Correlation of Photophysical Properties with the Photoacoustic

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Correlation of Photophysical Properties With the Photoacoustic Emission for a Selection of Established Chromophores Maryam Hatamimoslehabadi, Stephanie Bellinger, Jeffrey La, Esraa Ahmad, Mathieu Frenette, Chandra S. Yelleswarapu, and Jonathan Rochford J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b07598 • Publication Date (Web): 02 Oct 2017 Downloaded from http://pubs.acs.org on October 4, 2017

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The Journal of Physical Chemistry

Correlation of Photophysical Properties with the Photoacoustic Emission for a Selection of Established Chromophores Maryam Hatamimoslehabadi†, Stephanie Bellinger‡, Jeffrey La†, Esraa Ahmad‡, Mathieu Frenette‡, Chandra Yelleswarapu†*, Jonathan Rochford‡* †

Department of Physics, University of Massachusetts Boston, 100 Morrissey Boulevard, Boston, MA 02125. ‡ Department of Chemistry, University of Massachusetts Boston, 100 Morrissey Boulevard, Boston, MA 02125.

* [email protected], [email protected] RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)

ACS Paragon Plus Environment

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ABSTRACT

An archetypal study is presented to correlate both the optical and photoacoustic (PA) properties for a diverse selection of dyes whose structural properties range across organic and inorganic, symmetric and asymmetric, neutral and cationic systems. Three distinct classes of molecular PA contrast-agents (MPACs) have been identified and classified according to their optical-PA response as either (i) linear absorbers (LAs)-linear PA emitter, (ii) saturable absorbers (SAs)weak PA emitter or (iii) reverse saturable absorbers (RSAs)-nonlinear PA emitter. The molecular characteristics instrumental in determining the nature of the dyes optical absorption properties, i.e. ground state molar extinction coefficient (εg), excited state molar extinction coefficient (εe) and excited state lifetime (τ), are discussed to aid in the interpretation of a molecule’s optical vs PA response. An excellent linear PA emitter is established in crystal violet, which exhibits the strongest possible PA signal under low laser fluence conditions in both PA z-scan and tomography experiments. Ultimately, however, nonlinear reverse saturable absorber (RSA) materials are anticipated to be the most promising dye category for generation of an enhanced nonlinear PA response. Effective RSA behavior is expected for materials showing a high ratio of their excited state vs ground state absorption (εe/εg) whilst also possessing a long-lived excited state lifetime (τ) permitting sequential two-photon absorption. ZnTPP, C60, and methylene blue each show a nonlinear PA response which correlates well with their RSA optical behavior. Relative to the linear PA emission profile of crystal violet, a 3.8-fold enhancement is observed for the PA emission of ZnTPP at the highest laser fluence of 366 mJ cm-2. Similarly, C60 and methylene blue exhibit nonlinear enhancements of 2.15-fold and 1.38-fold, respectively. Finally, to investigate the practical pros and cons with respect to application of these dyes in PA imaging


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applications, a concentration dependence of their PA emission is presented at both low and high laser fluences, in addition to a complimentary photoacoustic tomography (PAT) study.

INTRODUCTION Nonlinear optical materials present a leading role in photonics and have become increasingly desirable by scientists in an aim to improve the efficiency of optical devices.1-7 Organic dye molecules are especially of great interest owing to their potential for high nonlinear optical properties, combined with their high degree of tunability by a wide availability of synthetic methods.8-10 Molecular dyes with large absorption cross sections or high fluorescence quantum yields (Φfl) can play an essential role in many research areas such as fluorescence imaging,11-12 optical power limiting,13-14 imaging microscopy,15-16 optical data storage,17-19 temporally and spatially resolved measurements,20 photodynamic therapy,21-23 photoacoustic (PA) imaging. 24-27 The PA effect involves the generation of acoustic waves in response to the absorption of electromagnetic radiation.28 PA spectroscopy is an effective tool to investigate non-radiative relaxation and multiphoton absorption processes for organic dyes in solution.29-31 A more recent incarnation of the PA phenomenon is PA imaging which takes advantage of the low scattering propensity of ultrasound waves in biological tissue and also maintains high optical contrast allowing for high contrast imaging of biological tissue up to 5 cm in depth.29, 32-37 This is in stark contrast to solely photon based modalities, such as fluorescence imaging, which are highly prone to scatter at biological tissue and typically have sub-millimeter thresholds.38 Additionally, a low fluorescence quantum yield is not an issue in acquiring at least a linear PA response. In fact, conventional wisdom dictates that low fluorescence quantum yields are actually favorable when it comes to generating a strong PA response, in that non-radiative S1→S0 vibrational relaxation is responsible for generating the PA signal and thus a molecule’s potential as a PA contrast agent


