optical imaging encompasses a multitude of techniques 
 for the elucidation of morphology , molecular function , and metabolism of tissue 
 with the general objective of detecting , diagnosing , staging , and treatment 
 monitoring of disease . progression of disease is usually accompanied by changes 
 in physiology and pathology that are manifested as location - specific changes in 
 optical properties thereby providing contrast for optical imaging to study 
 disease . optical imaging techniques span the range from surface 
 to bulk imaging systems with applications ranging from  optical biopsies  to 
 full human breast imaging with resolutions that cover the microscopic to 
 macroscopic . some important imaging techniques for superficial tissue imaging 
 are confocal microscopy , two - photon microscopy , and optical coherence 
 tomography ( oct ) . techniques that permit subsurface to deep imaging are diffuse optical imaging 
 ( dot )   and 
 photoacoustic imaging . the interaction of visible and near - infrared ( nir ) light 
 with tissue is dominated by 


 absorption 
 processes which are due to the presence of various chromophores such as 
 hemoglobin , oxy - hemoglobin , melanin , water , and lipids ; scattering 
 processes due to the cell membrane and cell structures such as the nucleus , 
 mitochondria , lysosomes . absorption 
 processes which are due to the presence of various chromophores such as 
 hemoglobin , oxy - hemoglobin , melanin , water , and lipids ;   scattering 
 processes due to the cell membrane and cell structures such as the nucleus , 
 mitochondria , lysosomes . penetration of light in tissue is dependent on the 
 extent of the two processes above and is low in the high - energy visible region 
 of the spectrum . this is due to high absorption by hemoglobin and severe light 
 scattering . in the wavelength regime between 600 nm and 1100 nm , absorption and 
 scattering losses this is the 
 so - called  optical imaging window  which is exploited for deep imaging in 
 tissue . the sensitivity and specificity of optical imaging 
 techniques to visualize a pathological disorder are governed by contrast : the 
 ability of the disease to differentially scatter or absorb light compared with 
 nonpathological tissue and background noise . this native or endogenous contrast 
 may not be sufficient and in any case , the interactions of light with tissue 
 are not disease - specific . therefore , there is a role for exogenously 
 administered contrast enhancing agents which have affinity for the disease site 
 through biochemical interactions , providing not only sensitive but also 
 disease - specific signals . contrast agents for optical imaging thus far have 
 near - infrared dyes based on cyanine dyes   such as indocyanine green , but in the last few years , 
 gold nanoparticles [ 1012 ] have emerged as prime candidates due to their unusual 
 optical properties and inherent biocompatibility . gold metal nanoparticles ( nps ) exhibit narrow and 
 intense absorption and scattering bands due to the phenomenon of plasmon 
 resonance . this occurs at the resonance condition of the collective oscillation 
 that the conduction electrons experience in an electromagnetic field of the 
 appropriate wavelength . the plasmon resonant condition of gold nps depends 
 upon their size , shape , structure ( solid or hollow ) , and upon the refractive 
 index of the embedding medium . spherical gold nanoparticles have a single 
 plasmon resonant extinction peak at around 520 nm , which does not shift 
 extensively with changes in size and refractive - index of the surrounding 
 medium . this is a wavelength at which light penetration in tissue is poor due 
 to strong scattering and absorption by hemoglobin , and gold nanospheres are not 
 useful in contrast enhancement for deep tissue imaging . rod - shaped nps exhibit two plasmon resonances due to 
 oscillation of the conduction electrons along the short axis as well as along 
 the long axis of the particles . the former plasmon band is called the 
 transverse resonance and the latter the longitudinal resonance . while the 
