semiconducting -conjugated polymers ( sps ) are optically and electronically active materials with many applications ranging from electronic devices and sensors , to tissue engineering . recently , sps have been transformed into nanoparticles as a new class of photostable fluorescent nanomaterials . these materials are bright , completely organic and absent from heavy metal ion - induced toxicity to living organisms , and thus have been applied for in vivo fluorescence ( fl ) imaging . however , fl imaging suffers from the general limitations of whole - body optical imaging techniques such as poor spatial resolution and shallow tissue penetration . in comparison , photoacoustic ( pa ) imaging allows imaging beyond the optical diffusion limit by integrating optical excitation with ultrasonic detection based on the pa effect , providing deeper tissue imaging penetration and higher spatial resolution . herein we report that near - infrared ( nir ) light absorbing semiconducting polymer nanoparticles ( spns ) can serve as an efficient and stable nanoplatform to allow photons to be used to generate ultrasound ( us ) waves , permitting in vivo pa molecular imaging . while endogenous molecules ( i.e. hemoglobin , melanin ) can generate pa contrast , many physiological and pathological processes often elicit little variation in these intrinsic pa signals , and thus it is essential to develop exogenous contrast agents for pa imaging in living subjects . many materials have been applied as effective pa contrast agents , including small - molecule dyes , metallic nanoparticles , carbon nanotubes and porphysomes . for most , signal production mechanism relies on simple accumulation through passive ( such as the enhanced permeability and retention effect ) or active ( binding to a receptor on the cell surface ) targeting to sites of interest . in contrast , activatable molecular imaging probes can undergo an intrinsic signal evolution upon detecting molecular targets or events , providing a real - time correlation between probe states ( i.e. activated vs. non - activated ) and pathological processes on a molecular level . activatable probes have been developed for other imaging modalities such as fl imaging , and widely used in biology and medicine . however , very few nir activatable probes have been reported for in vivo pa molecular imaging except for a very recent example demonstrating imaging of matrix metalloproteinase activity in thyroid tumor xenografts . the full utilization of the potential of pa imaging at a depth and spatial resolution that is unattainable by fl imaging requires new materials amenable to the construction of activatable pa probes . as sps typically used for solar cells were designed to have high nir absorption , we evaluated these photovoltaic sps as building blocks for the development of pa molecular imaging nanoprobes . we found that , depending on the molecular structure of sps , spns can show better photostability and generate higher pa signal output than two of the best current pa contrast nanoagents , single - wall carbon nanotubes ( swnts ) and gold nanorods ( gnrs ) , on a per mass basis . the high pa brightness in the nir region and favorable small size of spns allowed for pa lymph node ( ln ) mapping in living mice after a single intravenous administration of low doses ( 50 g ) . by virtue of their facile yet efficient formulation , an spn - based dual - peak ratiometric pa probe was synthesized for both in vitro and in vivo imaging of reactive oxygen species ( ros ) , which are key integral chemical mediators playing vital roles in the onset and progression of pathology ranging from cancer to neurodegenerative and cardiovascular diseases . this study thus provides one of the very few examples of nir activatable pa probes capable of reporting the progression of pathological processes in real time , and demonstrates the great potential of spns for advanced pa imaging in biomedical research . two photovoltaic sp derivatives with strong absorption in the nir region , poly(cyclopentadithiophene - alt - benzothiadiazole ) ( sp1 ) and poly(acenaphthothienopyrazine - alt - benzodithiophene ) ( sp2 ) ( fig . 1b ) afforded water - soluble sp1-based ( spn1 ) and sp2-based ( spn2 ) nanoparticles as clear dark - green and olive - green solutions ( fig . 1c ) , respectively . transmission electron microscopy ( tem ) revealed the spherical morphology of both spn1 and spn2 , and average diameters of 412 and 432 nm , respectively ( fig . moreover , dynamic light scattering ( dls ) confirmed their narrow size distribution with a polydispersity index of 0.160.03 for spn1 and 0.190.02 spn2 ( fig . spn1 and spn2 possessed large zeta potentials of 352 and 323 mv in pbs ( ph = 7.4 ) , respectively , attributable to the presence of dppc . s1 ) . spn1 and spn2 had nir absorptions with maxima at 660 and 700 nm ( fig . 