Optical detection of gold nanoparticles in a prostate-shaped … · Optical detection of gold nanoparticles in a prostate-shaped porcine phantom Serge Grabtchak, a,b,c Elena Tonkopi,

Optical detection of gold nanoparticles ina prostate-shaped porcine phantom

Serge GrabtchakElena TonkopiWilliam M. Whelan

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Optical detection of gold nanoparticles in aprostate-shaped porcine phantom

Serge Grabtchak,a,b,c Elena Tonkopi,d,e and William M. Whelana,f

aUniversity of Prince Edward Island, Department of Physics, 550 University Avenue, Charlottetown, Prince Edward Island C1A 4P3, CanadabDalhousie University, Department of Electrical and Computer Engineering, 1459 Oxford Street, Halifax, Nova Scotia B3H 4R2, CanadacDalhousie University, Department of Physics, 6310 Coburg Road, Halifax, Nova Scotia B3H 4R2, CanadadQEII Health Sciences Centre – Victoria General Hospital Site, Department of Diagnostic Imaging, 1276 South Park Street, Halifax,Nova Scotia B3H 2Y9, CanadaeDalhousie University, Department of Radiology, P.O. Box 9000, Halifax, Nova Scotia B3K 6A3, CanadafUniversity of Prince Edward Island, Atlantic Veterinary College, Department of Biomedical Sciences, 550 University Avenue, Charlottetown, PrinceEdward Island C1A 4P3, Canada

Abstract. Gold nanoparticles can be used as molecular contrast agents binding specifically to cancer sites and thusdelineating tumor regions. Imaging gold nanoparticles deeply embedded in tissues with optical techniques pos-sesses significant challenges due to multiple scattering of optical photons that blur the obtained images. Both diag-nostic and therapeutic applications can benefit from a minimally invasive technique that can identify, localize, andquantify the payloads of gold nanoparticles deeply embedded in biological tissues. An optical radiance technique isapplied to map localized inclusions of gold nanorods in 650- to 900-nm spectral range in a porcine phantom thatmimics prostate geometry. Optical radiance defines a variation in the angular density of photons impinging on aselected point in the tissue from various directions. The inclusions are formed by immersing a capillary filled withgold nanorods in the phantom at increasing distances from the detecting fiber. The technique allows the isolation ofthe spectroscopic signatures of the inclusions from the background and identification of inclusion locations in theangular domain. Detection of ∼4 × 1010 gold nanoparticles or 0.04 mgAu∕mL (detector–inclusion separation10 mm, source–detector separation 15 mm) in the porcine tissue is demonstrated. The encouraging results indicatea promising potential of radiance spectroscopy in early prostate cancer diagnostics with gold nanoparticles. © 2013

Society of Photo-Optical Instrumentation Engineers (SPIE) [DOI: 10.1117/1.JBO.18.7.077005]

Keywords: spectro-angular mapping; gold nanoparticles; gold nanorods; localized inclusion; radiance spectroscopy; optical contrastagents; porcine phantom; deep tissue imaging; interstitial fiber-optic diagnostics; prostate cancer.

Paper 130136PRR received Mar. 8, 2013; revised manuscript received Jun. 18, 2013; accepted for publication Jun. 20, 2013; publishedonline Jul. 17, 2013.

1 IntroductionNanotechnology utilizes nanoscale-size materials that offer newattractive properties not available in bulk form and is positionedto revolutionize cancer medicine. In a plethora of existingorganic and inorganic nanoparticles (NPs), gold NPs (Au NPs)enjoy an unprecedented growth in popularity. For cancer appli-cations, the use of Au NPs has advanced along several majorfronts covering both diagnostic and therapeutic aspects: (1) inter-stitial NP-mediated laser thermal therapy,1,2 (2) cancer diagnos-tics via delineation of cancer affected areas with NPs conjugatedto monoclonal antibodies,3–5 (3) enhanced radiation sensitivityand toxicity in cancer cells containing Au NPs,6 (4) drug deliv-ery vectors.7–9 Such functional diversity even prompted coininga special term, the theranostic NP meaning that a NP combinesboth therapeutic and diagnostic properties and this duality ena-bles simultaneous or sequential applications of imaging andtherapeutic modalities.

