current biomedical 
 nanotechnology has been aimed at effectively 
 imaging and characterizing abnormal biological processes at the cellular 
 or even molecular level in living subjects . successful progresses in the fundamental molecular 
 imaging research including instrumentations and image construction / registration 
 techniques greatly motivate the innovations of imaging probes . as 
 a relatively new molecular imaging technique , photoacoustic imaging 
 ( pai ) pai takes advantage of individual strengths of 
 both optical and acoustic imaging while largely overcoming the weaknesses 
 associated with each modality , providing functional and molecular 
 information of abnormalities with deep tissue penetration , high sensitivity , 
 and excellent spatial resolution . in addition 
 to several endogenous contrasts ( such as melanin and hemoglobin ) , various light - absorbing nanoparticles ( including 
 au - based nanostructures , carbon nanotubes , and nanodroplets ) have 
 been developed for pai contrast enhancement . although there are some nanoparticle - based exogenous agents particularly 
 promising for pai , their potential toxicity is still 
 under investigation , and the improvement of their in vivo behavior remains a challenge since most attempts to enhance the 
 contrast effect compromise their pharmacokinetic profile . as 
 one class of strong optical - absorption nanomaterials , colloidal 
 metallic nanostructures ( nps ) have recently attracted significant 
 attention from diverse disciplines for many biomedical applications . many synthetic methods have been developed for the construction 
 of a variety of plasmonic nanostructures with controllable size and 
 uniform shape ( for instance , triangles , prisms , rods , cubes , shells , 
 stars , and cages ) , largely because the effect of 
 induced - shape anisotropy of these nanostructures results in the splitting 
 of the underlying surface plasmon enhancement ( spr ) into several shape - dependent 
 modes , which could be accordingly tuned within the near - infrared window 
 ( nir , 650950 nm ) . as representative anisotropic nanogeometries , au - nanorods and -nanocages 
 have shown great promise as pai contrasts ; however , they generally 
 have relatively large particle sizes ( 50 nm ) , which could 
 result in unfavorable in vivo behavior and severely 
 limit their application for targeted cancer imaging . by making a compromise by trading contrast 
 effects for the ability to have preferable in vivo behaviors , strenuous efforts have been made recently to improve 
 their targeting efficiency , and to control pharmacokinetics and biodistributions 
 through variations of their sizes , shapes , and surface properties . although some metallic nanostructures with complex shapes showed desired 
 tumor targeting efficiency without compromising optical properties , 
 great challenges still remain in the precise control of their geometries 
 and monodispersity . recently , many colloidal hybrid nanostructures with sophisticated 
 architectures have been constructed from multiple functional components 
 either assembled using linkers or fused together by solid - state interfaces . in order to build novel hybrid nanostructures 
 with optimal structure and morphology for spr , we herein developed 
 a synthetic strategy to construct a series of anisotropic gold - based 
 nanomultipods , including dipods , tripods , and tetrapods ( figure 1 ) , with improved yield and excellent quality in 
 a predictable , controlled , and stepwise manner . among them , the gold 
 tripods ( au tripods ) have relatively small sizes with a narrow size 
 distribution and display stringently controlled morphology and , more 
 importantly , have well - defined absorptions in visible and nir regions . as a proof of concept , we further conjugated v3 integrins targeting the peptide , cyclic c(rgdfc ) peptide , to pegylated au - tripods ( rgd - au - tripods ) and 
 used this novel nanoprobe as a pai contrast agent to image tumor angiogenesis . 
 the tumor targeting efficacy and in vivo profile 
 of pegylated au - tripods ( modified with different sizes of pegs and/or 
 rgd ) labeled with radionuclide cu were evaluated in a 
 subcutaneous v3-positive u87 mg 
 glioblastoma xenograft model using small animal positron emission 
 tomography ( pet ) . finally , pai was performed to investigate the targeting 
 and imaging performance of rgd - au - tripods as photoacoustic contrast 
 agents . pt dumbbell 
 nps , au - dipods , au - tripods and au - tetrapods ) . ( a ) schematic showing 
 the stepwise syntheses of various gold multipods via a set of known 
 nucleation reactions and epitaxial growth processes . various gold 
 multipods are modeled by lumerical fdtd solution ( lumerical solution 
 inc . ) . considering the regioselectivity , several possible regioisomers 
 are shown in the bottom - left panel . hrtem images of representative 
 gold multipods are shown in the bottom - right panels . by 
 sequentially applying a set of nucleation reactions and epitaxial 
 growth processes , we successfully built up four gold - based nanostructures , 
 comprising au pt dumbbell , dipods , tripods , and tetrapods , 
 with predetermined composition and morphology ( figure 1a ) . in this structure - guided approach , we chose colloidal 
 platinum ( pt ) nps as core seeds ( or starting materials ) because they 
 are highly monodisperse and their sizes can be facilely tuned from 
 3 to 7 nm , and more importantly , they have 
 definite and rigid cubic shapes ( supporting information [ si ] , figure s4 ) . in order to tune the sizes of pt nanocrystals , 
 we applied stepwise seed - mediated growth processes to obtain monodisperse 
