This journal is c the Owner Societies 2013 Phys. Chem. Chem. Phys.

Cite this: DOI: 10.1039/c3cp52193a

The evaluation of NIR-absorbing porphyrin derivativesas contrast agents in photoacoustic imaging

Akram Abuteen,a Saeid Zanganeh,a Joshua Akhigbe,b Lalith P. Samankumara,b

Andres Aguirre,a Nrusingh Biswal,a Marcel Braune,c Anke Vollertsen,c Beate Roder,c

Christian Bruckner*b and Quing Zhu*a

Six free base tetrapyrrolic chromophores, three quinoline-annulated porphyrins and three morpholino-

bacteriochlorins, that absorb light in the near-IR range and possess, in comparison to regular porphyrins,

unusually low fluorescence emission and 1O2 quantum yields were tested with respect to their efficacy as

novel molecular photo-acoustic imaging contrast agents in a tissue phantom, providing an up to B2.5-fold

contrast enhancement over that of the benchmark contrast agent ICG. The testing protocol compares

the photoacoustic signal output strength upon absorption of approximately the same light energy.

Some relationships between photophysical parameters of the dyes and the resulting photoacoustic

signal strength could be derived.

Introduction

Photoacoustic imaging (PAI) is a non-invasive biomedical ima-ging modality that combines optical and ultrasound imaging insuch a way that its key characteristics are superior to each of thecomponent imaging techniques.1–3 The absorption of a lightpulse by a chromophore causes a rapid and transient rise intemperature (in the order of mK), leading to a localized thermal-elastic expansion. As a laser beam pulsed in the nanosecond rangeis scanned through an object to be imaged, the emitted ultrasonicwave profile is acquired using standard ultrasonic transducers.The data are used to reconstruct 2D or 3D optical absorptionmaps. PAI is non-invasive and combines the advantages of highoptical contrast and ultrasound (sub-mm) spatial resolution.2,4–6

Using the high optical absorption of the endogenous chromo-phore hemoglobin, PAI proved itself as a powerful tool for imagingthe blood content of, for instance, the vascular network of cancersin rodent brains or ovary tissues, in mesoscopic biological objects,or present in whole animals.7 However, to achieve the good signalto noise ratio in reasonably short times that would make PAI ofdeeply-seated lesions practical, the sensitivity of PAI at greatertissue depths needs to be improved.4 Furthermore, many cancers,particularly in their early stages, cannot be detected by their

intrinsic vascular contrast.8 These current shortcomings in PAIsuggest, inter alia, the use of exogenous contrast agents.

The native light absorption of tissue is wavelength-dependent,with the least absorbance in the NIR region. For instance, thewavelength of maximum penetration of breast tissue is B725 nm;whole blood has an absorption minimum at B710 nm.9 Thus,using near-IR wavelengths within the ‘spectroscopic window’(B700–1000 nm) allows tissues to be imaged at deeper depth(several cm) than most other optical imaging techniques.

It thus follows that a good PAI contrast agent ought to have astrong absorption in the NIR, particularly in the region betweenB700 and 800 nm. The FDA-approved ocular angiographicdye indocyanine green (ICG) and ICG derivatives fulfill thisspectroscopic requirement.10 ICG and ICG derivatives wereutilized as rare molecular NIR PAI contrast agents even thoughthere are other photophysical parameters – like the significantfluorescence of ICG derivatives, see below – that do notmake them ideal PAI contrast agents.11 Irrespective of theshortcomings, the utilization of ICG suggests its use as thebenchmark dye.

ICG is confined to the vasculature space and it clears rapidly(t1/2 o 3 min), complicating longitudinal in vivo studies.

a Department of Electrical and Computer Engineering, University of Connecticut,

Storrs, CT 06269, USA. E-mail: zhu@engr.uconn.edu; Fax: +1 860-486-2447;

Tel: +1 860-486-5523b Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA.

E-mail: c.bruckner@uconn.edu; Fax: +1 860-486-2981; Tel: +1 860-486-2743c Humboldt-Universitat zu Berlin, Institut fur Physik, AG Photobiophysik,

Newtonstrabe 15, 12489 Berlin, Germany

Received 24th May 2013,Accepted 4th September 2013

DOI: 10.1039/c3cp52193a

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This led to the development of other contrast agents, such asmetal-based nanoparticles combined with and without organicdyes, nanotubes, and porphyrin-based liposomes.2,12 Sincethe safety of the use of nanoparticles in medicine is as yetunclear,13 it led to the search for novel ways to, for instance,generate dyes in tissue.6,14 For instance, photoacoustic probesactivatable by cancer markers were developed.3 Notably rarefrom all these reports are novel small molecular dyes as PAIcontrast agents.15

The annulation of carba- and heterocycles to the peripheryof porphyrins is an established method to red-shift the opticalspectra of porphyrins (regular porphyrins generally do notabsorb much past 650 nm).16 We, and independently others,17

recently reported the synthesis of quinoline-annulated porphyrins,such as parent compound 1, its N-oxide 2, and bisquinoline 3.18

As a result of the extended p-conjugation within these chromo-phores, they are endowed with, for porphyrins, unusually red-shifted lmax bands in the 750 nm range.