Immunogold labeling reported by Faulk and Taylor10 in 1971can be considered one of the first applications of Au NPs in bio-medical imaging. Nowadays, a number of imaging techniqueshave benefited from the use of Au NPs. In particular, with Au

NPs serving as contrast agents computed tomography (CT) wasshown to demonstrate molecular specificity toward certain typesof cancer.4,11,12 Since X-ray photons do not scatter as much asoptical photons, CT delivers good image quality thus allowingexcellent deep tissue imaging capabilities. While providingobvious benefits for nonoptical imaging modalities, Au NPs canbe regarded as a disruptive technology because none of theexisting clinical medical imaging modalities are light-based13

and thus, cannot explore the primary strengths and benefits ofAu NPs. As opposed to nonoptical imaging modalities, opticaltechniques exploit unique optical properties of Au NPs (plas-mon enhanced scattering and absorption cross-sections leadingto an enhanced optical contrast14) which differentiate Au NPsfrom other chromophores used as contrast agents. In addition,optical diagnostics may offer a number of advantages comparedto traditional medical modalities: it uses nonionizing radiation,provides biochemical information via spectroscopic identifica-tion, allows the control of light penetration depth in tissues viawavelength selection and offers more portable and less expen-sive technical solutions. While there has been significantprogress in using optical microscopy for cellular-level imagingby labeling cancer cells with Au NPs3,15 deep tissue optical im-aging and cancer diagnostics using Au NPs for delineatingmalignant regions do not share the same degree of success. ItAddress all correspondence to: Serge Grabtchak, University of Prince Edward

Island, Department of Physics, 550 University Avenue, Charlottetown, PrinceEdward Island C1A 4P3, Canada. Tel: +(902)566-6078; Fax: +(902)566-0483;E-mail: [email protected] 0091-3286/2013/$25.00 © 2013 SPIE

Journal of Biomedical Optics 077005-1 July 2013 • Vol. 18(7)

Journal of Biomedical Optics 18(7), 077005 (July 2013)


http://dx.doi.org/10.1117/1.JBO.18.7.077005






is mainly due to the detrimental impact of multiple scattering ofphotons on the image quality that results in a loss of resolutionand image blurring16 making deep tissue imaging a formidabletask. In some cases, optical diagnostics can be performed in anoninvasive way by confining both illumination and detection tosuperficial layers [as in a diffuse optical tomography (DOT)].16,17

However, even with a few cm of penetration depth some regionsin a human body are difficult to reach (e.g., prostate) and one hasto resort to interstitial illumination and detection while stillattempting to minimize the degree of invasiveness. Regardlessof the applications, an ability to identify, locate, and quantifyfixed loads of Au NPs in the prostate gland via noninvasiveor minimally invasive methods would be highly beneficial.18

For prostate cancers, typical applications of Au NPs includeinterstitial NP-mediated laser thermal therapy,19 enhanced radi-ation sensitivity and toxicity in prostate cancer cells containingAu NPs,20 and brachytherapy implants of radioactive 198Au NPsplaced into a prostate gland to deliver the radiopharmaceuticaldose directly to the tumor site.21 In addition to the therapeuticapplications listed above, Au NPs can be used for prostatecancer diagnostics or imaging purposes. To achieve it, eitheractive targeting of Au NPs conjugated to monoclonal antibodiesor passive targeting by relying on the enhanced permeability andretention effect can be employed. Both procedures will lead tothe accumulation of Au NPs in tumor-affected areas inside theprostate gland.

Rather than battling optical scattering and employing multi-ple source–detector configurations (as in DOT) to detect AuNPs-based inclusions, we have pursued a different approach.By employing a single light source and a directional detector(based on optical radiance), we relied on optical scattering todeliver information on localized inclusions of Au NPs deeplyembedded in tissues to the detector. Using the optical radiancemethod,22 we detected localized inclusions of Au NPs, identifiedtheir spectroscopic signatures, and localized them in the angulardomain, presenting information in a compact form of spectro-angular maps.23,24 The approach has been tested extensively onIntralipid-based liquid phantoms that have scattering propertiessimilar to biological tissues. However, actual biological tissuesdemonstrate various degrees of heterogeneity (as opposed tohomogenous Intralipid-based phantoms) that may affect thedetection procedure.

In this work, we have applied, for the first time, the devel-oped approach to actual biological tissues aiming to detectinterstitially Au NPs deeply embedded in tissues in a prostate-relevant geometry for a porcine-based phantom.