 pt nanocubes ( si section c.1.1 . and 4.9 nm pt nps with truncated cubic shapes could 
 preferably grew into thermodynamically more stable morphologies ( cubic 
 6.5 nm pt nps ) after an extra seed - mediated growth process . similarly , 
 7.5 nm pt nps were obtained when 5.8 nm pt nps were used as seeds . all pt nanocubes have a very narrow size and shape distribution ( figure 
 s5 and table s2 ) . due to excellent lattice coherence between pt and 
 au crystals in the fcc phase , the epitaxial growth of gold preferentially 
 occurs at the vertices of cubic pt seeds ( si section c.1.2 ) , resulting in the formation of various au heterostructures 
 ( figure 1 ) . typically , the epitaxial growth 
 of au on 4.9 nm pt seeds produced dumbbell - like au  pt nps . there are two 
 geometrical isomers ; one is linear and the other is bent , corresponding 
 to para and meta configurations , respectively . interestingly , we found 
 that , unlike 7.5 nm pt nps as seeds resulting in a mixture of tripods 
 and tetrapods , 6.5 nm pt seeds exclusively led to the formation of 
 tripods with a narrow distribution of size and shape ( figure 1b ) . the statistical analysis suggested that a majority 
 of resultant nps were au - tripods ( si figure 
 s7s9 ) . to the best of our knowledge , it is the first time 
 for constructing novel au tripods with high yield and improved quality 
 by sequentially applying a set of known nucleation reactions and epitaxial 
 growth processes . there are two geometrical isomers identified 
 in the resultant tripods ; namely one with c3v symmetry 
 is called as tripod - a and the other with c2v symmetry is tripod - t ( figure 1a ) . the steric accessibility determines the spatial distribution 
 of au crystals on the pt nanocubes ( si scheme 
 s2 and s3 ) . there are two meta positions favorable for the nucleation 
 of incoming au crystals when an intermediate has a bent shape , thereby 
 resulting in the formation of tripod - a ; there are six nucleation positions 
 equal to the incoming au crystals in the case of a linear intermediate , 
 producing the other type of tripods ( tripod - t ) . the statistical analysis 
 results showed that an amount of tripod - t was finally obtained slightly 
 higher than that of tripod - a ( 60% vs 40% in the resultant tripods , the sizes and 
 shapes of various au - multipods and each component within nps were 
 determined by dynamic light scattering ( dls ) and tem ( si table s3 and figure s9 ) . the au - tripods showed 
 the lowest circularity among the branched nps , largely due to their 
 anisotropic nanostructures and surface roughness . the representative 
 tem and scanning transmission electron microscope 
 ( stem ) images ( figure 2b  e ) of au - tripods 
 showed that well - crystallized interfaces were formed between the pt 
 seeds and grown au branches . the lattice fringes between pt seeds 
 and au branches shown in si figure s11 
 are 0.23 nm and related to ( 111 ) planes of either pt or au in the 
 fcc phase [ 2.260  for ( 111 ) plane of pt , 2.355  for ( 111 ) 
 plane of au ] . stem images clearly showed that each au branch epitaxially 
 grew at one of the vertices of cubic pt seeds . two types of tripods , 
 tripod - t and tripod - a , were definitively identified in the stem images 
 ( figure 2d  e ) . the forward and inverse 
 fast fourier transforms ( fft ) were further applied to their hr - tem 
 images to distinguish au branches and pt core . the right panel of 
 figure 2 shows the hr - tem images of three typical 
 tripods ( the first two belong to the type  tripod - t ; 
 the third one is the inset in diffractogram 
 patterns shows the splits of the ( 222 ) and ( 331 ) peaks into two spots : 
 one ( blue ) for pt and the other ( black ) for au crystal , because of 
 slightly different planar distances of the pt and au crystals . inverse 
 fft reconstructions of the pt ( bottom ) or au ( top ) nps using only 
 their own reflections , , , , and , provided the 
 real - spatial distributions of pt and au in tripods , respectively . therefore , the lattice images coupled with the forward and inverse 
 fast fourier transform further confirmed the spatial configuration 
 of au branches and pt core within au - tripods . hrtem and stem images 
 of au - tripods , and their fourier transform 
 and inverse fourier transform analyses . there are two types of tripods : tripod - a ( b ) and tripod - t ( c ) . ( f  h ) hrtem images of 
 typical tripod - t and its fast fourier transform ( fft ) and inverse 
 fast fourier transform ( inverse fft ) analyses . the insets in ( g ) show 
 the splits of the ( 222 ) and ( 331 ) peaks into two spots : one for pt 
 and the other for au crystal . inverse fft reconstructions of the pt 
 ( bottom ) or au ( top ) nps using only the superlattice reflections , 
 , , , and , are shown in ( h ) . ( i  k ) hrtem , 
 fft , and inverse fft reconstruction of the other tripod - t with different 
 orientation . inverse fft reconstruction of the pt ( bottom ) or au ( top ) 
 nps using the superlattice reflections , , , and . ( l  n ) hrtem , fft , and inverse fft reconstruction of tripod - a . the split of the ( 222 ) peaks is attributed to the difference between 
 pt and au crystals and is shown in the inset of ( m ) . inverse fft reconstructions 
 of the pt ( bottom ) or au ( top ) nps using only the superlattice reflections 
 , , and   are shown in ( n ) . a conventional uv  vis  nir spectrometer can 
 measure the extinction spectra of nanostructures , which comprise two 
 components : scattering and absorption , eventually providing the extinction 
 cross section ( e , e = a + s , where a is absorption 
 cross section and s is scattering cross section ) . 