Bacteriochlorins (2,3,12,13-tetrahydroporphyrins) are thechromophores of the photosynthetic pigments of anoxygenicphoto-autotrophic cyanobacteria and are the light antenna andelectron-transfer pigments in strictly anaerobic heliobacteria.19

Bacteriochlorins are characterized by UV-visible spectra withintense absorption bands in the near infrared (NIR) region(>700 nm).20 These optical properties and their ability to generatesinglet oxygen make bacteriochlorins very attractive for use as,for instance, photochemotherapeutics21 or light-harvestingsystems.22 Preliminary reports on their use as PAI imagingagents have also appeared.15

We prepared the fully synthetic bacteriochlorin 4 bydihydroxylation/alkylation of the corresponding porphyrin.23

Further functional group manipulation leading to ring expan-sion reactions in the closely related dihydroxy-, dimethoxy- andtetrahydroxy-substituted bacteriochlorins resulted in the forma-tion of the bacteriochlorin analogues 5 and 6, containing one ortwo morpholino moieties in place of the pyrroline moieties.24

These chromophores are endowed with particularly red-shiftedand broadened optical spectra, whereby much of the spectralmodulation in the morpholinobacteriochlorins 4 and 6 is

presumed to be derived from a conformational modulation ofthe macrocycle; they are particularly ruffled and are believed tobe conformationally flexible.24

We report here the evaluation of the chromophores 1through 6 as PAI contrast agents in direct comparison to ICG(Fig. 1). We also compare the photophysical characteristics ofthe chromophores (longest wavelength absorption bands, extinc-tion coefficients, fluorescence and singlet oxygen quantum yields,singlet state lifetimes, and intersystem crossing yields) to theirrelative efficacy as PAI contrast agents. We are not aware of anystudy in which a correlation between the photophysical charac-teristics of a range of NIR dyes and their efficacy as PAI agentsusing a near-identical irradiance energy was reported. However,the efficacy of a dye as a PAI contrast agent is related to a largenumber of physical factors, including – but certainly not limitedto – the key photophysical properties listed above. Indeed, thisstudy cannot establish any correlation that would possess goodpredictive values. Nonetheless, we can identify molecular PAIcontrast agents that provide better PAI contrast enhancementsthan the benchmark dye ICG, and we can glean some trends inthe photophysical characteristics of a good imaging agent thatmay guide further investigations.

Results and discussionChromophore selection and their optical properties

We selected the chromophores 1 through 6 for our investiga-tion because of their UV-Vis-NIR absorption properties. Theirlmax values (the wavelengths of the band of longest absorption)in DMF between 726 and 790 nm fall into the spectroscopicwindow of tissue, and all fall within a range of 70 nm of those ofICG in the same solvent (Fig. 2), allowing for a meaningfuldirect comparison of the PAI data accrued (for further details,see below).

A number of general requirements for a chromophore to rendera strong photoacoustic effect can be predicted:3,25 inter alia, thechromophore should possess a high extinction coefficient (e) inthe NIR range and fast kinetics of the non-radiative deactiva-tion of the chromophore are required so that all absorbed light

Fig. 1 Structures of the NIR chromophores investigated.

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energy is rapidly converted to heat, resulting in a strong localizedthermo-elastic expansion.

Upon light absorption, porphyrins generally undergo a p- p*transition into an excited singlet state. From there the systemeither relaxes back to the ground state through fluorescence orthermally through internal conversion (IC). It can also undergoan inter-system crossing (ISC) into an excited triplet state. In turn,the triplet state relaxes either thermally, through phosphorescenceor, frequently observed in porphyrins, through interaction withtriplet oxygen (3Sg), generating singlet oxygen (1Dg). Thus, thesinglet state lifetime (tS1), the fluorescence quantum yield(FFluo), the ISC quantum yield (FISC), and the singlet oxygenquantum yield (FD) are crucial parameters to assess the relaxa-tion pathways of the chromophores investigated.

The photophysical characteristics of the quinoline-annulatedporphyrins 1 through 3, in comparison to the correspondingdata of the benchmark porphyrin TPP and the precursorto the quinoline-annulated porphyrin, oxime 7, are listed inTable 1.

A comparison of the data reveals that the annulated porphyrinsare characterized by parameters that are expectedly favorablefor their utilization as PAI contrast agents. Specifically, thequinoline-annulated porphyrins 1, 2, and 3 possess comparablyhigher extinction coefficients at lmax (that are, however, still afactor of B4 smaller than e of ICG), they possess low fluores-cence quantum yields (FFluo), low intersystem crossing quantumyields (FISC), short S1 state life times (tS1

), and generate littlesinglet oxygen (see also below why this is important). Theseparameters suggest that their excited S1 states relax rapidly alongprimarily thermal pathways, converting more than 98% of theincident light into heat. Therefore, using a good PAI signalgeneration can be expected upon irradiation of the quinoline-annulated porphyrins 1 through 3.

The use of porphyrins in diagnostic applications is potentiallyhampered by the photosensitization of toxic singlet oxygenupon their irradiation in oxygenated environments.26 However,chromophores 1 through 3 are characterized by very low singletoxygen quantum yields (FD). Applied in vivo, this reduces therisk of photosensitization-induced oxidative stress upon extendedperiods of irradiation and also of oxidative chromophorephotobleaching.

Some metalloporphyrins possess theoretically better photo-physical properties than their corresponding free bases: forinstance, the nickel(II) or copper(II) complexes are expected topossess high non-radiative decay rate constants from the excitedsinglet state and they are expected to not generate any singletoxygen.27 However, the metal complexes of bacteriochlorins aregenerally less studied because their preparation poses multiplechallenges.20 Moreover, the porphyrinoid metal complexesfrequently possess blue-shifted optical spectra, reduced longwavelengths absorptivities, and lower solubilities compared totheir free bases.28 We thus opted here for the study of the freebase compounds.