 transverse plasmon band occurs in the neighborhood of 520 nm , the longitudinal 
 band is red - shifted . the extent of the red - shift depends on the aspect ratio of 
 the nanorod ; the higher the aspect ratio , the further the shift . thus by 
 tailoring the length and/or width of these particles , their extinction peaks 
 may be made to cover the low - energy visible to infrared wavelength regions . the intense scattering and absorption of light , that 
 occurs under the plasmon resonant condition coupled with the ability to tune 
 the resonance into the near - infrared ( nir ) by manipulating the aspect ratio , 
 make gold nanorods extremely attractive as contrast agents for optical imaging 
 techniques . further , gold - protein chemistry is well developed and several 
 bioconjugation protocols are available in the literature , which allows the 
 combination of the targeting functionality of antibodies with such gold nps . the inertness and biocompatibility of gold in general hold promise the use of 
 gold nps for in vivo imaging applications . gold nps can be synthesized using wet chemical methods 
 which are based on the reduction of gold salts by reagents such as sodium 
 borohydride and ascorbic acid . these involve the reduction of gold using weak reducing 
 agents onto small nanospheres of gold as seed , in the presence of 
 shape - directing surfactants usually cetyl trimethylammonium bromide ( ctab ) . these methods may be distinguished into those that use silver ion assistance in 
 growth solutions and those that do not . murphy and coworkers described the three - step growth 
 protocol [ 14 , 15 ] , where medium to high 
 aspect ratio nanorods could be synthesized , without the use of silver nitrate . seed particles are generated by reducing gold salt using sodium borohydride in 
 the presence of sodium citrate . the spheres are coated with a layer of 
 negatively charged citrate ions that maintain colloid stability against 
 aggregation by electrostatic repulsion . these spheres seed a growth solution 
 comprising gold salt , ctab , and ascorbic acid in three steps thereby slowing 
 down reduction . murphy et al .   proposed that the polar cta 
  head group of the surfactant 
 binds with greater preference to certain crystallographic faces thereby 
 passivating them to the deposition of gold . the other faces , on the other hand , 
 would be exposed for gold to be reduced on , thereby producing anisotropic 
 growth into rods . the methods using silver nitrate in the growth 
 solutions were proposed by jana et al . , but modified 
 by nikoobakht and el - sayed   to achieve spectacular yields of nanorods with 
 excellent monodispersity . importantly , they also showed that changing the 
 quantity of ag ions in the growth solution allows for fine - tuning of the 
 aspect ratios of the nanorods . the mechanism at work in this protocol has been 
 debated in the recent past . one mechanism postulates ctab as a soft template 
 which elongates on addition of ag ions which occupy regions between the cta 
 head groups to reduce the repulsion between the head groups . a second mechanism invokes 
 the ctab passivation concept with additional adsorption of silver bromide on 
 facets slowing down reduction and producing rods shorter than those made 
 without using ag . a third mechanism which has appeared recently 
  proposes the 
 underpotential deposition of ag on certain faces , followed by ctab binding , 
 which serves to stabilize the faces , and allows gold reduction on other faces 
 resulting in rod formation . in this article , we present our experiences in 
 synthesizing gold nanospheres and nanorods using slight modifications of the 
 protocols discussed above . our goal is to obtain nanorods whose aspect ratios 
 can be tuned to obtain plasmon peaks between 650 nm850 nm . next , we conjugate 
 the gold nanospheres and gold nanorods to the her81 monoclonal antibody using 
 electrostatic and hydrophobic interactions . the conjugation does not use 
 modifications of the bilayer charge of nanorods nor does it use any linkers . we 