 as seen in figure 3a , the au - tripods have a 
 much stronger extinction peak in the nir region , compared with the 
 other au - multipods . there are two plasmon resonances at the 540 and 
 700 nm peaks in the region of 400 to 1000 nm . the 540 nm feature corresponds 
 to a quadrupole resonance out of the plane of the gold tripods , and 
 the 700 nm feature is attributed to a dipole resonance in the plane 
 of the au - tripods . optical properties of 
 au - tripods , and measurement and simulation 
 of optical absorption cross sections of tripods . ( a ) uv  vis 
 extinction curves of various gold multipods ( including au pt 
 dumbbell nps , au - dipods , au - tripods , and au - tetrapods ) at the same 
 sample weight ( based on icp - ms ) . ( b ) uv  vis extinction curves 
 of gold nanospheres , cubic platinum nps , and gold nanorods ( 54 nm 
 length and 18 nm diameter , more information in the si , sections c.2 and c.3 ) . ( c ) the calculated absorption cross 
 section of tripod - t as a function of x ( the incident beam is polarized along the z - axis , and the tripod - t is rotated around the x - axis . x is the angle between the e - field 
 and the long axis of tripod - t . ( d ) the calculated absorption cross 
 section of tripod - a as a function of x ( the incident beam is polarized along the z - axis , and the tripod - a is rotated around the x - axis . x is the angle between the e - field 
 and the side of tripod - a . polarization dependence of the average electric 
 field intensity of tripod - t ( e ) and tripod - a ( f ) . electric field intensity 
 contours in xz plane , xy plane , 
 and yz plane at 700 nm were obtained from the fdtd 
 calculations on both tripod - t and tripod - a . the long axis of tripod - t 
 is parallel to the z - axis ; one side of tripod - a is 
 parallel to the z - axis . the excitation polarization 
 relative to the z - axis is 0. x and y represent the horizontal and vertical lengths 
 of the calculated area . the a of au - tripods was calculated using 
 combined 
 uv  vis  nir spectrometer and photoacoustic measurements . while uv  vis  nir spectrometer 
 measures the total extinction coefficient , in photoacoustic imaging , 
 the detected signal is directly proportional to the absorption coefficient 
 ( a ) of the nps . because the spr peak of the tripods 
 is tuned to 700 nm , methylene blue is suitable as a reference dye 
 to obtain the calibration curve . on the basis of the linear relationship 
 between a and photoacoustic signal amplitude and 
 the known a of methylene blue ( si figure s15 ) , the photoacoustic signals from au - tripods 
 with different concentrations were converted into a , and the a of au - tripods was calculated by dividing 
 the a by the corresponding concentration of the 
 au - tripods . in the calculation , the a and e of the tripods at 670 nm were 2.02  0.03  10 and 2.06  0.03  10 m , respectively ( a/e = 0.98 ) . as a result , on a per - weight basis , the au - tripods 
 are generally able to generate more contrast ( 33% ) on pa images within 
 the nir region compared to gold rods ( si table s4 ) . to gain more insight into the localized surface 
 plasmon resonance 
 ( lspr ) spectra and to obtain better structural optimization , we performed 
 numerical analysis on various gold multipods using a commercial finite 
 difference time domain ( fdtd ) simulation package ( lumerical solution 
 inc . , the au - multipods are modeled as a cubic pt core with 
 au spheres ( see detailed modeling in the si sections b.3 and c.4 ) . the corresponding geometrical parameters 
 were obtained from the previous results in si tables s2 and s3 . the contributions of absorption cross sections 
 of two types of au - tripods ( tripod - t and tripod - a ) as a function of 
 the rotated angle around the x - axis were obtained 
 using the fdtd simulation and are shown in c and d of figure 3 . the calculated absorption cross section maxima 
 of tripod - t were at 530 and 710 nm , in good agreement with experimental 
 data ( a and c of figure 3 ) , while theoretical 
 absorption maximum peaks of tripod - a occurred at 520 and 900 nm ( very 
 broad peak centered at 900 nm ) . using the fdtd method , we have further 
 studied the near - field optical properties of all gold multipods , gold 
 rods , and nanospheres ( si section c.4 , 
 figures s16s21 ) . the electric field intensity contours were 
 calculated at the excitation wavelength of 700 nm as the excitation 
 polarization was varied gradually from the longitudinal to transverse 
 directions . similar to gold rods , the au - tripods have a strong polarization - dependent 
 cross section . it was clear that under the resonance excitation , the 
 maximum field enhancement regions were observed to rotate away from 
 vertical tripod as the excitation polarization rotated around the y - axis ( si figure s18 ) . it is 
 worth noting that the electric fields on the surface of au - tripods 
 ( especially au - tripod - t ) are 23 orders of magnitude 
 higher when compared to fields around spherical gold nanoparticles 
 at the same weight in the nir range . as seen in the figure 3e , f and si figure s18 , 
 the edges of tripods and the junctions between two au au nps 
 on the pt cores are locations of enhanced fields ( also called hot 
 spots ) , due to the occurrence of coupled plasmons . in order to stabilize 
 the au - tripods in the aqueous solution and provide the capability 
 for subsequent surface modification , we developed a facile , versatile 
 pegylation strategy which can significantly increase in vivo circulation time of resultant nps and reduce their reticuloendothelial 
 system ( res ) accumulation versus uncoated counterparts . thiolate bonds 
 at the gold  sulfur interface and the self - assembly of a monolayer 
 on gold surface ( si scheme s1 ) . the bidentate thiol - terminated polyethylene 
 glycol ( peg ) chains facilitated subsequent immobilization of various 
 biological molecules via bioconjugation chemistry . the c(rgdfc ) was 
 efficiently and site - specifically conjugated on the maleimide - terminated 
 nps in an oriented and homogeneous fashion . in order to track the 
 rgd - au - tripods in vivo by pet , the radioactive metal 
 chelator , 2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic 
 acid ( p - scn - bn - nota ) was further conjugated to the surface of rgd - au - tripods 
 in a well - defined manner for cu radiolabeling . the hydrodynamic 
 size change and the corresponding change in  potentials of 
 modified au - tripods clearly confirmed the efficient conjugation of 