A comparison of the photophysical data for oxime 7 andquinoline-annulated system 1 also clearly shows that the imine-type nitrogen substituent or the ketone functionality at theporphyrin b-positions are not the primary origin of the batho-chromic spectrum of chromophore 1 or the observed change inthe photophysical parameters. A comparison of the data forquinolines 1 and 2 reveals that the presence of the N-oxide onlyaccentuates the trends established by quinoline annulation.Bis-fusion in 3 causes an additional red-shift of lmax but doesnot improve other key parameters.

Fig. 2 UV-Vis spectra (DMF) of the chromophores used in the PAI experiments.The concentrations were adjusted to an absorption value of 1.0 � 0.05 at therespective lmax wavelength, 1 cm glass cuvette.

Table 1 Photophysical properties of the dyes investigated

lmax/nmlog e atlmax/cm�1 M�1 FFluo/%

FISC/%(�3%)

FDn/%

(�10%)tS1

/ns(�10%)

ICG 776,d 794 j 5.06 8g 0.6h —i 0.15h

TPPa 648 3.61 9 (11)c 50 45 0.241k 728 4.30 o1 2 5 0.042k 726 4.49 o1 b.d.e o1 b.d.e

3m 773 4.44 0.004 —b 4o 0.74, 1.70 f

7k, j 667 3.75 o1 27 7 1.334k, j 704 4.71 17 45 40 5.405k, j,l 745 4.54 3 85 —b 3.776k, j 790 4.36 0.01 87 12 0.45

a In toluene. b Not determined. c G. H. Barnett, M. F. Hudson, andK. M. Smith, J. Chem. Soc. Perkin Trans. I, 1975, 1401–1403. d At 6.5 mMICG in H2O, M. L. J. Landsman, G. Kwant, G. A. Mook, W. G. Zijlstra,J. Appl. Physiol., 1976, 40, 575–583. e Below the detection limit.f Biexponential decay. g J. Pauli, R. Brehm, M. Spieles, W. A. Kaiser,I. Hilger, and U. Resch-Genger, J. Fluoresc., 2010, 20, 681–693. h InO2-free H2O. T. Luo, MSc thesis, University of Waterloo, 2008. i WhileICG does not appear to generate much, if any, 1O2 upon radiation, itgenerates medicinally relevant reactive oxygen species: S. Fickweiler,R.-M. Szeimies, W. Baumler, P. Steinbach, S. Karrer, A. E. Goetz,C. Abels, and F. Hofstadter, J. Photochem. Photobiol. B, 1997, 38,178–183. j Ref. 22. k lmax and log e in CH2Cl2, all else in DMF. l Datafor the closely related meso-tetraphenyl derivative. m lmax and log e inCH2Cl2, all else in DMSO. n lexcitation = lSoret.

o At lexcitation = 625 nm,FD = 21%, a finding rationalized by the presence of non-sensitizingdimers that are excited at lSoret but that do not get excited at 625 nm;instead, strongly sensitizing monomers are addressed. A. Vollertsen,MS thesis, Humboldt-University, Berlin, 2012.

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The bacteriochlorins 4 through 6 cover a lmax range fromslightly above 700 nm for the idealized planar compound 4 to790 nm for the strongly ruffled bismorpholinobacteriochlorin 6,with 5 occupying a middle position.18 The more planar andpresumably conformationally more rigid chromophore 4 pos-sesses a reasonably high fluorescence yield, its ISC yield ishigher than those of all other chromophores investigated,it possesses a relatively long S1 state lifetime and correspondinghigh singlet oxygen quantum yield (that is, however, lower thanthose reported for other bacteriochlorins suggested as PAI/PDTtheranostic agents).15 However, with increasing pyrroline-to-morpholine replacement, increasing nonplanarity and confor-mational flexibility, the fluorescence yields degrade, and thesinglet oxygen yields are diminished (though still high comparedto those of the quinoline-substituted chromophores 1 through 3).Also, while the singlet lifetimes are reduced with increasingmorpholine substitution, they are still 1–2 orders of magnitudelonger than those of the quinoline-annulated systems 3through 6. In totality, this may suggest that even though theirstrong absorbance in the NIR is enticing, their long excitedstate lifetimes may not result in good PA signals.

Evaluation of the chromophores as PAI contrast agents

The Gruneisen coefficient describes the proportionality betweenthe thermal expansion coefficient and the heat capacity of amaterial (at constant pressure), amounting to the proportionalityof the absorbed light energy and the resulting photoacousticpressure.25 While known for tissues and other bulk materials,this number is not known for chromophore solutions. However,since something equivalent to the Gruneisen parameter couldbe very useful for the comparison of a number of dyes in dilutesolutions, we assessed the NIR-absorbing chromophores 1through 6 as PAI contrast agents in phantom studies in com-parison to ICG in a standardized fashion and to the PAI signalobtained from a pure blood sample. The data derived fromthese experiments approximate the comparative value of theGruneisen parameter.

