. INTRODUCTION Surface-enhanced Raman scattering (SERS) is a plasmonic effect where the molecular reporters are in close proximity to nano-roughened noble metal surfaces, which enhances the Raman signal by 7−10 orders of magnitude.1 The incorporation of gold or silver nanoparticles with a strong SERS response dramatically broadens the medical applications of the Raman imaging in diagnosis,2 prognosis3 and image-guided surgery4 by providing real-time, quantitative and chemically specific information of the target molecules in cellular or tissue samples.5 Compared to optical imaging approaches such as fluorescence imaging, SERS possesses higher sensitivity,6 less susceptibility to photobleaching and better multiplexing capability.7 Near-infrared (NIR) light excited SERS received wide interest for in vivo studies because of the low absorption of the endogenous molecules, reduced phototoxicity and auto-fluorescence background from the living tissues. Gambhir et al non-invasively imaged mouse liver with up to five SERS nanoprobes and obtained distinguishable, fingerprint-like Raman spectra after excitation with 785 nm laser light.8 With the assistance of an NIR light excited SERS probe, Garside et al visualized inflammatory lesions in animal models using Raman imaging that showed a higher sensitivity than a range of fluorescence-based imaging technologies including two-photon fluorescence microscopy.9 Currently, most in vivo SERS studies are conducted by confocal Raman spectroscopy because of its high sensitivity and spatial resolution.8 The static Raman microscope, however, is designed for bench-top experiments and is difficult and hardly to be used in clinical settings because of its immobility, insufficient operating space, fixed scanning angle, long acquisition time and narrow field of view (FOV). To overcome the limitations of instrumental apparatus, handheld Raman detectors with high portability, convenient handing and rapid acquisition rates have been applied in examining cervical tumors,10 imaging malignant skin cancers11 and delineating intraoperatively brain tumor margins.12 However, current applications of the handheld Raman detector in clinical settings are restricted to lesions located in superficial tissues. Strong scattering of turbid dermal tissue sharply decreases both the resolution and contrast of Raman imaging when the light propagates into deep tissues. Suppression of light scattering in the dermal tissue could therefore increase detectability and extend applications of the handheld Raman imaging system.

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The optical clearing technique is promising for reducing the light scattering of turbid tissue through application of osmophilic optical clearing agents (OCAs) that increase optical homogeneity of the tissues through matching the refractive indexes of different tissue components. Due to the remarkable increase of light penetration depth in living tissues, this technique has been applied for improving sensitivity and spatial resolution of multiple optical imaging techniques including fluorescence imaging,13 photoacoustic imaging,14 optical coherence tomography15 and laser speckle contrast imaging.16 Recent reports showed that a mixture of FDA- approved polypropylene glycol (PPG) and polyethylene glycol (PEG) increased the optical “transparency” of human skin in vivo.17 Gallwas et al proposed that OCA composed of a mixture of DMSO and PEG400 significantly clarified cervical epithelium specimens.18 The optical clearing technique, however, has not been introduced to SERS imaging. Neither has it assisted handheld Raman detectors to visualize subcutaneous lesions. We developed a gold nanostar-based SERS nanoprobe that demonstrated robust colloidal stability, spectral fingerprint and femtomolar sensitivity upon excitation by the NIR light. A home-made apparatus mimicking a vasculature embedded in a simulated turbid “tissue” confirmed that the optical clearing significantly increased the tissue imaging depth and detectability of the handheld Raman detector. FPT, a multicomponent OCA comprising fructose, PEG400 and thiazone demonstrated the highest efficacy in reduction of light scattering in the dermal tissue. In vivo studies confirmed that FPT could remarkably improve the detectability of the subcutaneous SERS nanoprobe. The subcutaneous tumor xenograft was non-invasively visualized by handheld Raman detector with high sensitivity shortly after intravenous injection of the SERS nanoprobe followed dermal treatment of FPT (Figure 1). To the best of our knowledge, this work is the first example to introduce the optical clearing technique into SERS imaging. Combining the optical clearing technique and SERS nanotechnology is a promising strategy for extension of clinical applications of handheld Raman detectors from superficial regions to subcutaneous or even deeper tissues.