2 Material and MethodsOptical radiance defines a variation in the angular density ofphotons impinging on a selected point in tissue from variousdirections. To obtain radiance data, a specially constructed opti-cal probe with a well-defined angular detection window must berotated along its axis. A schematic of the experimental setup isshown in Fig. 1(a). A tungsten halogen white light source(20 W) provided uniform tissue illumination through a fiberwith a 2-mm spherical diffuser at the end. The total power outputfrom the spherical diffuser was estimated to be ∼18 mW.Detection was performed with a 600-μm side-firing fiber (theradiance detector). The side-firing fiber (Pioneer Optics) wasproduced by polishing a fiber tip at ∼43- deg angle that providesa total internal reflection at the glass/air interface. When coupledto a light source, this fiber would emit light sideways at an angleclose to 90 deg within a well-defined angular aperture of∼10 deg in water [Fig. 1(b)]. The fiber can also be used forside light collection as in the current work. To prevent any sec-ondary rays that do not undergo the total internal reflection tocouple back to the fiber from other directions, a cap from a heat-shrink white polymer tube with a hole aligned to the entrancewindow was placed at the end of the side-firing fiber. Both illu-minating and detecting fibers were threaded through 15-gaugeneedles for mechanical stability. The side-firing fiber wasmounted on a computer-controlled rotation stage (Thorlabs).Radiance data were acquired by rotating the side-firing fiberover a 360-deg range with a 2-deg step. The side firing fiberwas connected to a computer-controlled USB 4000 spectrometer(Ocean Optics) that collected spectra at every angular step.Depending on the number of averages, it took between 4 and8 min to acquire a complete single angular profile.

The porcine phantom was prepared by cutting a prostate-sizepiece (∼4 cm diameter, ∼3 cm thick) from pork (loin) withsome representative images shown in Fig. 2. The phantom wasplaced into a black wall plastic holder to keep the cylindricalshape and provide absorptive type boundaries [as seen inFig. 2(a)]. Prior to inserting the illuminating and detecting fibers

Fig. 1 (a) Schematic of experimental setup for optical radiance measurements. (b) A light cone coming from the side-firing fiber.


Grabtchak, Tonkopi, and Whelan: Optical detection of gold nanoparticles in a prostate-shaped porcine phantom


and the capillary with Au NPs solution, holes were made in thedesired locations in the phantom [Fig. 2(b)].

In most experiments, a localized inclusion was formed byfilling a 2-mm diameter quartz capillary with ∼0.1 mL ofunconjugated Au nanorods water solution (Nanopartz). On asingle occasion indicated in the text, the inclusion was formedby syringe injection of 0.1 mL of the solution halfway into thetissue. Two types of Au nanorods were used: (i) 12 nm diameter,35 nm length, 5.7 × 1011 particle∕mL, 0.04 mgAu∕mL,700 nm plasmon resonancewavelength [Fig. 3(a)] and (ii) 12 nmdiameter, 48 nm length, 4 × 1011 particle∕mL, 0.04 mgAu∕mL,756 nm plasmon resonance wavelength [Fig. 3(b)]. The molarabsorption and scattering coefficients (at the wavelength of plas-mon resonance) were as follows: 8.09 × 108 M−1 cm−1 (absorp-tion) and 8.99 × 107 M−1 cm−1 (scattering) for 700-nm Au NPs;

4.6 × 108 M−1 cm−1 (absorption) and 9.4 × 107 M−1 cm−1

(scattering) for 756-nm Au NPs. Thus, all particles can be con-sidered as dominant absorbers. The tube was inserted at the fulldepth into the phantom at a selected location as can be seen inFig. 2(c). Measurements were performed in the dark [Fig. 2(d)].

For an average prostate with a diameter of 4 to 5 cm, illumi-nating through the rectal wall and detecting with a urethra-placed fiber will ensure that the prostate is probed in a minimallyinvasive way. Figure 4 shows a top-view cross-section of theporcine phantom with positions of the detecting fiber, illuminat-ing fiber and Au NPs-based inclusion (i.e., the target) along witha superimposed contour of a virtual prostate. The selected loca-tion of the Au NPs target at −90 deg corresponds to the mostdistant probing of the prostate when the targeted area is locatedon the periphery of the prostate. Detecting Au NPs in this

Fig. 2 (a) Porcine phantom in a plastic holder, (b) porcine phantom with holes prepared for inserting the fibers and the capillary tube, (c) porcinephantom in the experimental setup with the detecting fiber (in the middle), the illuminating fiber (in the metal holder) and the capillary tube with AuNRs at −90 deg angle, and (d) porcine phantom during measurements.

Fig. 3 (a) Absorption spectrum of Au nanorods (35 nm length) with a plasmon resonance at 700 nm. (b) Absorption spectrum of Au nanorods (45 nmlength) with a plasmon resonance at 756 nm.




location was of primary goal in the current work. One cansee that for a relevant prostate geometry, source–detector anddetector–target separations usually do not exceed ∼2 cm. Theseconsiderations determined the geometry for measurements inthe phantom with the Au NPs target. Since the distance betweenthe rectum and urethra is fixed and puncturing the prostate ishighly undesirable in a minimally invasive detection, wefixed the separation between the source and the detector at15 mm. With the fixed concentration of Au NPs in the target,the target was incrementally moved along −90 deg directionaway from the detector toward the periphery until the measuredsignature of the target was lost. Hence, our aim was to detect theAu NPs based inclusion of the fixed concentration as close aspossible to 2-cm separation as in Fig. 4.