 the c(rgdfc ) peptide to the nanoparticles ( si figure s25 and table s5s8 ) . there were no significant 
 size changes or aggregation in the presence of the mouse serum after 
 48 h at 37 c ( si figure s27 ) . we 
 also validated the targeting ability of rgd - au - tripods to v3-positive u87 mg cells in vitro by determining their cellular uptake using tem and inductively coupled 
 plasma mass spectrometry ( icp - ms ) analysis ( si figures s28 and s29 ) . in order to obtain a better understanding 
 of the in vivo behavior of au - tripods and study their 
 potential toxicity , we carried out a pilot preclinical animal toxicity 
 study to assess the potential toxicity of tripods to pave the way 
 for their clinical translation . both hematology and serum biochemistry 
 analyses , and histologic and microscopic examination revealed that 
 no evidence of significant acute toxicity was observed and the tripods 
 are likely highly biocompatible in small living subjects ( si figures s35s37 ) . the pharmacokinetics , biodistribution , 
 and tumor - targeting ability 
 of au - tripods were investigated in small living animals . as described 
 in the previous section , the water - soluble au - tripods were coated 
 with a layer of functional peg chains . quantification of grafted peg 
 density on pegylated au - tripods was performed using spectrophotometric 
 analyses of free amines on the pegylated au - tripods ( si table s6 ) . typically , the density of amine groups on au - tripod 
 ( peg 3400 coating ) was 0.966  0.07 number / nm ; the 
 ratio of amine groups to a single tripod was 757  56 . the chelating 
 agent nota was attached to the terminal of peg chains for pet radionuclide cu . importantly , the cu radiolabels on the tripods 
 remained intact on the tripods even though they were incubated in 
 the mouse serum over 24 h. the u87 mg tumor - bearing mice ( n = 4 ) were tail - vein injected with cu  au - tripod , 
 followed by small animal pet scans at different time points ( 1 , 2 , 
 4 , 24 , and 48 h ) . pet imaging results revealed nonspecific uptake 
 of pegylated tripods by the liver , spleen , and even kidney ( figure 4a and si figures s30s32 ) , 
 but minimum accumulation in the muscle or other major organs . although 
 most of the tripods were eliminated through hepatic excretion , the 
 kidney retention of radio - labeled tripods over time suggested that 
 the renal excretion could be an additional clearance route for tripods 
 in mice . since the heart acted as a cardiac blood pool , the signals 
 from the heart were used as indicators to calculate the blood circulation 
 time . the pegylated tripods showed slow systemic clearance because 
 of a long blood circulation time ( t1/2 = 2.29 h ) , making themselves more available for distribution to 
 target tissues via the enhanced permeability and retention ( epr ) effect . small 
 animal pet images and pet quantification of intravenous injected 
 tripods with different surface functionalization in mice bearing the 
 u87 mg human glioblastroma tumor . ( a  c ) targeting of integrin 
 v3-postitive u87 mg tumor in mice 
 by rgd - functionalized tripods . decay - corrected whole - body coronal 
 pet images of nude mice bearing human u87 mg tumors at 1 , 4 , 24 , and 
 48 h after injection of 3.7 mbq of cu - rgd - au - tripod , cu - rgd - au - tripod with a blocking dose of c(rgdfc ) ( 21 mol 
 of c(rgdfc)/kg of mouse body weight ) , and cu  au - tripod 
 ( 200 pmol / kg of mouse body weight , or 2 mg / kg of mouse body weight ) . ( d , f ) pet quantification of tumors and major organs after intravenous 
 injection to mice bearing subcutaneous u87 mg glioma xenografts ( n = 4 per group , data represent means  sd ) . ( g  i ) , 
 comparison of tumor and major organ uptake of cu - rgd - au - tripod , cu - rgd - au - tripod with a blocking dose of c(rgdfc ) , and cu  au - tripod for a time period up to 48 h after intravenous 
 injection to u87 mg tumor - bearing mice ( n = 4 per 
 group ) . 0.01 , * p < 
 0.05 ( two - sided student s t - test ) . in order 
 to target the integrin v3 for 
 imaging tumor angiogenesis and metastasis , the au - tripods were conjugated 
 with both p - scn - bn - nota for cu labeling and c(rgdfc ) 
 for tumor targeting . the numbers of nota on the rgd - tripod were determined 
 by an isotope dilution assay and typically equaled 54.5  3.3 . the conjugated rgd was actually determined by 
 measuring the differences in concentration of rgd using hplc before 
 and after addition of au - tripods during the coupling reaction . the 
 ratio of rgd to nota on au - tripods was 8.2  0.5 . the targeting 
 ability and specificity of rgd - au - tripods for the v3 integrin were evaluated in the u87 mg tumor - bearing 
 mice ( n = 4 ) . the representative coronal and transverse 
 pet images of a mouse acquired at 1 , 4 , 24 , and 48 h after tail vein 
 injection of cu - rgd - au - tripods , cu  au - tripods , 
 or cu - rgd - au - tripods coinjected with a blocking dose 
 of c(rgdfc ) are shown in figure 4 . quantitative 
 analysis showed that tumor uptake of cu - rgd - au - tripods 
 gradually accumulated in the tumor between 1 and 24 h , reaching a 
 plateau at about 24 h post - injection , and then leveled off in the 
 next 24 h. the cu - rgd - au - tripods exhibited a significantly 
 high tumor uptake of 7.9% id / g after 24 h post - injection , 
 which was more than 3 times higher than that of plain cu  au - tripods ( 2.6% id / g ) . such high tumor accumulation was 
 attributed to the tumor specific binding affinity of rgd functionalization 
 on the tripods and their long blood circulation time . in the control 
 group , the tumor uptake was significantly blocked when a blocking 
 dose of c(rgdfc ) was injected into tumor - bearing mice along with cu - rgd - au - tripods . the reduced tumor uptake was observed 
 ( 3.8% id / g after 24 h post - injection ) , which is significantly lower 
 than that of unblocked one ( p < 0.05 ) . interestingly , 
 compared to plain tripods , cu - rgd - au - tripods displayed 
 much longer blood circulation time ( t1/2 = 10.3 h for cu - rgd - au - tripods vs t1/2 = 6.4 h for cu  au - tripods ) and 
 less res uptake . cu - rgd - au - tripods were also found to 
 be excreted by both hepatobiliary and renal routes , as indicated by 
 the deposition of radioactivity in the kidney and the gastrointestinal 
 tract ( b and e of figure 4 ) , which were also 
 confirmed by the tissue histology following in vivo imaging ( si figure s40 ) . to confirm 
 the photoacoustic detection of tripods in tumor cells , we incubated 
 u87 mg cells with rgd - au - tripods for 2 h under standard conditions . 