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can be quantified as 1-Φfl or simply the quantum yield for non-radiative decay Φnr. The common assumption is that any dye with a low fluorescence quantum yield will make for a suitable PA contrast agent.39-40 In this respect, cyanine dyes primarily designed for in vivo NIR fluorescence imaging applications have been commonly applied as molecular PA contrast agents (here after referred to as MPACs) with metallic and polymeric nano-dimensional materials also attracting much interest of late.39, 41-49 This linear PA response induced by a single photon absorption is described by Eq. 1 PA = Γ I Φ

(1)

where εg is the ground state molar extinction coefficient at the incident wavelength, Cg is the ground state concentration of dye molecules, Γ is the Grüneisen coefficient, I is the incident photon fluence, and Φnr is the quantum yield for non-radiative decay. The Grüneisen coefficient,

Γ, is a constant that quantifies a medium’s ability to conduct sound efficiently that is defined by Eq. 2

=

(2)

where Vs is the velocity of sound, α is the thermal expansion coefficient of the medium, and Cp is the specific heat of the medium at constant pressure.50 While Eq. 1 holds true in a linear optical absorption regime, we recently demonstrated27 for a bis-styryl derived BODIPY dye that a high fluorescence quantum yield can give rise to a nonlinear optical absorption and an ensuing amplified PA response as described by Eq. 3 PA = Γ I Φ Γ …

(3)

where εe is the first excited state molar extinction coefficient at the incident wavelength and Ce is the concentration of excited state dye molecules.27, 51 In this case, consistent with Kasha’s rule,


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Φnr should equal unity for any excited state relaxation process equivalent to Sn→S1 or Tn→T1, hence Φnr is only relevant for the initial linear term in Eq. 3. Thus nonlinear absorption processes contribute quantitatively to the PA response resulting in a nonlinear PA amplification for a fluorescent dye even with a significantly large fluorescence (Φfl), or indeed phosphorescence (Φph), quantum yield, pending that the excited state absorption cross section is non-negligible. It is also important to note that the concentration of excited state dye molecules will vary with time and thus the laser pulse width is also an important consideration when optimizing such a nonlinear PA amplification with respect to the lifetime of the excited state chromophore. The PA technique is extremely advantageous as an imaging modality, however due to the current lack of established PA contrast agents its application has not advanced as broadly or rapidly as expected. Since there are vast varieties of commercially available chromophores, a desire for investigating their optical and corresponding PA properties is therefore warranted to bridge this gap. The work herein describes the PA response for a select yet diverse set of readily available visible dyes. Selection of these dyes was based upon their commercial accessibility and established, yet diverse, photophysical properties as discussed below. This study also utilizes photoexcitation at the 532 nm first harmonic of a common Nd:YAG nanosecond pulsed laser. The goal of this study is not only to characterize the PA response for these established dyes but also to correlate the nature of each PA response with the dye’s photophysical properties thus establishing the photophysical pathway of each dye for generating its PA signal. The selection of dyes investigated is illustrated in Figure 1.


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Figure 1. Molecular structures of dyes investigated in this study.