We performed two series of experiments. In one, we pre-pared solutions of the dyes 1 through 3 and ICG in PBS–1%DMF–1% Cremophore ELs at concentrations that resulted allsolutions to possess an equal absorption at lmax (O.D. of 1.0 at1 cm path length, see Fig. 2), and we irradiated at lmax. Since allsolutions absorbed within a narrow window, the energy inputdifference between the extremes of lmax is less than 9%.Empirically, we find the data of the PAI experiments to varyupon replication by up to 7%, i.e. the simplification of usingdifferent irradiation wavelengths/energies at equal absorbancevalues is within acceptable limits. We then performed thephantom studies described below, the results of which arelisted in Table 2.

To allow a much better direct comparison of the PAI signalstrength generated by ICG, a blood sample, and any given dyeand to allow a correlation to the photophysical data measuredprimarily in DMF (Table 1), we set up a second series ofexperiments. The spectra of the dyes 1 through 6 all overlapwith the spectrum of ICG. Thus, DMF solutions of the dyes and

ICG were prepared and the isoabsorbance point for each dye–ICG pair was determined. Then the concentration of the solutionswas adjusted to an O.D. of 1.0 at the isoabsorbance wavelength(at 1 cm path length; see Table 2 for the lirradiation used), thusassuring that each ICG–dye pair would receive the identical energyinput upon irradiation at this wavelength. The blood sample wasused as is and irradiated at 750 nm, an arbitrary wavelength in themiddle of the range of the lirradiation used.

The phantom studies to evaluate the dyes relative to ICGwere performed using the set-up shown in Fig. 3. A translucentpolyethylene tube with an inner diameter of 0.38 mm was filledwith the standard DMF solutions of the dyes 1 through 6 andICG. The tube was immersed at 1.0 and 2.0 cm depths in a tankfilled with a 4% intralipid suspension, a white opaque emulsionof soy bean oil, egg phospholipids and glycerin. This emulsion iswidely used to simulate the scattering properties of biologicaltissues at wavelengths in the red and infrared ranges where tissuehighly scatters but has a rather low absorption coefficient.29

Upon illumination with an NIR laser tuned to the lirradiation

chosen and spread to 2 cm diameter width at the point where itintersected with the tube, the photoacoustic measurements wererecorded for each sample solution using a commercial low-frequency transducer array. The array was held at a fixed position1.0 cm and 2.0 cm away from the center of the intersection of the

Table 2 Comparison of the PAI signal strengths in the phantom studya

lirradiation/nmRelative PAI signalstrength at 1 cm depth

Relative PAI signalstrength at 2 cm depth

ICG 790 1.0b (1.0)c 1.0b (1.0)c

By definition By definitionBloode 750 1.1 1.01 765 2.5b (1.6)d 2.4b (1.5)d

2 755 1.9b (1.4)d 1.2b (1.4)d

3 780 1.7b (1.0)d 1.1b (1.1)d

4 715 — f — f

5 765 0.5b 0.5b

6 741 0.8b 0.7b

a The concentrations of the dyes were adjusted to an equal absorbancevalue of A = 1.0 in a 1.0 cm path length cell. b Solvent DMF. c SolventPBS. d Solvent PBS–1% DMF–1% Cremophore ELs and lirradiation =lSoret, see Table 1. e Day-old whole rat blood, used as is, lirradiation =750 nm. f PAI signal strength below the detection limit.

Fig. 3 PAI experimental setup. Details: Nd:YAG-pumped tunable Ti:sapphire laser,average power of 18 mJ cm�2; ultrasound transducer of central frequency 1.3 MHzand bandwidth of 80%, connected to a 64 channel ultrasound PAI system.

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NIR beam and the tube, respectively. The PA signal was collectedand processed using a PAI system. Photoacoustic images werereconstructed using a typical ultrasound beam forming algorithmbased on the transducer array geometry (for details of the system,see Experimental section). The data thus obtained were plottedusing a color scale, providing the photoacoustic images shown inFig. 4. The PAI signal strengths relative to those obtained for ICGin the same solvents are listed in Table 2.

At 1 cm depth–detector distance, all dyes tested provide aclear image of the target. However, the intensity of the signalvaried. The signal strengths recorded for the quinoline-annulatedporphyrins 1 through 3 are 1.7 to 2.5-fold stronger than the signalfor the benchmark compound ICG or the blood sample (thatshowed very similar PAI signal strengths to those obtained withICG), with compound 1 providing the best signal. The signal

strength recorded for 1 is still nearly 2.4-fold stronger at 2 cmimmersion depth–detector distance, though the relative advantagesof these dyes in DMF over ICG are largely eroded. A comparison ofthe photophysical data for the dyes (Table 1) with the PAI data(Table 2) reveals that the best PAI contrast agent 1 is mostlydifferentiated from the other two structurally similar compounds(and ICG) by possessing the shortest singlet state lifetime,suggesting that this may have been the crucial factor for itsrelatively high efficacy.30

Using an aqueous solvent for the comparison of the threedyes with ICG, the absolute signal strength and the relativesignal strength advantages of the dyes 1 through 3 are not asstrong. Given the lower heat capacity of DMF (1.88 J K�1 cm�3)compared to that of water (4.19 J K�1 cm�3) (and ignoringfactors such as their varying thermal pressure coefficients),30

the lower light absorption-induced photoacoustic pressure inwater does not surprise. The fact, however, that the relative dropin the signal strengths with the phantom immersion depth–detector distance is not as steep when using the aqueous solventversus DMF does not find a simple explanation.