 the cells with internalized rgd - au - tripods after incubation were washed 
 and placed in agarose phantom at increasing cell concentrations from 
 11.5  10 to 368  10 cells per 
 well ( n = 3 per group ) . quantitative analysis of 
 the photoacoustic signals from the phantom revealed that the minimum 
 detectable number of tumor cells exposed to rgd - au - tripods could be 
 as low as 11.5  10 ( a and b of figure 5 , and figure s17 of si ) . we observed 
 a linear correlation between the number of rgd - au - tripod - loaded cells 
 and the corresponding photoacoustic signal . the photoacoustic signal 
 from the phantom when excited at 700 nm provided the best sensitivity 
 to detect tumor cells . even at 900 nm , ( a ) the top view of three - dimensional ( 3d ) volume rendering of photoacoustic 
 images of an agarose phantom containing decreasing number of u87 mg 
 cancer cells exposed to rgd - au - tripod at different wavelengths ( 670 , 
 700 , 725 , 750 , 800 , 850 , 900 nm ) . ( b ) quantitative analysis 
 of the photoacoustic signal ( relative to the background signal ) from 
 the phantom ( n = 3 ) . ( c ) rgd - au - tripod ranging in 
 concentrations from 390 pm to 12.5 nm were injected subcutaneously 
 into the flank of living mice ( n = 3 ) and scanned 
 with photoacoustic instrument . ( d , e ) picomolar photoacoustic detection 
 of tripods in living mice . the coronal view ( d ) of 3d volume rendering 
 of photoacoustic images of subcutaneous inclusions . the skin is visualized 
 in the ultrasound image ( gray - scale images ) , which is overlaid with 
 photoacoustic images ( green - scale images ) . ( f , g ) three - dimensional 
 volume rendering of photoacoustic images ( green ) and ultrasound images 
 ( brown ) of subcutaneous inclusion . ( h ) photoacoustic 
 signals recorded in vivo increased linearly with the tripod concentration 
 ( r = 0.96 , n = 3 mice , 
 data represent mean  sd ) . ( i ) quantitative analysis 
 of the photoacoustic signal ( relative to the background signal ) ( mice n = 3 ) . to test the tripod s 
 pai sensitivity in living mice , we 
 subcutaneously injected the right hind side of a mouse with 30 l 
 of rgd - au - tripods mixed with matrigel ( n = 3 ) at 
 increasing concentrations of 0.39 , 0.78 , 1.56 , 3.125 , 6.25 , and 12.5 
 nm . after the incision was solidified in place , the mouse was placed 
 on its left side ( left lateral recumbent ) and partially embedded in 
 the agarose gel covered with a water bath , and was then scanned under 
 the photoacoustic system . while the ultrasound signals were used to 
 reconstruct the mouse anatomy , including skin and inclusion edges , 
 the photoacoustic signals showed the tripods contrast in the mice 
 ( figure 5c  h ) . quantitative analysis 
 of the photoacoustic signals from each inclusion using a 3d region 
 of interest drawn over the inclusion revealed a linear correlation 
 between the tripod concentration and the corresponding photoacoustic 
 signal . compared to the tissue background , about 200 pm of rgd - au - tripods 
 extrapolated from the signal - concentration curve provided the equivalent 
 photoacoustic signal as the tissue background . similar to pet 
 imaging , the pai imaging ability of rgd - au - tripods 
 to v3 integrin - positive tumor 
 was evaluated in the u87 mg tumor - bearing mice ( n = 
 3 ) ( see figure 6 ) . before the injection , the 
 photoacoustic and ultrasound images of 
 the mice were taken . photoacoustic images with lateral step size of 
 0.25 mm were acquired at 700 nm wavelength . following the photoacoustic 
 scan , an ultrasound image of the entire tumor area was acquired . the 
 u87 mg tumor - bearing mice were then injected with 100200 l 
 of rgd - au - tripods ( 200 pmol per kg mouse body weight ) in pbs through 
 the tail vein . for the receptor - blocking experiment , mice were coinjected 
 with 21 mol of c(rgdfc)/kg of mouse body weight and 100200 
 l of rgd - au - tripods ( 200 pmol per kg mouse body weight ) in 
 pbs through the tail vein . after injection , photoacoustic images were 
 acquired at 700 nm at 0.5 , 1 , 2 , and 4 h post - injection . quantitative 
 analysis of the photoacoustic signal from the tumor was done by drawing 
 three - dimensional ( 3d ) regions - of - interest ( rois ) around tumors on 
 the basis of the ultrasound images . since there was the background 
 blood signal in each photoacoustic scan , a subtraction image calculated 
 at the 2-h post - injection image minus the preinjection image is shown 
 in si figure s39 and was used to subtract 
 out the background hemoglobin signal and examine the differences between 
 groups . mice injected with rgd - au - tripods showed significantly higher 
 photoacoustic signal in the tumor compared with the blocking group 
 coinjected with rgd after 2 h post - injection . quantitative analysis 
 showed that tumor uptake of rgd - au - tripods gradually accumulated in 
 the tumor between 1 and 4 h , reaching a plateau at about 2 h post - injection , 
 and then leveled off in the next 2 h. the mice injected with rgd - au - tripods 
 at 2 h post - injection showed more than 3 times higher photoacoustic 
 signal in the tumor than the mice coinjected with rgd and rgd - au - tripods . the tumor tissue histology combined with silver staining further confirmed 
 the specific targeting ability of rgd - au - tripods toward u87 mg tumors 
 ( si figure s40 ) . targeting of integrin 
 v3-postitive 
 u87 mg tumors in mice by rgd - au - tripod . ( a ) the coronal , sagittal , 
 and transverse views of 3d volume rendering of photoacoustic images 
 and ultrasound images of nude mice bearing u87 mg tumors were obtained 
 before injection or at 1 , 4 , 24 , and 48 h after intravenous injection 
 of rgd - au - tripod ( 200 pmol / kg of mouse body weight , or 2 mg / kg of 
 mouse body weight ) . ( b ) the coronal , sagittal , and transverse views 
 of 3d volume rendering of photoacoustic images and ultrasound images 