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Figure 1. The Handheld Raman detector non-invasively visualizes subcutaneous tumor xenograft in vivo aided by an optical clearing agent that suppresses light scattering from turbid skin tissue.

2. MATERIALS AND METHODS Materials and Instruments. Unless otherwise specified, all chemical reagents were purchased from Aladdin Chemistry Co. Ltd. (China) and were used without further purification. Intralipid (20%) was from Sichuan Kelun Pharmaceutical Co. Ltd. (China). mPEG-SH (2.0 kDa) was obtained from Tuoyang Co., Ltd. (China). FPT was prepared by mixing aqueous fructose solution (78%, w/w), PEG400 and thiazone at a volume ratio of 11:8:1.19 The refractive indexes of the OCAs, including the saturated aqueous solutions of glucose (52%, w/w), fructose (78%, w/w), glycerol, DMSO, 1,4-butanediol, 1,2-propanediol and PEG400, were measured using an Abbe refractometer (WAY-2WAJ, Shanghai INESA Physico-Optical Instrument Co. Ltd, China). The gold nanostar-based SERS nanoprobe was characterized by UV-Vis spectroscopy (UV-2550, Shimadzu, Japan), transmission electron microscopy (TEM) (Tecnai G2spirit Biotwin, FEI, USA), dynamic light scattering (Zetasizer 3000, Malvern Instruments, USA) and Fourier transform infrared (FT-IR) spectroscopy (Nicolet, Thermo Nicolet Corporation, USA). Molar concentrations of AuNS-Cy7 were determined by a nanoparticle tracking analyzer (Nanosight NS300, Malvern Instruments, UK). Ex vivo confocal Raman spectroscopic images were obtained from a Raman microscopic system (XploRA confocal microRaman system, Horiba Jobin Yvon, France). Unless otherwise noted, SERS spectra were recorded with a handheld Raman detector (QE65Pro, Ocean Optics, USA). An optic probe (Inphotonics) equipped with a 785 nm laser (Ocean Optics, USA) was used for both excitation and collection purposes. This handheld Raman

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detector possesses a 200 µm collection fiber and a 105 µm excitation fiber. The numerical aperture (NA) is 0.22. SERS spectra were processed by LabSpec5 software (v2.02, 2010). Synthesis. Detailed synthetic route and supplemented spectra including 1H and 13C NMR spectra of Cy7SH (3) are presented in Supporting Information. Molecular reporter Cy7-SH (3) was synthesized according to the Dinish’s method.20 Gold nanostar (5) was prepared by a previous method with minor modifications.21 Briefly, addition of 15 mL citrate solution (1%) into 100 mL boiling chloroauric acid aqueous solution (1.0 mM) yielded gold seeds (4) with an average diameter of 12 nm. The mixture of 4.0 mL gold seed solution and 800 µL HCl (0.1 M) was added into 200 mL chloroauric acid (0.25 mM). 2.0 mL AgNO3 (3.0 mM) and 1.0 mL ascorbic acid (100 mM) were then added simultaneously to the previous mixture to yield gold nanostar (6) with vigorous stirring (1100 rpm) for 30 s. Cy7-SH and mPEG-SH were successively added to the gold nanostar solution and stirred gently for 2 h (molar ratio of Cy7-SH/mPEG-SH/Au nanostars: 1500/10000/1) to give the desired SERS nanoprobe AuNS-Cy7 (7) after purification in a 10,000 MW cutoff dialysis bag. Sensitivity of AuNS-Cy7. Working solutions of AuNS-Cy7 with concentrations of 400, 200, 100, 50, 25, 20, 10 and 5 fM in distilled water were prepared. The Raman signal intensities of AuNS-Cy7 as a function of concentration were determined by quantifying the characteristic Raman peak assigned to C−S vibration (~541 cm-1). The incident laser power was 80 mW with a wavelength of 785 nm. Acquisition time for each measurement was 1.0 s (100 times). The confocal Raman spectroscopic images of the AuNS-Cy7 solutions with concentrations of 100, 50, 20 and 5 fM were collected with a step width of 3 µm and an acquisition time of 1.0 s for each mapping point (100 times). Stability of AuNS-Cy7. The colloidal stability of AuNS-Cy7 (1.0 nM) was studied in 0.01 M HCl, 0.01 M NaOH, 1.0 M NaCl and PBS by measuring the SERS spectra collected at 0, 1, 2, 4, 8, 16, 24 and 48 h postincubation (laser power: 400 mW, acquisition time: 200 ms). In vitro Raman Phantom Imaging. A glass capillary with an inner diameter of 0.3 mm used to mimic a “blood vessel” was immersed at selected depths into an intralipid medium (20%) mimicking turbid skin tissue. Selected OCAs and water were supplemented to keep the final concentration of intralipid (5%, v/v) unchanged. SERS spectra of a flowing AuNS-Cy7 (1.0 nM) solution in a glass capillary with a flow rate of 2.0 mm/s driven by a syringe pump were collected by a handheld Raman detector (laser power: 400 mW, acquisition