1f ) , respectively . due to the difference in the molecular structures of sp1 and sp2 , the peak mass extinction coefficient of spn1 ( 93 cm mg ml ) was 4.65-times higher than that of spn2 ( 20 cm mg ml ) . while spn1 was fluorescent upon excitation at 680 nm with emission maximum at 838 nm and a quantum yield of 0.1% in 1pbs at ph = 7.4 ( supplementary fig . with strong absorption in the nir region , both spns efficiently generated pa signals upon nir pulsed laser irradiation ( fig . 1f ) . the narrower pa spectral profile of spn1 was due to narrower absorption above 600 nm relative to spn2 ( fig . the maximum pa signals were observed at 690 and 705 nm for spn1 and spn2 , respectively , both of which were red - shifted as compared to their maximum absorption . the pa amplitudes of both spns at 700 nm were determined at different concentrations in an agar phantom , displaying a linear relationship between pa signal and concentration for spn1 and spn2 ( fig . the narrower pa spectral profile and 5-fold greater pa amplitude of spn1 , attributable to its larger extinction coefficient , as compared with those of spn2 , make spn1 a better platform for the construction of pa molecular imaging probes . the capability of spn1 to produce both fl and pa signals enabled a direct comparison of pa and fl imaging in terms of spn performance . two polyethylene ( pe ) tubes filled with spn1 were implanted in a tissue - mimicking gelatin phantom ( supplementary fig . pa imaging with spn1 provided better spatial resolution and much larger penetration depths relative to fl imaging . the pa properties of spn1 were compared with high - performing pa contrast agents , swnt and gnr , in both agar phantoms and living mice . the swnt produced strong pa amplitude at 690 nm and the gnr had a pa maximum at 705 nm ( supplementary fig . s5 ) , which were spectrally similar to spn1 ( 690 nm ) and thus permitted direct comparison with a 700 nm pulsed laser . at the same mass concentration , the pa amplitude of spn1 was 5.2 and 7.1-times higher than swnt and gnr , respectively ( fig . although this result was qualitatively consistent with the rank order of their mass extinction coefficients ( 93 , 50 and 45 cm mg ml for spn1 , swnt and gnr , respectively ) , the large differences in their pa signal amplitude suggested that additional mechanisms , such as the difference in heat conductance and heat capacity , should be relevant . at equal molar concentrations , gnr provided the highest pa amplitude ( fig . 2a ) on account of its larger molar mass and molar extinction coefficient ( 5.0  10 , 6.0  10 , and 3.0  10 m cm for spn1 , swnt and gnr , respectively ) . however , after exposure to 2.4  10 pulses ( corresponding to the total laser exposure time of 120 s ) at 9 mj cm fluence , the pa signal of gnr decreased by 303% ( fig . in contrast , no pa signal was lost for spn1 under the same conditions , revealing its superior photostability and suitability for long - term pa molecular imaging . the ability to detect nanoparticle pa signal in living subjects was tested through subcutaneous injections of matrigel - containing solutions of spn1 , swnt or gnr at different concentrations into the dorsal area of mice . linear correlations between the concentration and the pa signal were observed for all nanoparticles ( fig . , the pa amplitude of spn1 was ~4.0 and 5.8-times higher than swnt and gnr , respectively , which were similar to that in agar phantoms . the limit of detection of spn1 in living mice was ~2 g / ml ( 3.8 nm ) , however neither swnt nor gnr was detectable at concentrations of 8 g / ml ( fig . 2d ) , with pa signals approaching that of tissue background as calculated from the average pa signal in areas free from nanoparticles . this coincides with previous reports that set the limit of detection for swnt and gnr in living mice by pa tomography close to 9 g / ml . nanoparticles with diameters ranging from 20 to 50 nm have favorable accumulation and retention in draining ln , and thus are promising for ln tracking , which is clinically important to guide surgical resection of tumor tissues . spn1 ( 50 g ) were administered intravenously ( i.v . ) to healthy mice ( n = 4 ) for ln imaging . at 24 h post - injection strong pa and fl signals were detected in the lymphatic networks of living mice , including accumulation in brachial lymph nodes ( blns ) , inguinal lymph nodes ( ilns ) and superficial cervical lymph nodes ( sclns ) , which were undetectable prior to nanoparticle administration ( supplementary fig . the high accumulation of spn1 in the ln was confirmed following necropsy , showing that the second highest nanoparticle accumulation occurred in the ln with a fluorescence intensity that is 63% of that from liver ( supplementary fig . ex vivo ln imaging in an agar phantom showed a 13.3-fold enhancement in pa signal following spn administration relative to ln from a mouse receiving saline ( fig . 