As a result of the higher signal-to-noise ratios, the PAIimages recorded with dyes 1 and 2 also show a better-definedtubing structure than the image recorded with ICG (Fig. 3; notethat the images shown were recorded using aqueous solvents).The signal strength recorded for 3 (no image shown) wasidentical to that recorded for ICG. However, the B4-fold higherabsorptivity of ICG compared to the porphyrinoids at around750 nm suggests that ICG is still a better contrast agent at equalconcentrations.

As, perhaps, could be predicted based on their higher fluores-cence quantum yields, larger ISC yields, longer-lived excited singletstates, and higher singlet oxygen quantum yields, the bacterio-chlorins 4 through 6 did not perform as well as the quinoline-annulated porphyrins or ICG in the PAI experiments. Thisillustrates well the need to fine-tune all photophysical propertiesof a potential PAI imaging dye, not just their optimal absorptionproperties. On the other hand, the ability to induce a PAI signalwhen irradiated with NIR light as well as to generate ROS suggesttheir utilization in theranostic applications, as suggested byArnaut and co-workers.15

Conclusions

We have shown the direct comparsion of a range of porphyrinoidswith NIR absorbing properties belonging to two different compoundclasses (quinoline-annulated porphyrins and bacteriochlorins)as exogenous PAI imaging contrast agents in phantom studies.We demonstrated the imaging of a sub-mm target at up to 2 cmdepth in a tissue-like matrix whereby some porphyrinoidsmatched or exceeded the performance of the benchmark dyeICG at equal absorbance or the performance of pure blood.This suggests the use of quinoline-annulated porphyrins aspotential contrast agents for in vivo use for multi-cm deeptissue PAI. While our data do not allow any obvious directcorrelations between the photophysical data and the PAI con-trast enhancement measured to be made, the need for the

Fig. 4 Photoacoustic images of the dyes indicated, dissolved in an aqueoussolution containing the surfactant Cremophore EL to a concentration resulting inan absorbance value of A = 1.0 at 1.0 cm path lengths. Left column: targetslocated at 1 cm depth, the right column shows targets located at 2 cm depth.Targets: a transparent polyethylene tube of inner diameter 0.38 mm and outerdiameter 1.09 mm filled with the dye solution, submersed in an opaque 4%intralipid suspension. To best visualize the contrast, the dynamic range of thedisplay was set to 10 dB.

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ability of the dyes to access a very efficient thermal relaxationpathway is once again highlighted. Moreover, as we are screeningmore members of NIR-absorbing porphyrinoids using the simpleand rapid screening tool used here, a direct correlation betweenthe PAI signal strength and key photophysical parameters maycome into focus, further assisting in the design of optimizedcontrast agents.

The best contrast agents identified in this study, the twoquinoline-annulated porphyrins, are currently undergoing a rangeof in cyto and in vivo cytotoxicity and PAI studies. Preliminary datasuggest that the dyes possess low light and dark toxicity; thedetails of these investigations will be reported in due course.

ExperimentalMaterials

The porphyinic dyes 1 through 3, as well as their precursor 7,were prepared from TPP as described in the literature.18 Thebacteriochlorins 4 though 6 were also prepared from TPP aspreviously described.23 All solvents were of spectroscopic gradeand used as received. Cremophore ELs was acquired from Acros.ICG was purchased from Sigma Aldrich.

The dyes 1 through 6 and ICG were dissolved in DMF at aconcentration so that they possess all identical absorbances(A = 1.0 at the wavelengths listed in Table 2 at 1 cm path length).The dyes 1 through 3 and ICG were also tested in an aqueoussolution using the FDA-approved excipient Cremophore ELs,also at A = 1.0 at lSoret.

31

Photophysical measurements

The UV/Vis spectra shown in Fig. 2 were recorded on a Cary 50spectrophotometer. The absorption data listed in Table 1 wererecorded at room temperature on a Shimadzu UV/VIS-160spectrophotometer. The molar extinction coefficients e(l) weredetermined by means of serial dilutions resulting in concentrationcurves. Steady-state fluorescence spectra were also recorded todetermine fluorescence quantum yields (FFl) using differentstandards (TPP in toluene, FFl,TPP = 0.11; TPPS in H2O, FFl,TPPS =0.09; Rhodamine 6G in H2O, FFl,Rh = 0.90). A Xe-lamp (XBO 150,OSRAM) with a monochromator was used for excitation. For theemission detection a polychromator with a cooled CCD-matrix(LotOriel, Instaspec IV, 77131) was used.

Time-resolved fluorescence spectroscopy was performed inlow-concentration samples (OD E 0.1 at excitation wavelength)via the time-correlated single photon counting (TCSPC) technique,thus yielding fluorescence decay curves. The excitation source usedwas a pulsed, frequency-doubled Nd:VO4 laser (Cougar, TimeBandwith Products) with lexc = 532 nm, a pulse width of 12 psand a repetition rate of 60 MHz. The response function of thesystem was determined using a Ludox scattering solution(Aldrich). Data were analyzed using a homemade program basedon the Nelder–Mead simplex algorithm to obtain fluorescencelifetimes tFl.