 discuss various aspects of these protocols and postulate a possible mechanism 
 for the bioconjugation of the antibody with the gold nanorods . we also discuss 
 the feasibility of using these molecular probes for contrast enhancement of 
 photoacoustic cancer imaging using simulations . as mentioned earlier , the seed - mediated protocol 
 requires the use of small gold nanospheres to seed growth solutions with silver 
 nitrate as per the protocol of nikoobakht and el - sayed . the following are the reagents used for the synthesis 
 of the gold seed and gold nanorods . tetrachloroauric acid ( haucl4  3h2o ) was purchased 
 from acros organics ( belgium ) , hexadecyltrimethylammonium bromide ( ctab 
  > 99% ) , sodium 
 borohydride ( nabh4 , 99% ) , and ascorbic acid ( 99% ) from aldrich ( the 
 netherlands ) and silver nitrate ( agno3 , 99.8% ) from merck ( germany ) . prior to 
 use , all glassware was cleaned with hydrofluoric acid ( hf ) , further with aqua 
 regia ( hcl / hno3 ) and rinsed twice with deionized water . the synthesis was done using protocols of nikoobakht 
 and el - sayed   with 
 slight modifications . a solution of ctab ( 5 ml ; 0.2 m ) was sonicated for 20 
 minutes at 40c in a water 
 bath . a solution of haucl4 3h2o ( 5 ml ; 0.0005 m ) was added with continuous 
 stirring under inert conditions ( nitrogen environment ) . then , an ice - cold 
 aqueous solution of nabh4 ( 0.6 ml ; 0.01 m ) was added at once with vigorous 
 stirring for 1 minute . this seed solution ( ctab - capped ) five identical conical flasks containing 10 ml of a growth solution that consists of ctab ( 5 ml ; 0.2 m ) and haucl4  3h2o ( 5 ml ; 0.001 m ) were prepared . agno3 ( 50 , 100 , 150 , 200 , and 250 l of 0.006 m ) 
 was added to the five identical growth solution flasks in an amount that was 
 chosen so as to yield desired aspect ratios for the resulting nanorods . 
 following this , the mild reducing agent ascorbic acid ( 70 
 l ; 0.1 m ) finally , 14 l of preformed 
 ctab - capped seed solution was added to each conical flask , and mixtures were 
 gently mixed . after 3 hours at 24c , the nanorod 
 suspension turned into a dark - blue solution with a brownish opalescence . these 
 solutions were concentrated by centrifuging twice at 12000 g for 20 minutes 
 which also enabled removal of the excess unbound ctab . the centrifuged gold 
 nanorods which are dispersed in water were stored at 4c electron microscopy of the nps was performed using a 
 cm 30 philips transmission electron microscope ( tem ) or a zeiss-1550 
 high - resolution scanning electron microscope ( hrsem ) . particle sizes were 
 estimated using ni vision module ( labview , national instruments ) on the digital 
 sem images with at least 250 particles considered in each case . extinction 
 spectra of nps ( and bioconjugated nps ) were measured using the shimadzu pc3101 
 uv - vis - nir spectrophotometer . the concentration of nanorods synthesized was 
 estimated using the relation 
 a = cd , where 
 a is the measured 
 absorbance , 
 c the 
 concentration in moles ( m ) , 
  the molar 
 extinction coefficient ( m cm ) , and 
 d the path length 
 of the cuvette used to record the spectra . the derived molar extinction 
 coefficients can be compared with 
  values from a 
 recent report , estimated for a range of aspect ratios of nanorods by measuring 
 the gold content in sols using inductively coupled plasma ( icp ) atomic emission 
 spectroscopy . the particles chosen for 
 bioconjugation were 25 nm citrate - capped gold spheres ( aurion , wageningen , the 
 netherlands ) , and silver - assisted surfactant mediated gold nanorods with aspect 
 ratios of approximately 2.85 ( see table 1 ) with the longitudinal plasmon peak 
 at 764 nm . the anti - her2 mouse monoclonal antibody ( mab ) ( immunicon , usa ) was 
 chosen as the targeting moiety . the antibody designated as her81 recognizes 
 human egf receptor 2 , her2 . her2 , also called erbb2 , is a member of the 
 epidermal growth factor receptor ( egfr ) family and is overexpressed in 2040% 
 of human breast cancers . in general , for optimum conjugation , it is recommended 