3c&d ) , which was similar to the observed fl signal enhancement ( 12.3-times ) . this result demonstrates that spn1 can be used for ln mapping with both pa and fl imaging . the imaging of ros is critical to understanding the etiology of disease and optimizing therapeutic interventions against potentially life - threatening conditions . s8 ) , and thus we coupled it to a cyanine dye derivative ( ir775s ) that is sensitive to ros - mediated oxidation ( supplementary fig . s9 ) to design a ratiometric pa probe ( rspn ) for ros imaging ( fig . one - pot nanoprecipitation of ir775s and sp1 with dppc yielded the water - soluble rspn ( fig . 4a ) , which possessed a small diameter ( 45 nm ) and good size stability similar to spn1 ( supplementary fig . the average number of ir775s per nanoparticle was estimated to be 50 based on the molar extinction coefficients of spn1 and ir775s . the pa spectrum of rspn showed three maxima at 700 , 735 , and 820 nm ( fig . 4b ) with nearly the same amplitude , which differed from its absorption spectrum ( supplementary fig . s11a ) : the peak at 700 nm corresponds to the sp1 , and the peaks at 735 and 820 nm correspond to ir775s , which has a concentration - dependent pa spectrum . while a single pa peak at 780 nm is observed at low concentrations ( 0.010.04 mg / ml ) , an additional peak at 820 nm occurs at higher dye concentrations ( 0.10 mg / ml ) ( supplementary fig . s11b ) ; this is likely due to dye aggregation at elevated concentrations and the photosaturation effect induced by the high - power pulsed laser excitation used for the pa measurements . accordingly , as the local concentration of ir775s within the compact inner space of the nanoparticle is very high ( corresponding to ~2.5 mm ) , the pa spectrum of rspn also gave the same two peaks at 735 and 820 nm . the pa response of rspn toward different ros was examined in solution under physiological conditions . in the presence of onoo and clo , the pa peak at 735 nm decreased significantly while that at 820 nm almost disappeared , however the peak at 700 nm remained nearly the same ( fig . 4b ) . in contrast , in the presence of other ros such as no , oh , o2 , o2 and h2o2 , the pa spectrum remained essentially unchanged ( fig . the pa spectral change at 820 nm was attributed to the ros - mediated rapid oxidative decomposition of ir775s ( supplementary fig . s9 ) , validating the amenability of rspn to dual - peak ratiometric detection of ros ( pa700/pa820 ) . the enhanced specificity of rspn toward onoo and clorelative to unencapsulated ir775s likely stems from its nanoencapsulation , which limits interference from other ros with shorter lifetimes . the pa ratio of rspn increased from 1.2 to 25 in the presence of onoo and clo , remaining less than 4 for other ros ( fig . 4c ) . with a good linear correlation between pa ratio and onoo / clo concentration and a limit of detection of ~50 nm ( supplementary fig . s12 ) , rspn showed a large dynamic signal range and high sensitivity to detect both ros at pathologically relevant concentrations . the capability of rspn to detect endogenously generated ros was then tested incultured murine macrophage raw264.7 cells . after validating that spns s13 ) , rspn were incubated with raw264.7 , and cell pellets were loaded into an agar phantom for pa imaging . for cells in the resting state , 4e ) . to mimic the inflammatory condition that stimulates resting macrophages to produce ros , raw264.7 cells were pre - treated with bacterial cell wall lipopolysaccharide ( lps ) and a dimerized soluble cytokine , interferon- ( inf- ) . following lps / inf- stimulation , when n - acetylcysteine ( nac ) , a free - radical scavenger with high membrane permeability , was used to treat the cells along with lps/ inf- stimulation , pa700/pa820 returned to a low value ( 3.30.78 ) , indicating the scavenging of macrophage - generated ros by nac and inhibition of rspn activation . 4d ) , where green and red represent pa signals at 700 and 820 nm , respectively , facilitated the monitoring of ros levels in cells . the dramatic ratiometric pa responses of rspn were easily discernible between the resting raw264.7 cells , the stimulated cells and the nac - protected cells , with yellow representing a pa ratio near unity and green representing a significant elevation of ros . rspn was applied for the in vivo pa imaging of ros in a murine model of acute edema . zymosan , a structural polysaccharide of the cell wall of saccharomyces cerevisiae , which , similarly to lps , can simulate the generation of ros such as onoo and clo 