In completing this study, three distinct classes of MPACs have been identified and classified according to their optical response as either (i) linear absorbers (LAs), (ii) saturable absorbers (SAs) or (iii) reverse saturable absorbers (RSAs). The optical LA, SA and RSA classification of dye molecules is well established in the field of nonlinear optics using the optical Z-scan method.52 This report is the first example, to the best of our knowledge, that attempts to correlate the nature of LA, SA, and RSA dyes to their PA properties. The molecular characteristics


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instrumental in determining the type of optical absorption properties displayed have been determined to be ground state molar extinction coefficient (εg), excited state molar extinction coefficient (εe) and excited state lifetime (τ). As discussed in detail below, the nature of a molecule’s optical and PA response is also highly dependent upon the laser fluence. In summary, three distinct types of absorption processes and corresponding PA signal responses can be categorized as follows: i) Linear absorber (LA) → linear PA emitter (εg ≠ 0, τ τlaser). Similar to a linear absorption process, saturable absorbers exhibit negligible or zero excited state absorption. However, excited state lifetimes longer than the laser pulse width results in ground-state photo bleaching and an increase in incident photon transmission over the pulse width at increasing laser fluence. iii) Reverse-saturable absorber (RSA) → nonlinear PA emitter (εg ≠ 0, εe ≠ 0, τ > τlaser). Nonzero absorption cross sections of both the ground and excited states, as well as an excited state lifetime longer than the pulse width, results in a nonlinear increase in absorption and PA response at increasing laser intensity as described by Eq. 3. Ground-state photobleaching occurs but does not impede excited-state absorption.51 Typical optical and acoustic responses for each of these three scenarios are discussed below in the results and discussion section of this manuscript. Suffice to say however that nonlinear


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reverse saturable absorber (RSA) materials are anticipated to be the most promising dye category for generation of an enhanced nonlinear PA response. Effective RSA behavior is expected for materials showing non-zero excited state absorption (εe) and long-lived excited state lifetimes (τ). Key to nonlinear PA enhancement is the ratio of εe: εg at the incident wavelength, which is of course dependent upon the relative concentrations of excited versus ground state molecules during the laser pulse, and hence the strong dependence of a dye’s PA response upon the incident laser fluence.

EXPERIMENTAL SECTION Materials All dyes were sourced commercially from Sigma Aldrich apart from BODIPY53 and curcuminBF254 which were prepared according to their literature procedures. Crystal violet chloride salt was purchased from Sigma Aldrich and recrystallized via salt metathesis with an aqueous solution with NH4PF6 to generate the crystal violet PF6 salt. Spectroscopy UV-Vis spectra were measured in spectroscopic grade solvent (Sigma Aldrich) at room temperature on an Agilent 8452 spectrophotometer using a 1 cm path length quartz cell. Steady state and time-resolved fluorescence measurements were carried out on a Photon Technology International Quantamaster 40 & 25 fluorimeter at room temperature. Quantum yields were calculated by the optically dilute technique with fluorescein (aqueous NaOH, pH = 1, λexc = 390 nm, Φref = 0.925) and rhodamine 6G (neat acetonitrile, λexc = 480 nm, Φref = 0.94) used as reference standards according to Eq. 4.

Φ = !"

"

#

%$'

& '

Φ

(4)


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The subscript “s” refers to the unknown sample and the subscript “ref” to the reference sample, A is the absorbance at the excitation wavelength, I is the integrated emission area, and η is the solvent refractive index. Excitation and emission slits were both set to 5 nm. Fluorescence lifetimes (τ) were recorded at room temperature at the emission maximum following LED excitation at 456 nm or 572 nm. The radiative rate constant (kr) and nonradiative rate constant (knr) were both calculated from τ and Φ by using Eqs. 5-8. ( =

(5)

)

(6)

) )*#

Φ = )

)*#

,

+ = + =

(7)

., -

(8)

Photoacoustic z-scan studies and low vs. high fixed irradiance studies All the samples were dissolved in the stated solvents using a 2.0 mm path length quartz cuvette in order to prepare solutions of 0.3 optical density (50% transmission) at 532 nm excitation wavelength. With an incident angle of 45° giving an effective path length of 2.83 mm, this is equivalent to a linear absorption coefficient (α) of 345 m-1 at 532 nm for each sample. The UVVis absorption spectra of each sample over the wavelength range of 200-800 nm were monitored before and after PAZ-scan measurements to ensure sample stability using an Agilent Cary 60 UV-Vis Spectrophotometer. Using a lens of 20 cm focal length, the light from a Q-switched, frequency doubled Nd:YAG laser (Minilite II, Continuum) with a repetition rate of 10 Hz producing 532 nm laser pulses of 3 ns pulse width was focused on the sample to perform PAZscan on the samples. The 2 mm path length quartz cell containing the sample solution was placed