 that the ph of the antibody and gold sol be maintained at or slightly higher 
 than the isoelectric point ( pi ) of the antibody . the isoelectric ( pi ) point of her81 mab was 
 determined using the pharmacia phastsystem isoelectric focusing ( ief ) . the ph 
 of the antibody was adjusted with dialysis in 5 mm sodium acetate buffer and the 
 ph of the colloidal gold was adjusted with 0.1 m koh , to approximately 0.5 ph 
 units above this value . next , the minimum protecting amount of antibody to be 
 used for the conjugation is determined . this is the amount of protein that is 
 required to maintain colloidal stability of the conjugated nps upon addition of 
 nacl   as judged by 
 colorimetric analysis ; as long as the conjugated nps turns blue , particle 
 aggregation takes place implying that the amount of protein is not sufficient 
 to stabilize the suspension . by trial , different amounts of antibody are added 
 to samples of the gold sol , gently mixed , and allowed to stand at room 
 temperature for 2 minutes . spectroscopic analysis reveals which sample remains 
 stable ; the minimum amount of protein added is then ascertained and is used for 
 subsequent conjugation of the gold sol . to block the free surfaces on the gold , 10% bovine 
 serum albumin ( bsa ) in dialyzed buffer ( sodium acetate ) , maintained at the same 
 ph as the antibody solution , the bsa was added to the conjugate to a 
 final concentration of 1%and was allowed to incubate for 5 minutes . the 
 resultant was then centrifuged for 30 minutes at 12000 g to remove excess 
 protein and incompletely stabilized particles . the resulting pellet is 
 dispersed in phosphate buffered saline ( pbs ) containing 1% bsa and stored at 4c . the her2 positive mammary adenocarcinoma ( skbr3 ) cell 
 line was used as a positive cell line ; chinese hamster ovary ( cho ) cells were 
 used as an her2 negative cell line . the cells were cultured in rpmi 1640 medium 
 ( invitrogen ) supplemented with glutamine , 10% fbs ( fetal bovine serum ) with 
 antibiotics . the cells were replated onto 12 mm glass cover slips in a 6-well 
 tissue culture plate , and allowed to grow for 2 days at 37c , 5% co2 . when the cells grew to 80% confluence on the cover slips , the cells were rinsed 
 with phosphate buffered saline ( pbs ) and fixed in 4% paraformaldehyde ( pfa ) for 
 15 minutes at room temperature . the cells were incubated with 100 
 l of conjugated 
 gold nanoparticles at a concentration of 
 9.7  10 particles per 
 ml for 2 hours followed by silver enhancement performed using a silver - staining 
 kit ( aurion , wageningen , the netherlands ) . confocal microscopy reflection images of the cells on 
 cover slips were recorded on a zeiss lsm 510 confocal laser scanning microscope 
 using a c - apochromat 63 x/1.4 numerical aperture ( na ) water - immersion 
 objective . an excitation wavelength of 543 nm was chosen and reflection images 
 recorded using a 500550 nm bandpass filter . care was 
 taken to ensure that the excitation intensity as well as detector and amplifier 
 gains were maintained at the same values for all images to facilitate 
 comparison . we used seed particles within about 8 minutes of 
 formation in the subsequent growth phase . the optical extinction spectrum of 
 8-minute - aged seed is shown in figure 1 . figure 2 shows the extinction spectrum and hr - sem 
 image of the nanorods synthesized using 50 
 l agno3 in the 
 growth solution . the peak at 675 nm can be attributed to longitudinal plasmon 
 resonance and the peak in the vicinity of 516.5 nm to transverse plasmon 
 resonance . examination of the sem image and determination of the 
 mean sizes confirm that the nps produced consist of monodisperse nanorods of 
 aspect ratio of 
 2.3  0.3 , with a small number of large spheres ; the latter 's 
 extinction peak coinciding with the transverse plasmon band of the nanorods . figure 3 shows the extinction spectrum and the sem image for the sample 
 produced using 250 
 l of silver 
 nitrate . it is seen that the longitudinal plasmon band is shifted to 850 nm . 