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time: 200 ms) as functions of OCA types and the immersed depths below the intralipid medium level (from 0.1 mm to 1.1 mm, 0.1 mm interval). The supplemented OCAs included glycerol, DMSO, 1,4-butanediol and 1,2propanediol. Establishment of Subcutaneous Tumor Xenografts. All animal experiments were carried out according to the Guide for the Care and Use of Laboratory Animals, Fudan University (Shanghai, China). ICR mice and athymic nude mice (15−20 g, 3−4 weeks old) were purchased from the Laboratory Animal Center of Fudan University (Shanghai, China). Briefly, murine glioblastoma C6 cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin and streptomycin (37 ℃, 5% CO2). The male nude mice were anaesthetized by intraperitoneal (i.p.) injection of 150 µL chloral hydrate (5%). The cancer cells (2×107 cells in 200 µL PBS) were subcutaneously injected into the dorsum. The animal models with tumor volume greater than 10 mm3 were used for imaging studies. Ex vivo Raman Imaging Studies. Hair of the euthanatized ICR mice was shaved and completely removed by depilatory cream. The dorsal skin was excised carefully and cut into pieces with an area of approximately 1.0 cm2 and immersed into solutions of selected OCAs including fructose (78%), glucose (52%), PEG400 (100%), 1,4-butanediol (100%), glycerol (100%), 1,2-propanediol (100%), FPT (a multicomponent OCA including fructose aqueous solution (78%, w/w), PEG400 and thiazone with a volume ratio of 11:8:1) and PBS. After immersion in the selected OCA for a designated time (from 0 to 60 min with an interval of 10 min), the murine skin pieces were carefully wiped and placed above AuNS-Cy7 (1.0 nM) that was embedded between two glass slides. The SERS spectra of the subcutaneous nanoprobe were collected by the handheld Raman detector with a laser power of 350 mW and acquisition time of 200 ms. In vivo Raman Imaging Studies. ICR mice were anaesthetized and the dorsal hair was shaved. AuNSCy7 (5 nM in 200 µL distilled water) was subcutaneously injected at the dorsum. FPT (2.0 mL) or fructose solution (2.0 mL) was gently smeared on the dorsal skin of the mice. SERS spectra were collected using the handheld Raman detector at selected time points post-OCAs treatment. For non-invasively visualizing subcutaneous tumor, AuNS-Cy7 (0.2 nmol in 200 µL distilled water) was injected intravenously into mouse models bearing the C6 glioblastoma xenograft. At 24 h post-nanoprobe injection, the mice were anaesthetized and the tumor site was locally treated with FPT (2 ml) via gentle smearing. The handheld Raman detector was