 in vivo , or saline was injected intramuscularly into the thigh of living mice , followed 20 min later by the injection of rspn ( 3 g in saline ) into the same location . the pa signal was simultaneously monitored at 700 and 820 nm , as illustrated by pseudo green and red colors , respectively . the pa amplitude at 700 nm for both saline- and zymosan - treated mice remained nearly unchanged over time ( fig . the progressive enlargement in signaling area was attributed to tissue diffusion of nanoparticles over time . in contrast , both the pa amplitude and area of signal production at 820 nm for zymosan - treated mice significantly decreased over time ( fig . the superposition analysis delineated a progressive pseudocolor variation from yellow to green for zymosan - treated mice but not for control mice ( fig . 5a ) , reporting the in situ generation of inflammatory ros during edema . quantitative analysis revealed that pa700/pa820 gradually increased to 2.70.31 at 120 min post - injection for zymosan - treated mice , which was significantly elevated relative to that for control mice ( 1.40.22 ) ( fig . 5b ) . thus , rspn effectively detected in vivo ros production using a dual - peak ratiometric pa contrast mechanism , demonstrating the potential of spns for activatable pa imaging of the progression of pathological processes in real time . two photovoltaic sp derivatives with strong absorption in the nir region , poly(cyclopentadithiophene - alt - benzothiadiazole ) ( sp1 ) and poly(acenaphthothienopyrazine - alt - benzodithiophene ) ( sp2 ) ( fig . 1b ) afforded water - soluble sp1-based ( spn1 ) and sp2-based ( spn2 ) nanoparticles as clear dark - green and olive - green solutions ( fig . 1c ) , respectively . transmission electron microscopy ( tem ) revealed the spherical morphology of both spn1 and spn2 , and average diameters of 412 and 432 nm , respectively ( fig . moreover , dynamic light scattering ( dls ) confirmed their narrow size distribution with a polydispersity index of 0.160.03 for spn1 and 0.190.02 spn2 ( fig . spn1 and spn2 possessed large zeta potentials of 352 and 323 mv in pbs ( ph = 7.4 ) , respectively , attributable to the presence of dppc . s1 ) . spn1 and spn2 had nir absorptions with maxima at 660 and 700 nm ( fig . 1f ) , respectively . due to the difference in the molecular structures of sp1 and sp2 , the peak mass extinction coefficient of spn1 ( 93 cm mg ml ) was 4.65-times higher than that of spn2 ( 20 cm mg ml ) . while spn1 was fluorescent upon excitation at 680 nm with emission maximum at 838 nm and a quantum yield of 0.1% in 1pbs at ph = 7.4 ( supplementary fig . with strong absorption in the nir region , both spns efficiently generated pa signals upon nir pulsed laser irradiation ( fig . 1f ) . the narrower pa spectral profile of spn1 was due to narrower absorption above 600 nm relative to spn2 ( fig . the maximum pa signals were observed at 690 and 705 nm for spn1 and spn2 , respectively , both of which were red - shifted as compared to their maximum absorption . the pa amplitudes of both spns at 700 nm were determined at different concentrations in an agar phantom , displaying a linear relationship between pa signal and concentration for spn1 and spn2 ( fig . the narrower pa spectral profile and 5-fold greater pa amplitude of spn1 , attributable to its larger extinction coefficient , as compared with those of spn2 , make spn1 a better platform for the construction of pa molecular imaging probes . the capability of spn1 to produce both fl and pa signals enabled a direct comparison of pa and fl imaging in terms of spn performance . two polyethylene ( pe ) tubes filled with spn1 were implanted in a tissue - mimicking gelatin phantom ( supplementary fig . pa imaging with spn1 provided better spatial resolution and much larger penetration depths relative to fl imaging . the pa properties of spn1 were compared with high - performing pa contrast agents , swnt and gnr , in both agar phantoms and living mice . the swnt produced strong pa amplitude at 690 nm and the gnr had a pa maximum at 705 nm ( supplementary fig . s5 ) , which were spectrally similar to spn1 ( 690 nm ) and thus permitted direct comparison with a 700 nm pulsed laser . at the same mass concentration , the pa amplitude of spn1 was 5.2 and 7.1-times higher than swnt and gnr , respectively ( fig . although this result was qualitatively consistent with the rank order of their mass extinction coefficients ( 93 , 50 and 45 cm mg ml for spn1 , swnt and gnr , respectively ) , the large differences in their pa signal amplitude suggested that additional mechanisms , such as the difference in heat conductance and heat capacity , should be relevant . at equal molar concentrations 2a ) on account of its larger molar mass and molar extinction coefficient ( 5.0  10 , 6.0  10 , and 3.0  10 m cm for spn1 , swnt and gnr , respectively ) . however , after exposure to 2.4  10 pulses ( corresponding to the total laser exposure time of 120 s ) at 9 mj cm fluence , the pa signal of gnr decreased by 303% ( fig . in contrast , no pa signal was lost for spn1 under the same conditions , revealing its superior photostability and suitability for long - term pa molecular imaging . the ability to detect nanoparticle pa signal in living subjects was tested through subcutaneous injections of matrigel - containing solutions of spn1 , swnt or gnr at different concentrations into the dorsal area of mice . linear correlations between the concentration and the pa signal were observed for all nanoparticles ( fig . the pa amplitude of spn1 was ~4.0 and 5.8-times higher than swnt and gnr , respectively , which were similar to that in agar phantoms . the limit of detection of spn1 in living mice was ~2 g / ml ( 3.8 nm ) , however neither swnt nor gnr was detectable at concentrations of 8 g / ml ( fig . 2d ) , with pa signals approaching that of tissue background as calculated from the average pa signal in areas free from nanoparticles . this coincides with previous reports that set the limit of detection for swnt and gnr in living mice by pa tomography close to 9 g / ml . nanoparticles with diameters ranging from 20 to 50 nm have favorable accumulation and retention in draining ln , and thus are promising for ln tracking , which is clinically important to guide surgical resection of tumor tissues . spn1 ( 50 g ) were administered intravenously ( i.v . ) to healthy mice ( n = 4 ) for ln imaging . at 24 h post - injection strong pa and fl signals were detected in the lymphatic networks of living mice , including accumulation in brachial lymph nodes ( blns ) , inguinal lymph nodes ( ilns ) and superficial cervical lymph nodes ( sclns ) , which were undetectable prior to nanoparticle administration ( supplementary fig . the high accumulation of spn1 in the ln was confirmed following necropsy , showing that the second highest nanoparticle accumulation occurred in the ln with a fluorescence intensity that is 63% of that from liver ( supplementary fig . ex vivo ln imaging in an agar phantom showed a 13.3-fold enhancement in pa signal following spn administration relative to ln from a mouse receiving saline ( fig . 3c&d ) , which was similar to the observed fl signal enhancement ( 12.3-times ) . this result demonstrates that spn1 can be used for ln mapping with both pa and fl imaging . the imaging of ros is critical to understanding the etiology of disease and optimizing therapeutic interventions against potentially life - threatening conditions . s8 ) , and thus we coupled it to a cyanine dye derivative ( ir775s ) that is sensitive to ros - mediated oxidation ( supplementary fig . s9 ) to design a ratiometric pa probe ( rspn ) for ros imaging ( fig . one - pot nanoprecipitation of ir775s and sp1 with dppc yielded the water - soluble rspn ( fig . 4a ) , which possessed a small diameter ( 45 nm ) and good size stability similar to spn1 ( supplementary fig . the average number of ir775s per nanoparticle was estimated to be 50 based on the molar extinction coefficients of spn1 and ir775s . the pa spectrum of rspn showed three maxima at 700 , 735 , and 820 nm ( fig . 4b ) with nearly the same amplitude , which differed from its absorption spectrum ( supplementary fig . s11a ) : the peak at 700 nm corresponds to the sp1 , and the peaks at 735 and 820 nm correspond to ir775s , which has a concentration - dependent pa spectrum . while a single pa peak at 780 nm is observed at low concentrations ( 0.010.04 mg / ml ) , an additional peak at 820 nm occurs at higher dye concentrations ( 0.10 mg / ml ) ( supplementary fig . s11b ) ; this is likely due to dye aggregation at elevated concentrations and the photosaturation effect induced by the high - power pulsed laser excitation used for the pa measurements . accordingly , as the local concentration of ir775s within the compact inner space of the nanoparticle is very high ( corresponding to ~2.5 mm ) , the pa spectrum of rspn also gave the same two peaks at 735 and 820 nm . the pa response of rspn toward different ros was examined in solution under physiological conditions . in the presence of onoo and clo , the pa peak at 735 nm decreased significantly while that at 820 nm almost disappeared , however the peak at 700 nm remained nearly the same ( fig . 