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in a sample holder at 45° with respect to the incident laser light while being surrounded by deionized water for ultrasound coupling. The sample holder was mounted on an automated XYZ translation stage (Thorlabs NRT 150) so that it could be moved gently in very small steps. As the sample translated along the laser beam direction through the focal plane of the lens, the sample confronted a moderately increasing laser intensity which reached its maximum at focus then moving the sample forward, it experienced a gradual decrease in laser energy. Depending on the absorption properties of each dye, the optical light transmittance and the generated photoacoustic signal changed by moving the sample along the laser beam direction due to changes in the laser energy transmitted through the sample. The photoacoustic response of each sample was detected by a 10 MHz focused water immersion transducer and the laser light transmitted through the sample was collected by a photodiode. The beam waist of the laser light was measured to be about 70 /m at focal point of the lens where the sample experiences the highest intensity of the laser. The energy of the laser pulses before it reaches the focusing lens was 60 / J. A neutral density filter of 1.00 OD was placed in front of the detector to ensure the linearity of the optical detector. For variable dye concentration experiments conducted at a low laser fluence of 13 mJ cm-2 (4.39 x 106 W cm-2 irradiance) PA data was acquired while placing the sample at a distance far from the focal plane (far field). For variable dye concentration experiments conducted at high laser fluence (360 mJ cm-2) PA data was acquired while placing the sample at the focal plane. Photoacoustic tomography Dye solutions were prepared similar to PAZ-scan experiments, i.e. 0.3 optical density (50% transmission) at 532 nm excitation wavelength in a 2.0 mm path length quartz cuvette, and subsequently transferred to 1 mm diameter borosilicate glass tubes giving a final optical density


10

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of 0.15. To perform photoacoustic tomography all borosilicate glass tubes were placed parallel inside a sample housing unit filled with water. A combination of two linear translation stages (Thorlabs NRT 150) with increment movement size of 0.2 mm in longitudinal direction and 0.5 mm in the direction along the capillary tube length was used to move the sample holder along a planar 2D trajectory. A 10 MHz focused transducer with 25.5 mm focal length (Olympus V311SU) was positioned at 2.54 cm vertical translation from the tubes. A right angle prism was situated in the setup to direct the laser beam of the Nd: YAG laser onto the tubes. The PA signal was amplified with 30 dB gain on a pulser-receiver (Olympus 5072PR) and digitized using an oscilloscope (WaveRunner 625Zi, Lecroy). At each scanning step, the signal was averaged over 20 laser pulses. A LabVIEW interface was used to acquire the signal data while communicating with the stage controllers to control over scanning movements. The PA image was then reconstructed using MATLAB to compute the absolute value of the Hilbert envelope of the acquired signal at each position.

RESULTS & DISCUSSION A diverse selection of dyes were chosen to encompass a wide variety of photophysical properties and ascertain their influence on the PA response upon 532 nm laser excitation. Structurally these dyes range across organic and inorganic, symmetric and asymmetric, neutral and cationic systems. Consequently, this provides for a diverse range of photophysical properties which are here correlated versus their PA response. For example, the BODIPY chromophore exhibits a sharp visible absorption and high fluorescence quantum yield whereas crystal violet exhibits a broad absorption and is non-emissive, while C60 and [Ru(bpy)3](PF6)2 provide access to a triplet T1 excited state. A detailed account of each dye’s photophysical properties is beyond


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the scope of this manuscript, however a summary of their UV-Vis electronic absorption and emission properties are summarized in Table 1 with a brief outline of their UV-Vis electronic absorption and emission properties to follow.