3 Results

3.1 Spectro-Angular Mapping of Light Distribution inthe Bare Porcine Phantom

To test the limits of light transmission in the porcine phantomand get its spectral characteristics, the source and the detectingfibers were inserted along the diameter of the phantom and eachwas 5 mm away from the outer boundaries. Such geometryresulted in the largest possible source–detector separation(i.e., 30 mm) and allowed testing illumination conditions forthe most distant regions of the phantom. Detection of AuNPs based inclusions depends greatly on the ability to provideadequate illumination in the inclusion’s location which could beanywhere in the phantom. Prior to introducing the localizedinclusion, it is desirable to know if the entire phantom is acces-sible to illumination. Extending the distance between the sourceand the detector almost to the size of the phantom corresponds tothe extreme possible separation and provides the necessarygeometry for obtaining the answer. Due to physical limitations,it was not possible to position both fibers directly at the circum-ference of the phantom which would give a 40-mm source–detector separation.

Radiance (I) is a function of the source–detector separation(r), the angle between the direction of propagation and directionof scattering (θ) and the wavelength of light (λ). Individualspectral profiles of radiance, measured every 2 deg over360 deg, were combined into the phantom spectro-angular

matrix IPhantomðr; θ; λÞ with columns corresponding to wave-lengths from 650 to 900 nm and rows corresponding to anglesfrom −178 deg to 178 deg. In a similar way, the water matrixIWater0ðr; θ; λÞ was assembled in which the same spectrum ofradiance measured at 0 deg in water was replicated for allrows (i.e., angles) with the subscript “0” emphasizing it.Constructing a ratio of these two matrices eliminates spectralresponses from the white light source, illuminating anddetecting fibers in addition to referencing phantom measure-ments to those in water. This procedure is similar to referencingto a blank in spectroscopic measurements. The ratio ofIPhantomðr; θ; λÞ∕IWater0ðr; θ; λÞ focuses on transmission proper-ties of the porcine phantom, has a clear physical definition andcorresponds to relative radiance that can be converted to theabsolute value with a proper normalization coefficient.24 Theratio is presented as a contour plot in Fig. 5(a) and demonstrateslight distribution in the porcine phantom measured in the loca-tion most distant from the light source. The maximum transmis-sion of light is marked with red and occurs in the forwardscattering direction (i.e., along 0 deg when the detector is facingthe light source). The relatively strong signal indicates that thecombination of the white light source and the spherical diffuserprovides adequate tissue illumination as far as 30 mm awayfrom the source. The highest transmission is observed for wave-lengths around 810 nm. In the angular domain, light remainsrelatively well localized such that photon density drops downto ∼30% of the total density in the backscattered direction(i.e., 178 deg). This is drastically different from the spectro-angular map of Intralipid-1% measured for the same 30-mmsource–detector separation where the intensity dropped downto only ∼80% of the total value.24 The origin of better angularconfinement of light is due to the presence of additional chro-mophores in the porcine phantom that increase absorption forlong-travelled photons in the backscattering direction.

Spatial distribution of light inside turbid media is a wave-length-dependent quantity based on absorption and scatteringof individual chromophores. While the presence of particularchromophores depends on the nature of biological tissue, themost significant chromophores in biological tissues (includingporcine phantoms) are oxygenated and de-oxygenated hemoglo-bin, water, and lipids. A combination of absorption spectra ofchromophores as well as scattering defines the most usable opti-cal range (the biomedical transparency window) where light canpropagate the most. To understand the nature of the spectralchanges from Fig. 5(a), the spectrum corresponding to 0 degwas plotted in Fig. 5(b) along with reciprocal molar extinctioncoefficients (mm−1 M−1) for oxygenated (blue) and de-oxygen-ated (red) hemoglobin.25 In the absence of knowledge of con-centration values for individual chromophores, the overlap ofblue and red curves from Fig. 5(b) agrees qualitatively withexperimentally measured radiance along 0 deg which gives thetransmission spectrum of the porcine phantom. The maximumtransmission occurs close to the isosbestic point around 800 nmwhile the dip at 760 nm is brought by the feature from Hb curve.The steep drop toward long wavelengths can be explained byincreasing water absorption (absent in the plot). The relativeradiance curve indicates that the optimal optical transmissionwindow for the porcine sample would be between ∼770 and∼850 nm. However, it should be noted that for in vivo tissuesblood circulation may change the ratio between oxygenated andde-oxygenated hemoglobin thus shifting the most optimal wave-length toward 690 nm.