Intersystem crossing quantum yields (FISC) were measuredby transient absorption spectroscopy. To measure transientabsorption spectra, a white light continuum was generated

(test pulse) in a cell containing a D2O–H2O mixture using highlyintense, 25 ps-wide pulses from a Nd3+:YAG laser (PL 2143A,Ekspla) of 1064 nm wavelength. On its way to the sample, thewhite continuum pulse was split to generate a reference spectrum.Both the transmitted and reference beam were focused into opticalfibers and recorded simultaneously at different traces on a CCD-matrix (LotOriel, Instaspec IV, 77131). Tunable radiation from anOPG/OPA (Ekspla PG 401/SH, tuning range: 200–2300 nm) pumpedby the TH (355 nm) of the same laser was used for excitation. Apropagation delay line enables the measurement of light-inducedchanges of the absorption spectrum at different delay times up to15 ns after excitation. The absorbance (OD) of all samples was 1.0 atthe maximum of the lowest-energy absorption band.

Photoacoustic phantom studies

A translucent polyethylene tube with an inner diameter of0.38 mm and outer diameter of 1.09 mm was filled with thestandard solutions of the dyes 1 through 6, ICG and day-oldwhole rat blood. The tube was immersed at 1.0 and 2.0 cmdepths in a tank filled with a 4% intralipid suspension. Uponillumination with an NIR laser tuned to the lmax of the dyeinvestigated (Table 1) and spread to 2 cm diameter width at thepoint where it intersected with the tube, the photoacousticmeasurements were recorded for each sample solution usinga commercial low-frequency transducer array (produced byVermon, France), and consisted of 64 elements with 0.85 mmpitch. The center frequency of the transducer was 1.3 MHz andbandwidth of 80%, connected to a 64 channel ultrasound PAIsystem. The array was held at 1.0 and 2.0 cm, respectively, awayfrom the center of the intersection of the NIR beam and thetube. The beam was provided by an Nd:YAG-pumped tunableTi:sapphire laser with an average power of radiant exposure of18 mJ cm�2, i.e., well below the ANSI limits of 21 mJ cm�2, forthe shortest wavelengths used.32 The signal was then collectedand processed by a 64 channel ultrasound PAI system with ascalable center frequency and bandwidth of the ultrasoundtransducer: imaging speed 5 frames per s and utilizing a uniquefield programmable gate array (FPGA) based reconfigurableprocessor that allows real-time switching and interlacingbetween the two imaging modalities (i.e. ultrasound and photo-acoustic imaging). The system features a modular design andthe ability of real-time parallel acquisition from 64 channelswith each channel sampled at 40 MHz.33 Photoacoustic imageswere reconstructed using a typical ultrasound beam formingalgorithm based on the transducer array geometry. Theseimages were then plotted using a color scale, providing thephotoacoustic images shown in Fig. 4.

Notes and references

1 M. Xua and L. V. Wang, Rev. Sci. Instrum., 2006, 77,041101–041122; M. A. Pysz, S. S. Gambhir and J. K. Willmann,Clin. Radiol., 2010, 65, 500–516; L. V. Wang, Nat. Photonics,2009, 3, 503–509.

2 C. Kim, C. Favazza and L. V. Wang, Chem. Rev., 2010, 110,2756–2782.

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3 J. Levi, S. R. Kothapalli, T.-J. Ma, K. Hartman, B. T. Khuri-Yakub and S. S. Gambhir, J. Am. Chem. Soc., 2010, 132,11264–11269.

4 R. O. Esenaliev, A. A. Karabutov and A. A. Oraevsky, IEEE J.Sel. Top. Quantum Electron., 1999, 5, 981–988.

5 R. A. Kruger, D. R. Reinecke and G. A. Kruger, Med. Phys.,1999, 26, 1832–1837.

6 A. S. Stender, K. Marchuk, C. Liu, S. Sander, M. W. Meyer,E. A. Smith, B. Neupane, G. Wang, J. Li, J.-X. Cheng,B. Huang and N. Fang, Chem. Rev., 2013, 113, 2469–2527.

7 C. G. A. Holen, F. F. M. de Mul, R. Pongers and A. Dekker,Opt. Lett., 1998, 23, 648–650; X. D. Wang, Y. J. Pang, G. Ku,X. Y. Xie and G. Stoica, Nat. Biotechnol., 2003, 21, 803–806;A. Aguirre, P. Guo, J. Gamelin, S. Yan, M. M. Sanders, M. Brewerand Q. Zhu, J. Biomed. Opt., 2009, 14, 0540141–0540149; R. Ma,A. Taruttis, V. Ntziachristos and D. Razansky, Opt. Express,2009, 17, 21414–21426; D. Razansky1, C. Vinegoni andV. Ntziachristos, Phys. Med. Biol., 2009, 54, 2769–2777.

8 T. L. Troy, D. L. Page and E. M. Sevick-Muraca, J. Biomed.Opt., 1996, 1, 342–355.

9 A. E. Cerussi, A. J. Berger, F. Bevilacqua, N. Shah, D. Jakubowski,J. Butler, R. F. Holcombe and B. J. Tromberg, Acad. Radiol.,2001, 8, 211–218.

10 S. Luo, E. Zhang, Y. Su, T. Cheng and C. Shi, Biomaterials,2011, 32, 7127–7138.

11 X. Wang, G. Ku, M. A. Wegiel, D. J. Bornhop, G. Stoica andL.-H. Wang, Opt. Lett., 2004, 29, 730–732; G. Ku and L. V.Wang, Opt. Lett., 2005, 30, 507–509; B. Wang, Z. Qing,N. M. Barkey, D. L. Morse and H. Jiang, Med. Phys., 2012,39, 2512–2517.