4b ) . in contrast , in the presence of other ros such as no , oh , o2 , o2 and h2o2 , the pa spectrum remained essentially unchanged ( fig . the pa spectral change at 820 nm was attributed to the ros - mediated rapid oxidative decomposition of ir775s ( supplementary fig . s9 ) , validating the amenability of rspn to dual - peak ratiometric detection of ros ( pa700/pa820 ) . the enhanced specificity of rspn toward onoo and clorelative to unencapsulated ir775s likely stems from its nanoencapsulation , which limits interference from other ros with shorter lifetimes . the pa ratio of rspn increased from 1.2 to 25 in the presence of onoo and clo , remaining less than 4 for other ros ( fig . 4c ) . with a good linear correlation between pa ratio and onoo / clo concentration and a limit of detection of ~50 nm ( supplementary fig . s12 ) , rspn showed a large dynamic signal range and high sensitivity to detect both ros at pathologically relevant concentrations . the capability of rspn to detect endogenously generated ros was then tested incultured murine macrophage raw264.7 cells . after validating that spns s13 ) , rspn were incubated with raw264.7 , and cell pellets were loaded into an agar phantom for pa imaging . for cells in the resting state , 4e ) . to mimic the inflammatory condition that stimulates resting macrophages to produce ros , raw264.7 cells were pre - treated with bacterial cell wall lipopolysaccharide ( lps ) and a dimerized soluble cytokine , interferon- ( inf- ) . following lps / inf- stimulation when n - acetylcysteine ( nac ) , a free - radical scavenger with high membrane permeability , was used to treat the cells along with lps/ inf- stimulation , pa700/pa820 returned to a low value ( 3.30.78 ) , indicating the scavenging of macrophage - generated ros by nac and inhibition of rspn activation . 4d ) , where green and red represent pa signals at 700 and 820 nm , respectively , facilitated the monitoring of ros levels in cells . the dramatic ratiometric pa responses of rspn were easily discernible between the resting raw264.7 cells , the stimulated cells and the nac - protected cells , with yellow representing a pa ratio near unity and green representing a significant elevation of ros . rspn was applied for the in vivo pa imaging of ros in a murine model of acute edema . zymosan , a structural polysaccharide of the cell wall of saccharomyces cerevisiae , which , similarly to lps , can simulate the generation of ros such as onoo and clo 
 in vivo , or saline was injected intramuscularly into the thigh of living mice , followed 20 min later by the injection of rspn ( 3 g in saline ) into the same location . the pa signal was simultaneously monitored at 700 and 820 nm , as illustrated by pseudo green and red colors , respectively . the pa amplitude at 700 nm for both saline- and zymosan - treated mice remained nearly unchanged over time ( fig . the progressive enlargement in signaling area was attributed to tissue diffusion of nanoparticles over time . in contrast , both the pa amplitude and area of signal production at 820 nm for zymosan - treated mice significantly decreased over time ( fig . the superposition analysis delineated a progressive pseudocolor variation from yellow to green for zymosan - treated mice but not for control mice ( fig . 5a ) , reporting the in situ generation of inflammatory ros during edema . quantitative analysis revealed that pa700/pa820 gradually increased to 2.70.31 at 120 min post - injection for zymosan - treated mice , which was significantly elevated relative to that for control mice ( 1.40.22 ) ( fig . 5b ) . thus , rspn effectively detected in vivo ros production using a dual - peak ratiometric pa contrast mechanism , demonstrating the potential of spns for activatable pa imaging of the progression of pathological processes in real time . photovoltaic sps have been made into nanoparticles that produce ultrasound signals upon pulsed nir laser irradiation . these purely organic pa nanoparticles have a unique set of advantages that derive from their precursor sps , including a large mass extinction coefficient and high photostability , providing stronger ( on a per mass basis ) and more photostable pa signals in the nir region when compared with swnt and gnr ( fig . the pa amplitude of spn1 at 700 nm was more than 5-times that of swnt and gnr ( fig . 