Fig. 4 A conceptual top-view diagram of the porcine phantomwith illu-minating and detecting fibers and Au NPs based inclusion (the target) inthe most distant area at −90 deg. The virtual prostate contour is out-lined with a green dashed line.




3.2 Spectro-Angular Mapping of Au NPs Inclusion inthe Porcine Phantom

Inserting the Au NPs localized inclusion into the porcinephantom perturbs the original light distribution. It was shownearlier22 that out of two qualities offered by plasmon NPs(increased absorption and/or increased scattering), distant mea-surements in turbid media can detect only a decrease in a signaldue to locally increased absorption thus excluding NPs withmostly scattering properties from a list of possible contrastagents. This is distinctly different from very thin slices of tissuesused in optical microscopy and OCT measurements (up to fewmms) where reduced light scattering can support the use of bothabsorption- and scattering-based contrast agents. Radiance canlocalize the Au NPs based inclusion via the extinction ratioapproach that effectively cancels the contribution from the tur-bid medium and ensures that any signal constructed from theratio comes from the presence of the Au NPs only.22–24,26

The extinction ratio approach relies on obtaining two datasets, for the phantom with the inclusion (or target)IPhantomþtargetðr; θ; λÞ and for the phantom without the inclusion,IPhantomðr; θ; λÞ. Then, the radiance extinction ratio (RER) isdefined as RERðr;θ;λÞ¼IPhantomðr;θ;λÞ∕IPhantomþtargetðr;θ;λÞ.This ratio emphasizes attenuation of light in the phantom withthe inclusion such that values of RER > 1 map regions of theincreased extinction (relative to the bare phantom) in the spectraland angular domains.

In all experiments with localized inclusions in the phantom,the source–detector separation was kept at 15 mm to be consis-tent with a typical urethra-rectum distance in the human pros-tate. Initially, the inclusion was placed at a 5-mm distance fromthe detector at −90 deg angle according to geometry shown inFig. 4. The spectro-angular map of RER(5, −90 deg, λ) for theinclusion formed with 756-nm plasmon resonance Au NPs sol-ution is shown in Fig. 6(a). Regions of high extinction aremarked by red color and low extinction by the blue. The maxi-mum of RER in the angular domain occurs around −85 deg[dotted line in Fig. 6(a)] and points to the direction of the actual

position of the inclusion, i.e., −90 deg. Considering �5 deg

positioning accuracy, the agreement between the values isgood. (High values of RER observed for ∼180 deg for almostall wavelengths and marked by red color is an artifact caused bythe fiber dragging the tissue while rotating.) In the spectraldomain, the maximum of RER occurs near 750 nm. The spec-trum taken along the dotted line is plotted in Fig. 6(b). The plas-mon resonance around 750 nm allows for a clear identificationof the Au NPs absorption spectrum that is presented in Fig. 3(b).

Due to the perturbative nature, the inclusion produces locallyincreased absorption in the background medium causing anincrease in RER values. Introducing the localized absorberdecreases the density of photons that originate from the source,pass through the inclusion’s location (in the absence of theinclusion), and get collected by the detector. Thus, the inclusionessentially allows to tag the photons that would have normallybeen collected by the detector but are removed by the interactionwith the inclusion. Due to multiple scattering, the absorptiveinclusion removes photons from the area that is larger thanphysical dimensions of the inclusion. It corresponds to angularbroadening when detection is performed in the angular domain.However, the maximum of interaction between the absorber andphotons occurs in the physical area occupied by the inclusion.Hence, the inclusion can be localized in the angular domain byidentifying the angle that corresponds to highest values of RERin spectro-angular maps.

When the phantom was measured with 700-nm Au NPsbased inclusion, a pattern shown in Fig. 7(a) was obtained.(Note that two different phantoms were used for testing thetwo types of Au NPs.) The spectrum taken along the anglecorresponding to the inclusion’s direction is shown inFig. 7(b). The position of the peak matches the one fromFig. 3(a) for the 700-nm Au NPs absorption spectrum.However, some new features can be seen in Fig. 7(a). In addi-tion to the main peak in RER observed at −90 deg, two morepeaks of smaller intensity are delineated by the contour lines:one at −130 deg and the other at −15 deg. Since this

Fig. 5 (a) Spectro-angular plot of IPhantom∕IWater0 (¼relative radiance) for 30-mm source–detector separation in the porcine phantom. (b) Transmissionthrough 30 mm of the porcine phantom [black, corresponds to the spectrum at 0 deg from (a)], oxygenated (blue) and de-oxygenated (red) hemoglobinfrom Ref. 25.