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placed approximately 1.0 cm above tumor site. The SERS spectra were collected at selected time points postFPT treatment and the integration time for each measurement was 200 ms. Laser power was 400 mW. The white light images of the tumor-bearing mouse before and at 30 min after FPT treatment were also captured. Ex vivo Confocal Raman Spectroscopy and TEM Studies. At 24 h after intravenous injection of AuNS-Cy7, mouse specimens with subcutaneous C6 glioblastoma xenograft were anaesthetized and perfused with saline and then paraformaldehyde (4%). The tumor was excised and then cyro-sectioned with a thickness of 200 µm. The Raman spectroscopic images of the tumor sections were collected by measuring the intensity of characteristic Raman peak at 509 cm-1 with 10 µm steps with an incident laser wavelength of 785 nm, laser power of 80 mW, and acquisition time of 1.0 s. The ultra-thin tumor sections (150 nm) were also prepared and used in TEM (Tecnai, G2spirit Biotwin, FEI, USA) imaging studies. Side-effects of Dermal OCA Treatment and AuNS-Cy7. White-light images of dorsal skin in nude mice before and at selected times post-FPT treatment and injection of AuNS-Cy7 were captured. At 7 and 21 days post-FPT dermal treatment, murine skin, liver and kidney tissues were harvested and cyro-sectioned with a thickness of 5 µm and then stained with hematoxylin and eosin (H&E) for pathological analysis.

3. RESULT AND DISCUSSION Design, Synthesis and Characterization of AuNS-Cy7. Surface-enhanced resonance Raman scattering (SERRS) occurs when the electronic excitation energy of the reporter molecules attached to a nanoparticle surface is close to that of incident laser, which results in more than 3 orders of magnitude signal enhancement compared to SERS.22 Due to its good biocompatibility and similar electronic excitation wavelength to the incident laser (785 nm) equipped in most handheld Raman detectors, heptamethine cyanine derivative Cy7-SH, with a maximum absorption at 790 nm, was chosen as the reporter molecule (RM). Meanwhile, gold nanostar (AuNS) was selected as the SERRS substrate because it possesses numerous sharp tips. They remarkably enhance Raman signal via a “hot spot” effect induced by tremendous enhancement of the local electromagnetic field.23

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Figure 2. Synthesis and characterization of SERS nanoprobe AuNS-Cy7. (A) Synthetic procedure of AuNS-Cy7. (i) n-BuOH/toluene, 120 ℃, (ii) lipoic acid, DCC/TsOH, dichloromethane, 70 ℃, (iii) sodium citrate/H2O, 100 ℃, (iv) silver nitrate, ascorbic acid, r.t., (v) Cy7-SH, r.t., (vi) mPEG-SH, r.t.. (B) TEM images of AuNS-Cy7. Red arrows indicate to PEG coating. (C) SERS signal increased linearly with AuNS-Cy7 concentration in a range of 5−400 fM (R2 = 0.9995). (D) Confocal Raman spectroscopic images of aqueous AuNS-Cy7 solutions at concentrations of 5, 20, 50 and 100 fM. Scale bar = 8.0 µm. (E) Representative Raman spectrum of AuNS-Cy7 in aqueous solution (5.0 nM). The characteristic twin peaks are pointed with arrows. r.t.: room temperature, DCC: dicyclohexylcarbodiimide. The covalent coating of Cy7-SH on the AuNS surface via Au−S bond kept the RM in proximity to the rough gold surface for maximizing the SERS effect. Polyethylene glycols (PEGs, 2.0 kDa) were fabricated on