2a ) , effectively reducing their detection limit ( 2 g / ml ) in living mice relative to both swnt and gnr ( 9 g / ml ) ( fig . their favorable size ( ~40 nm ) enabled efficient pa imaging of major ln in living mice with a high signal - to - noise ratio of 13.3 after a single low dose ( 50 g ) intravenous administration ( fig . although detailed toxicity investigations are required for clinical translation , our preliminary studies have shown no overt spn cytotoxicity ( supplementary fig . others have also reported that the implantation of sps in living animals invoked little adverse tissue response with no evidence of acute and subacute toxicity . this level of biocompatibility was comparable to the fda - approved poly(lactic acid - co - glycolic acid ) , suggesting their great promise for clinical applications . spns are readily prepared through a nanoprecipitation - based bottom - up approach , allowing for facile adjustment of pa properties by choosing different constituent photovoltaic sps without significantly affecting particle dimensions . for example , spn1 and spn2 displayed similar sizes ( ~40 nm ) but different pa spectral profiles ( fig . this independence of photophysical properties from particle size differentiates spns from many of the metallic nanoparticles with strongly size - dependent spectral properties . this attribute of spns is beneficial to in vivo multiplexed imaging , as nanoparticles can be formulated to have distinct pa wavelengths without altering pharmacokinetic profiles . additionally , other molecules , such as dyes and drugs , may be simultaneously doped into spns during nanoprecipitation , potentially endowing properties to spns beyond sole pa imaging . the synthetic and structural flexibility of spns permitted the further development of spn1 into an activatable nir ratiometric pa probe ( rspn ) for in vivo ros imaging . to our knowledge , this study is the first demonstration of a pa imaging probe to detect ros , since existing ros probes are mainly based on fl imaging . among nanoparticle agents , spns uniquely possess properties of a narrow pa spectral profile , good photostability and a ros - inert pa signal . s5b ) nor gnr ( poor photostability and poor stability to ros , fig 2b & supplementary fig . s8a , respectively ) possess all of these characteristics necessary for designing ratiometirc ros probes . porphysomes are purely organic and photostable pa nanoagents with pa brightness similar to gnrs , however the porphyrin - derived generation of singlet oxygen under laser irradiation limits their application for ros detection and makes them more well - suited to photo - induced therapy applications . coupling of the excellent resistance to oxidation of spn1 with the ros sensing ability of ir775s , ros were effectively detected by rspn with pa ratiometric signal enhancements of 25 , 7.3 , and 2.7-times in solution ( fig . 4c ) , in cells ( fig . 4e ) and in living mice ( fig . 5b ) , respectively , comparable to that reported for fl probes . as pa imaging has the advantages of deep tissue penetration , high spatial resolution ( supplementary fig . s3&s4 ) , and the capability to simultaneously acquire anatomical and molecular information , rspn can further outperform existing fl probes , allowing interrogation of the key role of ros in the etiology of diseases at levels not achievable by fl imaging . in summary , we have introduced spns as a new class of nir pa contrast agents for in vivo pa molecular imaging . they can serve not only as simple accumulation - based probes ( as demonstrated for ln mapping ) but also as a nanoplatform to develop activatable probes ( as shown by ratiometric pa imaging of ros in living mice ) . given the many key merits of spns , we believe that spns can provide opportunities for advanced pa molecular imaging , from facilitating the preclinical investigation of physiological and pathological processes in living subjects to enhancing the pa imaging modality now in the process of clinical translation .
photoacoustic ( pa ) imaging holds great promise for the visualization of physiology and pathology at the molecular level with deep tissue penetration and fine spatial resolution . to fully utilize this potential , pa molecular imaging probes have to be developed 
 . 
 herein we introduce near infrared ( nir ) light absorbing semiconducting polymer nanoparticles ( spns ) as a new class of contrast agents for pa molecular imaging . 
 spns can produce stronger signal than commonly used single - wall carbon nanotubes and gold nanorods on a per mass basis , permitting whole - body lymph node pa mapping in living mice at a low systematic injection mass . 
 furthermore , spns possess high structural flexibility , narrow pa spectral profiles , and strong resistance to photodegradation and oxidation , which enables development of the first nir ratiometric pa probe for in vivo real - time imaging of reactive oxygen species 
 vital chemical mediators of many diseases . 
 these results demonstrate spns an ideal nanoplatform for developing pa molecular probes .