experimental setup was shown earlier to produce single RERpeaks when applied for detection of localized inclusions inhomogeneous Intralipid phantoms, appearance of multipleRER peaks in the porcine phantom might be due to well-pronounced heterogeneity of the phantom. Indeed, Fig. 2(a)and 2(b) shows distinct regions of white-colored structuresincorporated between porcine muscle fibers. These structuresrepresent fat. Scattering on these structures would change theangular distribution of photons especially when photonsencounter them on the way from the inclusion’s location to

the detector. As a result, anisotropic scattering by localheterogeneities would occur at some preferential angles thatappear in spectro-angular maps as secondary or satellitepeaks. Since every piece of phantom has its own degree ofheterogeneity and local organization, the presence and angu-lar positions of such secondary peaks may differ from phan-tom to phantom.

Next, using the fresh phantom the inclusion was placed at10 mm distance from the detector keeping all other parametersthe same, and the measurements were repeated for both types of

Fig. 7 (a) Contour plot of the spectro-angular map of the RER of capillary formed Au NPs target with 700-nm plasmon resonance in the porcinephantom. (b) The spectrum corresponding to −90 deg angle. Source–detector separation 15 mm, detector–target separation 5 mm, target angle−90 deg.





Au NPs solutions. For this distance, the same phantom was usedfor measurements of both types of Au NPs (756 and 700 nm)with the capillary tubes inserted to the same location. Thespectro-angular map along with the spectrum measured alongthe angle of the maximum extinction is presented in Fig. 8(a)and 8(b) for 756-nm Au NPs and in Fig. 9(a) and 9(b) for700-nm Au NPs. The spectro-angular map for the 756-nm AuNPs inclusion [Fig. 8(a)] shows two peaks: the main peak ofhigh intensity around −90 deg pointing to the physical location

of the inclusion (dotted line) and the secondary peak of lowerintensity around 60 deg that can be linked to local hetero-geneities present in the sample. The spectrum taken along theangle corresponding to maximum extinction [Fig. 8(b)] showsthe characteristic plasmon peak around 750 nm and allows iden-tification of the inclusion although with reduced values of theRER and increased level of noise.

Replacing 756-nm Au NPs inclusion with 700-nm Au NPsinclusion resulted in expected changes in the spectral signature






of the inclusion by shifting the maximum of RER from∼750 nm to ∼700 nm [Fig. 9(a) and 9(b)]. However, the angularpattern remained virtually the same as in Fig. 8(a) demonstratingthe highest peak around −90 deg and the smaller peak around60 deg. It confirms the previously made assumption thatobserved secondary peaks are phantom specific and are pre-served for the same phantom.

To mimic the effect of actual in vivo distribution of Au NPs inbiological tissues, we injected Au NPs directly into the porcinetissue with a syringe. Such injections are known to produceirregular diffused distributions which depend on the tissue

morphology as well as injection procedure.27 The volume ofinjection was kept the same as the volume of the localized inclu-sion in the capillary tube. The spectro-angular plot for the 756-nm Au NPs based inclusion obtained by a direct injection to thephantom at −90 deg and 10 mm distance from the detector isshown in Fig. 10(a). Figure 10(b) contains the spectrum takenalong the angle corresponding to the maximum value of RER.Figure 10(a) demonstrates that in addition to the absolute maxi-mum of RER observed around −90 deg a few satellite peakscan be seen for RER at −135, −80, −55, −25 and 125 deg.While the location of the main maximum at −90 deg agreeswell with the injection point [shown with the dotted line inFig. 10(a)], all other maxima produce satellite signatures ofthe inclusion in some other directions. The spectrum taken alongthe direction of the main maximum (−90 deg) demonstrates anoticeable spectral broadening and a possible red shift in theposition of the plasmon resonance (from ∼756 to ∼770 nm).The spectra (not shown) taken along the directions of the sat-ellite peaks produce similar spectral signatures but with reducedRER values. Spectral broadening and a red shift indicate a pos-sible aggregation of NPs and the change of the dielectric envi-ronment around them, correspondingly. We believe that theorigin of satellite peaks observed in this measurement can belinked not only to local heterogeneities as discussed earlier butto the uneven spatial distribution of Au NPs. Since it is known27

that diffusion of the injected material (Au NPs in our case) pro-duces dendritic-like 3-D structures, such structures may contrib-ute to the multiangular signatures observed in the current work.

Once the position of the inclusion was moved to a 15-mmdistance from the detector, no signature of plasmon resonancesin spectro-angular maps could be detected indicating that theability to detect the presence and localize the inclusion in theangular domain was lost. We also checked all other angles inthe entire 360-deg interval around the detector, and detectioncapabilities with some differences were found to be similarto the reported values.