 of nude mice bearing u87 mg tumors were obtained before injection or 
 at 1 , 4 , 24 , and 48 h after coinjection of a blocking dose of c(rgdfc ) 
 ( 21 mol of c(rgdfc)/kg of mouse body weight ) and rgd - au - tripod 
 ( 200 pmol / kg of mouse body weight , or 2 mg / kg of mouse body weight ) . subtraction images were calculated at the 2-h post - injection image 
 minus the preinjection image ( si figure 
 s34 ) . ( c ) mice injected with rgd - au - tripod showed significantly higher 
 photoacoustic signal than mice with coinjection of a blocking dose 
 of c(rgdfc ) and the same amount of rgd - au - tripod ( p < 0.001 , two - sided student s t - test ) . ( d ) the perspective views of 3d volume rendering of photoacoustic 
 images ( green ) and ultrasound images ( brown ) of tumors . we showed the ability 
 to engineer and manipulate constitutional 
 nanocrystals at the nanometer - scale to build novel nanotripods in 
 a predictable and controlled manner . the construction of au - tripods 
 involved a set of nucleation reactions and epitaxial growth processes , 
 which are controlled by surface diffusion . as solid - state analogues of multifunctional organic molecules , pt 
 nanocubes could be used as building blocks to construct sophisticated 
 hybrid architectures . although sun and co - workers provided 
 an important insight in the formation of various branched nps , the stochastic simulation in two dimensions 
 inevitably overlooked certain configurations of constructed nanostructures . the selective growth of a heterogeneous nanocrystal phase onto certain 
 regions of nanocrystal seeds depends on the surface potential and 
 lattice matching . we found that regioselectivity 
 of the heterogeneous nucleation on cubic pt seeds plays an important 
 role on the formation of nanotripods ( si schemes s1 and s2 ) . considering the possible nucleation regions 
 and steric effect , 6.5 nm cubic seeds exclusively resulted in the 
 formation of uniform au - tripods with high yield and improved quality 
 by sequentially applying a set of known nucleation reactions and epitaxial 
 growth processes . the theoretical calculation coupled with experimental 
 results on 
 such anisotropic tripod nanostructures we successfully identified 
 tripods with two possible configurations as high absorbance nanomaterials 
 from various gold multipods using a numerical simulation analysis . the enhancement of the electric field on the surface of au - tripods 
 is due to the occurrence of coupled plasmons at the edges of tripods 
 and the junctions between gold nanocrystals on the pt cubes . optimizing 
 the geometrical configurations of gold nanocrystals on the pt cube 
 could improve the plasmon resonances of au - tripods . the measured absorption 
 spectrum of au - tripods was identified by the calculated fdtd spectrum . 
 due to an obvious increase in the cross section of au - tripods on a 
 per - weight basis compared to that of the gold rods , au - tripods could 
 generate more contrast on pa images within the nir region . among nanoparticle - based 
 pa contrast agents , au - tripods exhibit superior optical properties 
 in the nir region , and more importantly , they have exceptionally small 
 sizes , which are distinctive advantages over traditional gold - based 
 nps or carbon nanotubes for in vivo molecular imaging . we have successfully validated 
 novel au - tripods as multimodality 
 probes for in vivo molecular imaging ( pai and pet ) . 
 the in vivo biodistribution of au - tripods favorable 
 for living subject imaging was confirmed by cu radiolabeling 
 and imaging their localization over time using pet . after intravenous 
 administration , the au - tripods accumulated in liver and spleen , suggesting 
 that the hepatic excretion is a major route of elimination of au - tripods . 
 interestingly , the kidney retention of au - tripods over time revealed 
 that the renal excretion could be an additional clearance route for 
 au - tripods . considering the anisotropic shape of au - tripods [ the thickness 
 of tripods is close to the renal clearance threshold ( less than 7 
 nm ) ] , it is possible for au - tripods to be cleared to some extent through 
 the renal system . intravenous administration of rgd - au - tripods to u87 mg tumor - bearing 
 mice showed remarkably higher contrast in tumors than competitive 
 injection controls 
 even at subnanomolar concentrations . quantitative biodistribution data revealed 
 that 7.9% id / g of rgd - au - tripods accumulated in the u87 mg tumor after 
 24 h post - injection , but an obvious decrease in res uptake ( low liver 
 and spleen accumulation , figure 4 ) of rgd - au - tripods 
 was observed during the imaging . it is clear that their tumor uptake 
 was higher than most of traditional gold - based nps for in 
 vivo molecular imaging , probably due 
 to their unique anisotropic shape and relatively small size . a pilot 
 mouse toxicology study confirmed that no evidence of significant acute 
 and systemic toxicity was observed in histopathological examination . by sequentially applying a set of nucleation reactions and epitaxial 
 growth processes , we herein developed a control and stepwise strategy 
 to build novel anisotropic au - tripods with predesigned shape , high 
 yield , and excellent quality . these au - tripods exhibit superior optical 
 and physical properties compared to their counterparts with regular 
 architectures . we showed the ability to tune their shape - dependent 
 plasmon resonances to the nir window without compromising their pharmacokinetic 
 profile . our strategy opens up novel ways to enable the creation of 
 sophisticated nanostructures with predetermined optical and physical 
 properties for specific biomedical applications . moreover , our study 
 suggests that highly selective and sensitive detection of cancer cells 
 in a living subject is possible using molecular specific au - tripods 