 sizing from the sem image yields an average aspect ratio of 
 3.6  0.6 . the values of the molar extinction coefficient for the 
 2 cases above are 3.30.310 and 
 5.50.310 m cm obtained by extrapolation of 
 the data as reported in . with this , we arrive at the concentration of the 
 nanorods with peak at 675 nm as 
 4.30.310 nr / ml ; for 
 nanorods with the peak at 850 nm as 1.30.710 nr / ml . figure 5 shows the consolidated normalized extinction 
 spectra of 5 nanorod solutions , having identical growth solutions with varying 
 silver nitrate volumes . the spectra were normalized to the peak at 516 nm , 
 which is due to a combination of the transverse plasmon resonance of the 
 nanorods and the signature peak of gold nanospheres . it is seen that with 
 higher silver nitrate volumes the extent of red - shifting increases . the details of the 
 observed changes in aspect ratios and plasmon bands are presented in table 1 . a signature for successful binding of protein to gold 
 nps is a red - shifted and amplitude reduced plasmon band . both these effects are 
 due to the formation of the inhomogeneous layer of protein on the gold particle 
 surface that leads to the modification of refractive index of the embedding 
 medium . figure 6(a ) shows the extinction spectrum of gold 
 nanospheres before and after incubation with her81 . figure 6(b ) is the 
 corresponding situation with gold nanorods before and after incubation with the 
 her81 mab . in both cases , the characteristic red - shift in the extinction peak 
 of the plasmon bands is seen . not too much should be read into the amplitude changes 
 of the extinction spectra since centrifugation of the bioconjugate to remove 
 unbound protein , redispersion in water , and other procedures results in a 
 change in the concentration of the nps used for spectroscopy . figure 7(a ) show confocal reflectance image ( on left ) 
 and bright field image ( on right ) of the her81 mab / gold sphere conjugates 
 incubated with skbr3 cells . as discussed in the experimental section , silver 
 enhancement was used by which silver is reduced onto the gold particles forming 
 large clusters around 500 nm in size . the high intensities in both images at the cell membrane are then 
 evidence of the preservation of the functionality of the antibody and also 
 illustrate successful conjugation . the images in figure 7(b ) , which show the 
 situation with the negative control using the cho cells , display no such 
 accumulation of gold particles . also , adding nonantibody conjugated gold nps to 
 the skbr3 cells did not result in accumulation of the nanorods , indicating the 
 specificity of the her81 antibody - nanorod conjugate ( data not shown ) . figures 8(a ) and 8(b ) are the results of the 
 corresponding controls using the her81 mab / gold nanorods . the end products of the seed - mediated growth protocols 
 are crucially dependent on the nature of the seed , upon their size and upon the 
 capping agents used . additionally , the constituents and their concentrations in 
 the growth solution influence the outcome of the synthesis products . the 
 addition of silver ions in the growth solution and the use of preformed ctab 
 stabilized seed in the protocol of nikoobakht and el - sayed   produced not only a high 
 yield of monodisperse nanorods but fine tunability of aspect ratios . there are many unanswered questions regarding the 
 mechanism of formation of gold nanorods using the silver - assisted protocol and 
 this has been the topic of several studies [ 15 , 1719 ] . recent reports of orendorff and murphy , and liu and guyot - sionnest 
  provide some 
 insights into the mechanisms that could be involved in the synthesis . it is 
 postulated that silver ions are reduced by ascorbic acid even though it is a 
 weak reducing agent , by the phenomenon of underpotential deposition ( upd ) . this 
 is reduction of silver in monolayers on the growing gold nanorod surface at a 
 potential less than the standard reduction potential . the deposition is not 
 uniform on the gold surface but occurs faster on the sidewalls compared with 
 the end faces . remarkably , the sidewalls in the case of nanorods produced with 
 silver assistance using ctab protected seed bear au{110 } faces , while the end 
 faces have au{100 } faces . this is in contrast to the rods prepared by using 
 citrate - capped seed without ag . this faster passivation of the sidewalls is 
 followed by ctab binding possibly via bromide ions . ultimately , the end faces are also 
 stabilized preventing the formation of very long nanorods . the model claims 
 also to explain the increase in aspect ratio of the nanorods produced with 
 higher concentration of silver ions used , by proposing that higher upd of 
 silver monolayers occur on the sidewalls which one assumes reducing the width 
 of the nanorods thus increasing the aspect ratios . we are able to synthesize gold nanorods only up to an 
 aspect ratio of 3.6 , as shown in figure 3 . addition of higher volumes of silver 
 nitrate produces no further increase in the aspect ratios of the particles . this supports the idea that 
 ultimately complete passivation of the entire nanorod surface occurs preventing 
 further gold deposition even though the reagents have not been exhausted . further , we observe that nanorods that are made with increasing ag volumes 
 have smaller diameters with the lengths practically unchanged or only slightly 
 increasing ( see table 1 ) . it must be 
 mentioned that the model does not have an appealing explanation regarding the 
 ability to tune the aspect ratios so precisely by ag variation . it is very 
 likely that the model will have to undergo refinements or even major changes 
 before it is universally accepted . the noncovalent conjugation of proteins to colloidal gold is usually due to a combination of electrostatic and hydrophobic interactions . citrate - capped gold nps are negatively charged due to a layer of negative 
 citrate ions . positively charged amino groups of the antibody will be attracted 
 to the gold surface , and when the protein comes close enough for binding , the 
 hydrophobic pockets of the protein will make contact and bind with the gold 
 . a general 
 guideline to optimize the bioconjugation is that the ph of the antibody and the 
 gold sol must be maintained at or slightly above the isoelectric point of the 
 antibody . 