Fig. 10 (a) Contour plot of the spectro-angular map of the RER of syringe injection formed Au NPs target with 756-nm plasmon resonance in the porcinephantom. (b) The spectrum corresponding to −90 deg angle. Source–detector separation 15 mm, detector–target separation 10 mm, target angle−90 deg.

Fig. 11 Calibration plot adapted from Hainfeld et al.12 with added dual-scale axis on the right and superimposed with data from other authorsand current work. Dual scale on the right corresponds to 15-nm diam-eter Au spheres (labeled as “spheres”) and 12 nm × 48 nm Au nanorods(labeled as “rods”). The size of nanorods from Popovtzer et al.4 wasnearly identical to the one used in calculation.




4 DiscussionGiven nearly the same concentration of both types of Au NPs inall experiments (∼0.04mgAu∕mL or ∼4×1011 nanoparticles∕mL) the detection level of ∼4 × 1010 particles in a ∼0.1-mLvolume located 10 mm from the radiance detector wasdemonstrated in the porcine phantom. These numbers havevery important implications for diagnostic imaging with AuNPs as contrast agents. With functionalization by proper surfaceligands, Au NPs have been reported to be used as contrast agentsin CT to detect cancer in cell cultures and soft tissues.4,11,12 InCT imaging contrast medium increases X-ray absorption result-ing in the enhancement of anatomical structures due to higherattenuation. The concentration of Au NPs in the tumor areashould be high enough to produce a noticeable difference inattenuation between normal and malignant tissues. In fact, toproduce an easily measured contrast (∼160 HU) in soft tissues,a very high density of Au NPs (2.5 mgAu∕mL) was required.4 Inanother article, the loaded density of Au resulted in 190 HU con-trast for the tumor while the actual density of Au was notreported.11 In micro-CT microlocalization studies,12 1.82mgAu∕mL produced 159 HU in the whole tumor area while tumorperiphery exhibited 3.48 mgAu∕mL with 310 HU. We addedall these data to the calibration plot from Hainfeld et al.12

and presented the resulting plot in Fig. 11. Since sometimes itis informative to present a concentration of Au NPs rather than adensity of Au used, we also added the additional dual-scaledaxis on the right for the plot in Fig. 11. Two different scalesconverted the density of Au (mg∕mL) from the axis on the leftto the concentration (NPs∕mL) of 15-nm spherical Au NPs(used in Ref. 12) and 12 nm × 48 nm Au nanorods (used inRef. 4 and the current work). The concentration of Au NPsused in our experiments (0.04 mgAu∕mL) was lower by a fac-tor of ∼60 than 2.5 mgAu∕mL used in CT measurements4 andcould not be detected using clinical CT. To confirm it, the por-cine phantom with the inserted 3 mm capillary tube filled withAu NPs solution was imaged with CT (GE Discovery STE 16).Images were acquired using scanning parameters of 120 kVp,250 mAs, and a reconstructed slice thickness of 1.3 mm.

The measured attenuation of the porcine tissue (68 HU) wasvery close to that of human prostate (40 to 60 HU).28 The attenu-ation of the capillary with Au NPs solution was 3 HU, whichcorresponds to the water range (�4 HU). According to the cal-ibration plot in Fig. 11, 0.04 mgAu∕mL should give 3 HUwhich agrees well with our value presented as a red circle. Whilenot confirmed experimentally, it was estimated by Hainfeldet al.12 that minimum 0.5 mg∕mL of Au NPs was required toproduce the minimally detected contrast of 8 HU in soft tissues,which can be detected on a typical CT scanner. This value is stillhigher by a factor of ∼12 than the density used in our opticalexperiments. Thus, our results indicate that the presented radi-ance-based approach can detect much smaller concentrations ofAu NPs in soft tissues meaning that with the same labeling effi-ciency and the number of overexpressed receptors on the cellu-lar surface much fewer cells can be detected. Simple estimationsprovide the supporting numbers as follows. The most abundantcancer-associated cell surface targets, such as prostate-specificmembrane antigen (PSMA) are expressed at approximately 106

sites per cell.29 Considering a broad typical range of uptake val-ues of 104 to 105 Au NPs per cell, detecting ∼1010 Au NPs as inthe current work would correspond to detecting about 106 to 105

malignant cells in the prostate. These numbers are very close tothe ideal target of 105 malignant cells that corresponds to a

transition from diffusion-limited nutrition to neovascularizationlabeled as angiogenic switch.13 Hence, the technique may offer apromising potential in early cancer detection in the human pros-tate with Au NPs serving as cancer-specific molecular contrastagents. To extend the distance at which Au NP based inclusion isdetected, several steps can be implemented. First, to take fulladvantage of the knowledge of the biomedical transparency win-dow for a specific tissue (porcine, in the current study), the plas-mon resonance of Au NPs can be fine-tuned to the most optimalwavelength (∼820 nm for the porcine phantom) as can be seenfrom Fig. 5(b). Second, the photon budget in the tissue can beincreased by employing more powerful broadband near-infraredlight sources.