 as pai contrast agents . the functionalized rgd - tripods showed significantly 
 enhanced photoacoustic contrast effect in both phantom and small animal 
 imaging experiments . functional and molecular information of the tumor 
 with high spatial resolution was further obtained by pai , which correlated 
 well with the corresponding pet quantification . due to their excellent 
 biocompatibility and stability in a biological environment , ease of 
 functionalization , passive and activated targeting capabilities and 
 potential hepatic and renal clearance , the au - tripods represent a 
 new generation of a nanoplatform for biomedical research and personalized 
 therapy . the 
 integrin v3 targeting peptide 
 cyclo ( arg - gly - asp - d - phe - cys ) 
 ( crgdfc ) was purchased from peptides international , inc . ( louisville , 
 ky ) . the p - scn - bn - nota was purchased from macrocyclics , inc . hydrogen 
 tetrachloroaurate(iii ) hydrate ( haucl4 ) and platinum(ii ) 
 acetylacetonate ( pt(acac)2 ) was ordered from strem chemicals , 
 inc . n - hydroxysuccinimide ( nhs ) , n - hydroxysulfosuccinimide ( sulfo - nhs ) , succinimidyl 4-(n - maleimidomethyl ) cyclohexane-1-carboxylate ( sulfo - smcc ) , and 1-ethyl-3-(3-dimethylaminopropyl ) 
 carbodiimide , hydrochloride ( edc ) were purchased from thermo fisher 
 scientific . the dialysis 
 membrane tubing ( mwco : 12,00014,000 , and 100,000 ) were purchased 
 from spectrum laboratories . the 
 size of various cubic 
 pt nps was successfully tuned by adjustment of the reaction conditions 
 in a precise manner . monodisperse pt seeds were synthesized by thermal 
 decomposition of platinum precursor [ pt(acetylacetonate)2 , pt(acac)2 ] , in 1-octadecene solution containing a trace 
 amount of iron pentacarbonyl [ fe(co)5 ] , which facilitates 
 a fast nucleation and improves the homogeneous growth of platinum 
 crystals . the shape and size of pt nps 
 are dependent on the reaction temperature at which a trace amount 
 of fe(co)5 was injected . multiple stepwise seed - mediated 
 growth processes were applied to obtain monodisperse pt nanocubes . the 
 gold precursor ( hydrogen tetrachloroaurate , haucl4 , 100 
 mg , 0.29 mmol ) was dissolved in 20 ml of 1-octadecene ( ode ) containing 
 2 ml of oleylamine under nitrogen protection . after stirring at room 
 temperature for 5 min , twenty 
 milligrams of 6.5 nm freshly synthesized pt nps ( dispersed in 1 ml 
 of hexane ) was quickly injected into the above solution . the resultant 
 mixture was then heated up to 110 c and kept at this temperature 
 for one hour before it was cooled down to room temperature . the solution 
 finally turned to gray - purplish color , indicating the formation of 
 gold branched nanostructures . the particles were precipitated out 
 by adding 30 ml of isopropanol and collected by a centrifuge ( 3000 
 rcf , 5 min ) . the resultant particles were redispersed in 5 ml of hexane 
 and then precipitated out by adding ethanol . this purification step 
 was repeated twice to remove the extra surfactant and ode . the final 
 product ( au - tripods ) was dispersed in 10 ml of hexane in the presence 
 of 0.01 ml of oleylamine for further use . surface pegylation 
 of au - tripods is described in detail in the si sections a.4 , b.2 , c.5 , and c.6 . briefly , the au - tripods ( 10 mg ) 
 were suspended in 3 ml of a chloroform solution of bidentate thiol - termined 
 peg chains ( o-(3-aminopropyl)polyethylene glycol 
 lipoate amide , lp - peg-3400-nh2 , 0.02 mmol , si section a.3 ) . after stirring at room temperature for 24 h , pegylated 
 au - tripods were precipitated by adding 20 ml of hexane , collected 
 by a brief centrifugation , and dried under the nitrogen gas flow . 
 pegylated au - tripods were then dispersed in water , and the unbound 
 peg and any other excess reagents were removed by dialysis against 
 water or phosphate - buffered saline ( pbs ) ( 10 mm , ph 7.4 ) by dialysis 
 tubing ( spectrum spectra / por dialysis membrane tubing , mwco = 12 kda ) . any impurity or precipitate was removed by a 0.22 m syringe 
 filter . the final gold or platinum concentration of pegylated au - tripods was measured by inductively coupled plasma mass spectrometry ( icp - ms ) 
 analysis . those nps were further characterized with transmission electron 
 microscopy ( tem ) , uv  vis absorption spectroscopy , and dynamic 
 light scattering ( dls ) ( si sections b.2 , 
 b.3 , b.5 , and sections c.4 , c.6 ) . conjugation of au - tripods 
 with crgd ( rgd - au - tripods ) is described 
 in detail in the si sections a.5 , c.6 , 
 and c.7 . typically , the cross - linker solution , the water - soluble au - tripods 
 ( 100 nm , 0.5 ml , 5  10 mol , see the determination 
 of concentrations of tripods and rods in si section c.3 ) in 10 mm pbs ( ph = 7.2 ) were incubated with the cross - linker 
 solution ( sulfo - smcc [ 0.5 mg , 1.5 mol ] , was freshly prepared 
 in 15 l of dmso ) for 2 h at room temperature . after removal 
 of excessive sulfo - smcc and byproducts using a pd-10 column ( ge healthcare , 
 piscataway , nj ) , the resultant thiol - active au - tripods were concentrated 
 to the final volume of 0.5 ml with a centrifugal - filter ( amicon centrifugal 
 filter device , mwco = 30 kda ) and were incubated with the crgdfc stock 
 solution ( 50 l of 5 mm in the degassed water , 0.25 mol , 
 the final rgd concentration in the mixture was 0.5 mm ) with stirring . after the 
 uncoupled rgd and byproducts were removed through pd-10 column , the 
 resultant product , rgd - au - tripods , was concentrated by a centrifugal - filter 
 ( amicon centrifugal filter device , mwco = 30 kda ) and stored at 4 
 c for one month without losing targeting activity . the final 
 rgd - au - tripods was reconstituted in pbs and filtered through a 0.22 m 
 filter for cell and animal experiments . the gold and platinum concentrations 