12 A. De La Zerda, C. Zavaleta, S. Keren, S. Vaithilingam,S. Bodapati, Z. Liu, J. Levi, B. R. Smith, T.-J. Ma,O. Oralkan, Z. Cheng, X. Chen, H. Dai, B. T. Khuri-Yakuband S. S. Gambhir, Nat. Nanotechnol., 2008, 3, 557–562;K. H. Song, C. Kim, C. M. Cobley, Y. Xia and L. V. Wang,Nano Lett., 2008, 9, 183–188; X. Yang, S. Skrabalak, E. Stein,B. Wu and X. Wei, Proc. SPIE, 2008, 6856, 6856601; B. Wang,E. Yantsen, T. Larson, A. B. Karpiouk, S. Sethuraman,J. L. Su, K. Sokolov and S. Y. Emelianov, Nano Lett., 2008,9, 2212–2217; S. Mallidi, T. Larson, J. Tam, P. P. Joshi,A. Karpiouk, K. Sokolov and S. Emelianov, Nano Lett., 2009,9, 2825–2831; D. Pan, M. Pramanik, A. Senpan, X. Yang,K. H. Song, M. J. Scott, H. Zhang, P. J. Gaffney,S. A. Wickline, L. V. Wang and G. M. Lanza, Angew. Chem.,Int. Ed., 2009, 48, 4170–4173; X. Yang, E. W. Stein,S. Ashkenazi and L. V. Wang, WIREs Nanomed. Nanobiotech.,2009, 1, 260–368; J. Chen, M. Yang, Q. Zhang, E. C. Cho,C. M. Cobley, C. Kim, C. Glaus, L. V. Wang, M. J. Welch andY. Xia, Adv. Funct. Mater., 2010, 20, 3684–3694; A. d. l. Zerda,Z. Liu, S. Bodapati, R. Teed, S. Vaithilingam, B. T. Khuri-Yakub, X. Chen, H. Dai and S. S. Gambhir, Nano Lett., 2010,10, 2168–2172; J. F. Lovell, C. S. Jin, E. Huynh, H. Jin,C. Kim, J. L. Rubinstein, W. C. W. Chan, W. Cao, L. V. Wangand G. Zheng, Nat. Mater., 2011, 10, 324–332; G. D.Moon, S.-W. Choi, X. Cai, W. Li, E. C. Cho, U. Jeong, L. V.Wang and Y. Xia, J. Am. Chem. Soc., 2011, 133, 4762–4765;

D. Pan, X. Cai, C. Yalaz, A. Senpan, K. Omanakuttan, S. A.Wickline, L. V. Wang and G. M. Lanza, ACS Nano, 2012, 6,1260–1267; J. V. Jokerst, A. J. Cole, D. Van de Sompel andS. S. Gambhir, ACS Nano, 2012, 6, 10366–10377; G. Ku,M. Zhou, S. Song, Q. Huang, J. Hazle and C. Li, ACS Nano,2012, 6, 7489–7496; J. V. Jokerst, M. Thangaraj, P. J.Kempen, R. Sinclair and S. S. Gambhir, ACS Nano, 2012,6, 5920–5930; A. de la Zerda, S. Bodapati, R. Teed, S. N. Y.May, S. M. Tabakman, Z. Liu, B. T. Khuri-Yakub, X. Chen,H. Dai and S. S. Gambhir, ACS Nano, 2012, 6, 4694–4701;K. A. Homan, M. Souza, R. Truby, G. P. Luke, C. Green,E. Vreeland and S. Emelianov, ACS Nano, 2012, 6, 641–650;J. R. Cook, W. Frey and S. Emelianov, ACS Nano, 2013, 7,1272–1280.

13 E. Heister, E. W. Brunner, G. R. Dieckmann, I. Jurewicz andA. B. Dalton, ACS Appl. Mater. Interfaces, 2013, 5, 1870–1891.

14 G. S. Filonov, A. Krumholz, J. Xia, J. Yao, L. V. Wang andV. V. Verkhusha, Angew. Chem., Int. Ed., 2011, 51, 1448–1451;Y. Zhang, X. Cai, Y. Wang, C. Zhang, L. Li, S.-W. Choi,L. V. Wang and Y. Xia, Angew. Chem., Int. Ed., 2011, 50,7359–7363.

15 F. A. Schaberle, L. G. Arnaut, C. Serpa, E. F. F. Silva,M. M. Pereira, A. R. Abreu and S. Simoes, Proc. SPIE, 2010,7376, 7376X; F. A. Schaberle, L. A. F. Reis, G. F. F. Sa,C. Serpa, A. R. Abreu, M. M. Pereira and L. G. Arnaut,Proc. SPIE, 2011, 8089, 8089Q.

16 J. E. Rogers, K. A. Nguyen, D. C. Hufnagle, D. G. McLean,W. Su, K. M. Gossett, A. R. Burke, S. A. Vinogradov, R. Pachterand P. A. Fleitz, J. Phys. Chem. A, 2003, 107, 11331–11339;S. Fox and R. W. Boyle, Tetrahedron, 2006, 62, 10039–10054.

17 C. Jeandon and R. Ruppert, Eur. J. Org. Chem., 2011,4098–4102; A. M. V. M. Pereira, S. Richeter, C. Jeandon,J.-P. Gisselbrecht, J. Wytko and R. Ruppert, J. PorphyrinsPhthalocyanines, 2012, 16, 464–478.