For prostate application, the initial step would be to take thereference measurement of the bare prostate before introducingAu NPs into the body of a patient. This measurement would bedone in a minimally invasive way by placing the radiance basedendoscope into the urethra and the illuminating fiber basedendoscope into the rectum. This is clinically feasible since thisstep would be very similar to standard intraurethral cystoscopyand transrectal ultrasound-guided prostate biopsy (TRUS-PB)procedures commonly performed in the urological office underlocal anesthesia (lidocaine) or without any anesthesia.30,31 Itwould take about 10 min or less to perform the complete radi-ance measurement which does not exceed typical times for per-forming cystoscopy. After administering Au NPs, it may take up6 h to see the accumulation in the targeted organ depending onthe delivery method, Au NPs surface preparation, and the tar-geted organ.32 Hence, we envision that measurements of the tis-sue with Au NPs can be performed after the selected timeinterval (i.e., 6 h) after the administration. Such time intervalsare not uncommon for clinical diagnostic procedures such as theradioactive iodine uptake test—RAIU (test is from 6 to 24 hafter uptake of radioactive iodine) or bone scan (test is from3 to 4 h after uptake of radioactive technetium). Also, in thetreatment of most cancers with the photodynamic therapy(PDT) the interval between drug and light administration canbe of the order of 24 to 72 h.33

While the current study demonstrates the 2-D map informa-tion, the proposed technique can be extended for 3-D applica-tions. For this purpose, the spherical diffuser in the light sourcehas to be replaced by a cylindrical diffuser to ensure a uniformillumination of the entire phantom (or prostate). Then, incre-mentally translating the detecting fiber in a vertical directionand collecting the spectro-angular map at every new positionwould generate a stack of 2-D maps assigned to a certaindepth inside the phantom. Such extension would provide addi-tional information about inclusion’s localization in a verticaldirection but will add extra time to the overall procedure.

Finally, we would like to add that in order achieve a completecharacterization of the Au NPs based inclusion, spectral iden-tification and the angular coordinate must be complementedwith the radial coordinate and a value of Au NP density. It isattractive to relate the measured values of RER to optical proper-ties of the localized inclusion in a similar way as was suggestedfor nonscattering inclusions (voids) in turbid media.26 LinkingRER to the absorption coefficient of Au NPs would allowextracting data on the concentration of Au NPs using theoreticalvalues of the absorption cross-section for a plasmon nanopar-ticle of known geometry. In order to obtain the precise relationbetween RER and optical properties of the localized inclusionfurther developments in the perturbation approach34,35 for




radiance are required. Alternatively, one can construct someempirical or semi-empirical relation between the concentrationand RER but it would require a priori knowledge of inclusion’slocation relative to the source and the detector. Indeed, the mea-sured values of RER depend not only on the concentration ofAu NPs but corresponding distances i.e., the detector–target sep-aration and the source–target separation. Varying the source–target separation would expose the same concentration of AuNPs to different illumination levels. Keeping the same source–target separation and varying the detector–target separationwould affect the collection efficiency of the detector. We arecurrently working on developing approaches to extract the dis-tance-related information from radiance measurements. Oncethe distance to the target is known, we will concentrate on con-structing an empirical relation between the measured parametersand the concentration of Au NPs.

5 ConclusionsThese proof of principle experiments demonstrated that a radi-ance-based approach can identify, through spectro-angular map-ping, the presence of Au NPs deeply embedded into porcinetissues (15 mm source–detector separation, 10 mm detector–target separation). The detection level of ∼4 × 1010 particlesin ∼0.1 mL volume corresponds to ∼0.04 mgAu∕mL or∼4 × 1011 NPs∕mL, that is lower by a factor of 60 than the con-centration of Au NPs required to produce a significant contrast(∼160 HU) relative to the typical soft tissue background level(20 to 70 HU) in CT images. The proposed method can beemployed for in vivo early diagnostics of prostate cancerwhen functionalized Au NPs bind specifically to cancer tissuesthus delineating cancer lesions.

AcknowledgmentsThe authors acknowledge financial support from NSERC,CIHR, and AIF.

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