 of rgd - au - tripods were measured by icp - ms analysis . the process 
 to conjugate the tripods with both rgd and nota was 
 similar to the conjugation of tripods and rgd , except that sulfo - smcc 
 solution was mixed with nota aqueous solution in the first step before 
 being added into the tripod solution . briefly , the sulfo - smcc ( 0.5 
 mg , 1.5 mol ) was dissolved in 15 l of dmso and mixed 
 with 4.5 l of p - scn - bn - nota solution in the water ( 33 mm , 0.15 
 mol ) . the ratio of smcc to nota was optimized according to 
 the specific activity of radioactive tracer - labeled nps ( si section c.7 ) . pet imaging was carried out 
 on a micropet r4 rodent model scanner ( manufactured by cti concorde 
 microsystems , knoxville , tn ) as previously reported . pet scans were performed at 1 , 2 , 4 , 24 , and 48 h post - injection 
 ( p.i . ) . u87 mg tumor - bearing mice were divided into several injection 
 groups ( 4 mice per each group ) to evaluate differences in specific 
 targeting and biodistribution of au - tripods and rgd modified tripods 
 ( si section a.5 and c.7 ) . for the tripod 
 groups , each mice was injected with 3.7 mbq of cu - rgd - au - tripod 
 ( 200 pmol / kg of mouse body weight , or 2 mg / kg of mouse body weight , 
 normally 5 pmol per each mouse ) in 100200 l pbs via 
 the tail vein . for the receptor - blocking experiment , u87 mg tumor - bearing 
 mice were coinjected with 12 mg of c(rgdfc)/kg of mouse body weight 
 and 100200 l of cu - rgd - au tripod ( 200 
 pmol per kg mouse body weight ) in pbs via the tail vein . prior to 
 imaging , mice were anesthetized with isoflurane ( 5% for induction 
 and 2% for maintenance in house oxygen at 2 l / min ) . mice ( 4 mice per 
 group ) were placed in the prone position and near the center of the 
 field of view ( fov ) of the scanner . the 5-min static scans were obtained 
 at the predetermined time points after post - injection . all the pet 
 images were then reconstructed by a two - dimensional ordered - subset 
 expectation maximization ( osem ) algorithm with a spatial resolution 
 of 1.661.85 mm . the custom - built 
 photoacoustic instrument was described previously and is shown in si figure s2 . the photoacoustic characterizations 
 of au - tripods were further determined by nexus 128 ( endra , mi ) . two 
 groups of female nude mice ( n = 3 in each group , 
 68 weeks old ) were inoculated subcutaneously at their right 
 hind side with 5  10 u87 mg cells in 50 l 
 of pbs . the mouse was placed on its left side ( left lateral 
 recumbent ) and partially embedded in the agarose gel covered with 
 a water bath ( si figure s2 ) , and was then 
 scanned under the photoacoustic system . before the injection , the 
 photoacoustic and ultrasound images of the mice were taken . a photoacoustic 
 image with lateral step size of 0.25 mm was acquired using the 5 mhz 
 transducer at 670 , 700 , and 725 nm wavelength . following the photoacoustic 
 scan , the u87 mg tumor - bearing mouse was then injected with 100200 
 l of rgd - au - tripods ( 200 pmol per kg mouse body weight ) in 
 pbs through the tail vein using a butterfly catheter to avoid any 
 position change during the injection ( 50 l of dead volume ) 
 ( si figure s2b ) . for the receptor - blocking 
 experiment , mice were coinjected with 21 mol of c(rgdfc)/kg 
 of mouse body weight and 100200 l of rgd - au - tripods 
 ( 200 pmol per kg mouse body weight ) in pbs through the tail vein . after injection , photoacoustic images ( 20 mm  20 mm ) were acquired 
 at 670 , 700 , and 725 nm with step size of 0.25 mm and at 0.5 , 1 , 2 , 
 and 4 h post - injection . following the photoacoustic scan , the photoacoustic and 
 ultrasound images were analyzed , coregistered , and displayed using 
 amide software . quantitative analysis of the photoacoustic signal 
 from the tumor was done by drawing three - dimensional rois around the 
 tumor on the basis of the ultrasound images . after 4 h post - injection , 
 the mice were sacrificed , and tumors and tissues of interest were 
 collected for tem ( si section b.1 ) , elemental 
 analysis ( si section b.5 ) , and histology 
 study ( si section b.6 ) .
anisotropic colloidal hybrid nanoparticles 
 exhibit superior optical 
 and physical properties compared to their counterparts with regular 
 architectures . 
 we herein developed a controlled , stepwise strategy 
 to build novel , anisotropic , branched , gold nanoarchitectures ( au - tripods ) 
 with predetermined composition and morphology for bioimaging . the 
 resultant au - tripods with size less than 20 nm showed great promise 
 as contrast agents for in vivo photoacoustic imaging 
 ( pai ) . 
 we further identified au - tripods with two possible configurations 
 as high - absorbance nanomaterials from various gold multipods using 
 a numerical simulation analysis . 
 the pai signals were linearly correlated 
 with their concentrations after subcutaneous injection . 
 the in vivo biodistribution of au - tripods favorable for molecular 
 imaging was confirmed using small animal positron emission tomography 
 ( pet ) . 
 intravenous administration of cyclic arg - gly - asp - d - phe - cys ( rgdfc ) peptide conjugated au - tripods ( rgd - au - tripods ) to 
 u87 mg tumor - bearing mice showed pai contrasts in tumors almost 3-fold 
 higher than for the blocking group . 
 pai results correlated well with 
 the corresponding pet images . 
 quantitative biodistribution data revealed 
 that 7.9% id / g of rgd - au - tripods had accumulated in the u87 mg tumor 
 after 24 h post - injection . 
 a pilot mouse toxicology study confirmed 
 that no evidence of significant acute or systemic toxicity was observed 
 in histopathological examination . 
 our study suggests that au - tripods 
 can be reliably synthesized through stringently controlled chemical 
 synthesis and could serve as a new generation of platform with high 
 selectivity and sensitivity for multimodality molecular imaging .