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the AuNS surface to improve the Au colloidal stability and prolong the circulation lifetime of the nanoprobes.24 Figure 2A illustrates the synthetic procedures of SERS nanoprobe AuNS-Cy7. Briefly, the reaction of 1-(2-hydroxyethyl)-2,3,3-trimethyl-3H-indol (1) and 1-cyclohexene-1-carboxaldehyde in a mixture of 1-butanol/toluene (7:3, v/v) yielded heptamethine cyanine derivative (2). The conjugation between compound 2 and lipoic acid with dicyclohexylcarbodiimide (DCC) as dehydrating agent and p-toluenesulfonic acid as catalyst gave the sulfydryl group modified molecular reporter Cy7-SH (3). The gold nanosphere acted as gold seed (4) and gold nanostar (AuNS) (5) were prepared according to a previously reported seed-mediated growth method.21 The modifications of Cy7-SH and PEG-SH on the AuNS surface gave the aiming nanoprobe AuNS-Cy7. Both naked AuNS and AuNS-Cy7 showed a maximum absorption centered at ~850 nm that was assigned to the longitudinal plasmon band (LPB) of the mono-dispersed AuNS and indicated a localized surface plasmon resonance (LSPR) in the NIR wavelength range (Figure S1). The molar concentration of the nanoprobe was determined by nanoparticle tracking analysis (NTA) that directly counted the nanoparticle number in a unit volume by an optical microscope equipped with a high-speed CCD camera. A good linear regression between the maximum absorption and molar concentration of AuNS-Cy7 in aqueous solutions was observed in Figure S2 (from 8 pM to 157 pM, R2 = 0.9997). The extinction coefficient of the AuNS-Cy7 was determined as 8.2  109 M-1 cm-1 according to the Lamber-Beer equation. The covalent conjugation of Cy7SH to the AuNS surface was verified by FT-IR spectra (Figure S3). AuNS-Cy7 demonstrated characteristic FT-IR peaks of Cy7-SH, including 2926 cm-1 (υCH2 ), 1735 cm-1 (υC=O), 1550 cm-1 (υC=C), 1393 cm-1 (δCH), 1141 cm-1 (υC-O) and 712 cm-1 (υC-Cl), which indicated the successful conjugation of Cy7-SH on AuNS. TEM images (Figure 2B, Figure S4) showed both mono-dispersed AuNS-Cy7 and AuNS possessing a highlybranched morphology and nearly same particle size distribution. A semi-transparent coating with a thickness of about 5.0 nm was observed in the TEM images of AuNS-Cy7 but not in those of AuNS, which indicated the PEG layer was modified on the nanoprobe (Figure S5). The diameters of AuNS-Cy7 and AuNS were found to be between 55 and 70 nm with an average of 60 nm. Each nanoprobe contained 6−16 prolate tips with an average of 10. The tip lengths were between 15 and 25 nm with an average of 20 nm. The number of the “protruding tips” has been reported to correlate proportionally with the excitation cross section of the nanostar plasmons. The multiple tips enhance the local electromagnetic field and hence SERS signal of the

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nanoprobes.21, 25The hydrodynamic diameter and Zeta potential of AuNS-Cy7 were determined to be ~80 nm and +25.4 mV in aqueous solution, respectively (Figure S6−7). The sensitivity of AuNS-Cy7 was determined by measuring its Raman signal intensity as a function of concentration (Figure 2C). Raman signal of AuNSCy7 in aqueous solutions increased linearly within the femtomolar concentration range (from 5 fM to 400 fM, R2 = 0.9995). Confocal Raman spectroscopic images showed that the distinguishable Raman signal of AuNSCy7 could be detected even at concentrations as low as 5 fM (Figure 2D). Compared to the non-resonant SERS nanoprobes, the sensitivity of AuNS-Cy7 was enhanced by 2 orders of magnitude.26 Raman spectrum of AuNS-Cy7 showed characteristic twin peaks located in 509 and 541 cm-1 that might be assigned to the S−S vibration and C−S vibration (Figure 2E). Colloidal stability of the AuNS-Cy7 under physiological conditions was determined by measuring the time dependent Raman intensity under neutral (pH 7.4), acidic (pH 2.0), alkaline (pH 12) and highly saline (1.0 M NaCl) conditions (Figure S8). Less than 15% Raman signal attenuation was observed after 48 h incubation under all above conditions, which indicated good colloidal stability. The femtomolar sensitivity, robust colloidal stability and the spectral fingerprint offered the advantages of AuNS-Cy7 to visualize subcutaneous lesions by handheld Raman detector.