 following this , procedure for citrate - capped gold nanospheres with the her81 
 antibody resulted in good bioconjugation as shown by the spectroscopy and 
 bioactivity studies ( figure 7 ) . with nanorods the sidewalls of the nanorods are expected to be 
 stabilized with a bilayer of ctab , which imparts a positive charge to the gold . huang et al . 
 first changed the positively charged surface to a negatively charged one by 
 exposing the nanorods to poly(styrenesulfonate ) pss polyelectrolyte solution . the pss - capped nanorods were then treated in the same way as in section 
 2.3 with the conjugation being done with anti - egfr monoclonal antibodies . zeta potentials of the gold nanorod solution as 
 originally prepared were determined to be 
 + 55 mv . centrifugation to remove the excess unbound ctab and redispersion of the rods 
 in water saw a reduction in the zeta potential to 
 + 7.5 mv , which also 
 points to a low stability . we surmise that inspite of the net positive charge , 
 the unpassivated end faces would be negatively charged due to the presence of 
 aucl ions . we 
 therefore performed the same protocol ( as in section 2.3 ) and found that the 
 bioconjugation indeed was achieved as demonstrated by the red - shifted extinction 
 spectra as seen in figure 6 . further , confocal microscopy successfully detected 
 the bioconjugates on the her2 positive cell line ( figure 8) , indicating the 
 success of the conjugation . we believe that the mechanism of conjugation is the 
 same as that in the case of gold nanospheres that is electrostatic and 
 hydrophobic physisorption . it is also likely that at the ph at which the 
 antibody is maintained , the fc fragment of the antibody that is rich in 
 positively charged amino acids such as lysine will bind to the negatively 
 charged chloride ion layer on the exposed end faces of the rods . further , we will perform the 
 protocol of first capping the nanorods with pss for example , and then comparing 
 the antigen binding affinity constants of the bioconjugates from the two 
 methods . the scattering and absorption bands of the synthesized 
 nanorods span the wavelength regime between 675850 nm that is of interest to 
 optical imaging . this occupies the most important part of the  optical imaging 
 window  where light penetration in tissue is high due to reduced scattering 
 and absorption coefficients . optical imaging techniques ( table 2 ) that rely on 
 scattering and/or absorption contrast to detect pathological tissue could 
 benefit from the use of such nanoparticles with or without targeting 
 capability . our goal is to employ these particles as contrast 
 agents for photoacoustic cancer imaging , which has been proposed earlier by 
 oraevsky and coworkers [ 26 , 27 ] . when photons are absorbed , nonradiative de - excitation of the 
 absorbed optical energy takes place with the release of localized heat . the 
 local thermal expansion that results produces pressure transients . when illuminated with 
 pulsed laser light , a tumor site by virtue of its higher absorption with respect 
 to the healthy background tissue , due to angiogenesis , will act as a source of 
 bipolar photoacoustic pulses this ultrasound propagates with minimal 
 distortion to the surface where it is detected using appropriate wideband 
 detectors . the time - of - flight , amplitude , and peak - peak time of the bipolar pa 
 pulse possess information regarding the location , absorption , and dimensions of 
 the source , thereby permitting a reconstruction of the tumor site [ 29 , 30 ] . it is known that the nir optical absorption contrast 
 of tumors versus healthy tissue , measured using optical mammographic methods , 
 is between 1.5 and 3 . clinical trials of optical mammography are being conducted 
 worldwide but at present , it seems implausible that intrinsic contrast alone 