18 J. Akhigbe, M. Zeller and C. Bruckner, Org. Lett., 2011, 13,1322–1325.

19 H. Scheer, in Chlorophylls and Bacteriochlorophylls, ed.B. Grimm, R. J. Porra, W. Rudinger and H. Scheer, Springer,Dordrecht, NL, 2006, pp. 1–26.

20 C. Bruckner, L. Samankumara and J. Ogikubo, in Handbookof Porphyrin Science, ed. K. M. Kadish, K. M. Smith andR. Guilard, World Scientific, River Edge, NY, 2012, (SyntheticDevelopments, Part II), ch. 76, vol. 17, pp. 1–112.

21 Y. Chen, W. R. Potter, J. R. Missert, J. Morgan andR. K. Pandey, Bioconjugate Chem., 2007, 18, 1460–1473;Y. Chen, G. Li and R. K. Pandey, Curr. Org. Chem., 2004, 8,1105–1134; P. Mroz, Y.-Y. Huang, A. Szokalska, T. Zhiyentayev,S. Janjua, A.-P. Nifli, M. E. Sherwood, C. Ruzie, K. E. Borbas,D. Fan, M. Krayer, T. Balasubramanian, E. Yang, H. L. Kee,C. Kirmaier, J. R. Diers, D. F. Bocian, D. Holten, J. S. Lindseyand M. R. Hamblin, FASEB J., 2010, 24, 3160–3170;Y.-Y. Huang, P. Mroz, T. Zhiyentayev, S. K. Sharma,T. Balasubramanian, C. Ruzie, M. Krayer, D. Fan,K. E. Borbas, E. Yang, H. L. Kee, C. Kirmaier, J. R. Diers,D. F. Bocian, D. Holten, J. S. Lindsey and M. R. Hamblin,J. Med. Chem., 2010, 53, 4018–4027; S. Fukuzumi,

PCCP Paper

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ishe

d on

05

Sept

embe

r 20

13. D

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ut o

n 27

/09/

2013

18:

44:4

7.

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Phys. Chem. Chem. Phys. This journal is c the Owner Societies 2013

K. Ohkubo, X. Zheng, Y. Chen, R. K. Pandey, R. Zhan andK. M. Kadish, J. Phys. Chem. B, 2008, 112, 2738–2746;R. Yerushalmi, I. Ashur and A. Scherz, in Chlorophylls andBacteriochlorophylls, ed. B. Grimm, R. J. Porra, W. Rudingerand H. Scheer, Springer, Dordrecht, NL, 2006, pp. 495–506;L. Huang, Y.-Y. Huang, P. Mroz, G. P. Tegos, T. Zhiyentayev,S. K. Sharma, Z. Lu, T. Balasubramanian, M. Krayer,C. Ruzie, E. Yang, H. L. Kee, C. Kirmaier, J. R. Diers,D. F. Bocian, D. Holten, J. S. Lindsey and M. R. Hamblin,Antimicrob. Agents Chemother., 2010, 54, 3834–3841.

22 J. W. Springer, P. S. Parkes-Loach, K. R. Reddy, M. Krayer,J. Jiao, G. M. Lee, D. M. Niedzwiedzki, M. A. Harris,C. Kirmaier, D. F. Bocian, J. S. Lindsey, D. Holten andP. A. Loach, J. Am. Chem. Soc., 2012, 134, 4589–4599.

23 L. P. Samankumara, M. Zeller, J. A. Krause and C. Bruckner,Org. Biomol. Chem., 2010, 8, 1951–1965.

24 L. P. Samankumara, S. Wells, M. Zeller, A. M. Acuna,B. Roder and C. Bruckner, Angew. Chem., Int. Ed., 2012,51, 5757–5760.

25 I. V. Larina, K. V. Larin and R. O. Esenaliev, J. Phys. D: Appl.Phys., 2005, 38, 2633–2639.

26 The strong fluorescence and generally good 1O2 photosensiti-zation ability of porphyrins is the basis of their utilization as

imaging and photochemotherapeutics: M. Ethirajan, Y. Chen,P. Joshi and R. K. Pandey, Chem. Soc. Rev., 2011, 40, 340–362.See also ref. 15.

27 C. M. Drain, S. Gentemann, J. A. Roberts, N. Y. Nelson,C. J. Medforth, S. Jia, M. C. Simpson, K. M. Smith, J. Fajer,J. A. Shelnutt and D. Holten, J. Am. Chem. Soc., 1998, 120,3781–3791; J. L. Retsek, C. M. Drain, C. Kirmaier,D. J. Nurco, C. J. Medforth, K. M. Smith, I. V. Sazanovich,V. S. Chirvony, J. Fajer and D. Holten, J. Am. Chem. Soc.,2003, 125, 9787–9800.

28 J. W. Buchler, in Porphyrins, ed. D. Dolphin, 1978, vol. 1,pp. 389–483.

29 I. Driver, J. W. Feather, P. R. King and J. B. Dawson,Phys. Med. Biol., 1989, 34, 1927–1930.

30 L. G. Arnaut, R. A. Caldwell, J. E. Elbert and L. A. Melton,Rev. Sci. Instrum., 1992, 63, 5381–5389.

31 Cremophor EL (BASF), a polyethoxylated castor oil withexcellent surfactant qualities.

32 ANSI Z136.1-2007: American National Standard for SafeUse of Lasers, American National Standards Institute Inc.2007.

33 U. Alqasemi, H. Li, A. Aguirre and Q. Zhu, IEEE Trans.Ultrason. Ferroelectr. Freq. Control, 2012, 59, 1344–1353.

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