Detection of AuNS-Cy7 in a Simulated Turbid Tissue. The use of OCAs to assist in the identification of AuNS-Cy7 immersed in turbid “tissue” using a handheld Raman detector was investigated using a homemade apparatus comprised of a glass capillary mimicking a “vasculature” and a container holding a 5% intralipid medium mimicking the turbid tissue (Figure 3A). The glass capillary with syringe pump-driven AuNS-Cy7 was immersed into the intralipid medium that has been widely used as a light scattering medium due to its similar scattering characteristics to living tissue.27 While no Raman signal of AuNS-Cy7 could be detected when the immersion depth of the glass capillary exceeded 0.1 mm, this nanoprobe could be visualized as deep as 0.9 mm after addition of OCAs (Figure 3B, Figure S9). The efficacy of OCA to suppress light scattering of the turbid media was further evaluated at a glass capillary immersion depth of 0.4 mm. While only background noise was detected before the addition of OCAs, the fingerprint spectrum of AuNS-Cy7 was evident after addition of various OCAs including DMSO, glycerol, 1,2-propanediol or 1,4-butanediol into the intralipid medium (Figure 3C). Quantification of the characteristic peak (509 cm-1) of the nanoprobe showed

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that DMSO offered the best optical clearing effect. The Raman signal for DMSO group was 1.2, 1.6 and 2.0 times the intensity of that of glycerol, 1,4-butanediol and 1,2-propanediol, respectively (Figure 3D).

Figure 3. The optical clearing agent increases detectability of handheld Raman detector. (A) Illustration of the homemade apparatus in which a glass capillary mimicking a subcutaneous vasculature was immersed into an intralipid medium (5%) mimicking the turbid skin tissue. The flowing AuNS-Cy7 aqueous solution in the glass capillary was driven by a syringe pump. (B) Raman signal intensities of AuNS-Cy7 as functions of immersion depth and OCAs added to the intralipid medium. Raman spectra (C) and Raman signal intensities (D) of AuNS-Cy7 in intralipid medium with absence or presence of OCAs. The capillary was placed at 0.4 mm below the medium surface. The characteristic twin peaks are highlighted by a red rectangle. The peak at 509 cm-1 was used for signal intensity quantification purposes. PG: 1,2-propanediol, BDO: 1,4-butanediol. Above studies revealed that OCAs effectively suppressed light scattering and increased the tissue imaging depth of the handheld Raman detector. No matter whether exogenous probes or endogenous biomolecules are visualized in subcutaneous lesions, the photonics from the excited laser first need to penetrate dermal tissue, complete inelastic collision with the target molecule and travel through the skin again to reach the detector. However, the scattering particles such as collagens, elastic fibers, intracellular organelles with high refractive indexes (RI) and their surrounding interstitial fluid, cytoplasm media with low RI make the incident light traveling with heterogeneous speeds and angles in turbid tissue, which leads to strong light scattering. The penetration of OCAs with high RI into turbid tissue helps to match the RIs between the scatters and the surrounding media, and therefore reduces the scattering coefficient. To further elucidate the mechanism of the

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OCA to enhance imaging sensitivity of the nanoprobe immersed into the turbid intralipid medium, a simple approximation of Mie calculation was used as presented below27:  .

 1 −  = 3.28 





 − 1 . 1 <  ≤ 1.1, 5