 will provide sufficient sensitivity and specificity , and targeted contrast 
 enhancement is likely to be required . since the same contrast mechanism of optical 
 absorption is operative in photoacoustic imaging as well , an impression of the feasibility of using the nanorods 
 synthesized for contrast enhancement is now discussed . the absorption 
 cross - section of a nanorod at a wavelength , say 800 nm , is estimated using 
 discrete dipole approximation ( ddscat ) simulations [ 32 , 33 ] as 
 cabs = 2.8  10 m . a typical average optical 
 absorption coefficient for an invasive ductal carcinoma is 
 a = 0.008 mm at 800 nm . in order to achieve 
 contrast enhancement , a certain number density of gold nanorods is required to 
 exhibit higher absorption than the intrinsic value and may be calculated 
 as 
 ( 1)nracabs . further photoacoustic signals 
 can be enhanced by a thermal nonlinearity mechanism to 3 orders of magnitude 
 higher , then the 
 modified number density of nanorods required is only 
 nr = 2.8  10 nr / cm . published studies report that most tumour cell 
 types express from 
 2  10 to 
 20  10 erbb2 
 receptors / cell . let us assume arbitrarily that 
 2  10 of these sites 
 per cell are occupied by conjugated nanorods . further , if we assume that 1% of 
 cells at a tumor site overexpress her2 results in a figure of 
 2  10 cancer cells this will then lead to an 
 estimation of the density of binding sites of the order of 
 10 cm . comparison of 
 nr and the 
 estimated figure of density of binding sites leads us to believe that contrast 
 enhancement will be possible . accumulation of the contrast agent at the tumor 
 site will depend on enhanced permeation and retention ( epr ) . active targeted 
 studies will follow , with conjugated nanoparticles administered to the animal 
 via the tail vein . in all studies , emphasis will be on ascertaining the 
 sensitivity / efficacy of the technique with and without contrast agent . we have synthesized gold nanorods with optical 
 extinction peaks in the region from 675850 nm making these eminently suited 
 for scattering and absorption contrast enhancements in optical imaging . we have 
 performed bioconjugation of these nanorods with her81 antibodies , which bind 
 with high efficiency to her2 receptors expressed by skbr3 breast carcinoma 
 cells . we demonstrated in fixed cell studies that the targeting functionality 
 of the antibody moiety remains viable . however , it must be mentioned that the 
 situation in vivo will be complex compared to the simplified situation in 
 vitro . one of these is regarding 
 toxicity and cellular uptake of these particles in vivo . further , 
 whether these molecular probes will be able to extravasate into the tumor 
 tissue through leaks in the vasculature has not yet been studied . these are 
 some lines of research that we intend to follow in the near future .
we have synthesized and characterized gold nanoparticles ( spheres and rods ) with optical extinction bands within 
	 the  optical imaging window . 
 the intense plasmon resonant driven absorption and scattering peaks of these nanoparticles 
	 make them suitable as contrast agents for optical imaging techniques . 
 further , we have conjugated these gold nanoparticles 
	 to a mouse monoclonal antibody specific to her2 overexpressing skbr3 breast carcinoma cells . 
 the bioconjugation 
	 protocol uses noncovalent modes of binding based on a combination of electrostatic and hydrophobic interactions of 
	 the antibody and the gold surface . 
 we discuss various aspects of the synthesis and bioconjugation protocols and the 
	 characterization results of the functionalized nanoparticles . 
 some proposed applications of these potential molecular 
	 probes in the field of biomedical imaging are also discussed .