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Table 1. Photophysical properties of all dyes including solvent, absorption and emission maxima, and calculated extinction coefficients at 532 nm. Compound

Solvent

Absorption

ε @ 532 nm

Emission

Stokes shift

λmax (nm)

(M-1 cm-1)

λmax (nm)

(cm-1)

Φfl

τ

kr

knr

Φph

(ns)

(s-1)

(s-1)

a

Crystal Violet

THF

595

45,600

-

-

-

-

-

-

BODIPY

THF

502

700

514

465

0.34

2.2

1.55 x 108

3.00 x 108

Nile Red

THF

529

3,900

594

2069

0.92

3.7

2.49 x 108

2.16 x 107

ZnTPP

THF

422

4,200

603, 654

2695

0.03

1.5

2.00 x 107

6.47 x 108

CurcuminBF2

THF

503

11,700

552

1765

0.43

1.2

3.58 x 108

4.75 x 108

Cy3

THF

555

53,500

575

627

0.16

0.3

5.33 x 107

2.80 x 109

Merocyanine 540

THF

564

55,100

584

607

0.33

2.3

1.43 x 108

2.91 x 108

Rhodamine 6G

THF:DCM (9:1)

540

65,700

568

913

0.54

2.7

2.00 x 108

1.70 x 108

Methylene Blue

THF:DCM (9:1)

660

1,200

678

402

0.14

1.6

8.75 x 107

5.38 x 108

C60

toluene

335

1,000

-

-

-

-

-

-

[Ru(bpy)3]2+

acetonitrile

452

1,300

616

5890

0.06

855

a

a

b

7.02 x 104

b

1.10 x 106

phosphorescence quantum yield b radiative and nonradiative rate constants were calculated using Φph as Φisc = 1.


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UV-Vis electronic absorption spectra Although the molecules in this study have been well characterized in the literature, their molar extinction coefficients have been confirmed here at the laser excitation wavelength of 532 nm in the appropriate solvents (Table 1). In an effort to limit solvent environment as a variable, tetrahydrofuran (THF) was identified as the best common solvent for the majority of dyes. Unfortunately, C60 and [Ru(bpy)3]2+ showed limited solubility in THF and were investigated in toluene and acetonitrile, respectively. It should be borne in mind that the Grüneisen coefficients of these samples may differ significantly from all dyes recorded in THF precluding a direct comparison to evaluate only the dye variable. Rhodamine 6G and methylene blue were also sparingly soluble in THF however it was here possible to use 9:1 mixtures of tetrahydrofuran:dichloromethane to achieve necessary concentrations (optical density = 0.3 at 532 nm). For reference, the visible absorption spectra for all investigated dyes are plotted as a function of molar extinction coefficient in figure 2. While C60 and ZnTPP possess sharp and intense high energy absorptions, relevant to this study are the symmetry prohibited and much less intense absorptions at lower energy (ε532 = 1.00 x 104 M-1cm-1 and ε532 = 4.20 x 104 M-1cm-1 respectively).55-56 The [Ru(bpy)3]2+ dye displays broad metal-to-ligand charge-transfer electronic transitions in the visible portion of the spectrum with an experimentally determined ε 532 = 1.30 x 103 M-1cm-1. It can be seen that curcuminBF2 (fwhm= 2991 cm-1), crystal violet (fwhm = 2424 cm-1) and methylene blue (fwhm= 958 cm-1) all display broad absorption peaks typical of increased access to S1 excited state vibrational modes. Conversely, more intense and narrow absorption profiles are exhibited by rhodamine 6G (ε532 = 6.57 x 104 M-1cm-1), which displays the most intense absorption in THF at 532 nm, followed by Cy3 (ε532 = 5.35 x 104 M-1cm-1), merocyanine 540 (ε532 = 5.51 x 104 M-1cm-1) and BODIPY (ε532 = 7.00 x 102 M-1cm-1).


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20

ZnTPP C60 Methylene blue [Ru(bpy)3]2+

15

ε (104 M-1 cm-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Crystal violet BODIPY CurcuminBF2 Cy3 Merocyanine 540 Nile red Rhodamine 6G

10

5

0 400

450

500

550

600

650

700

Wavelength (nm)

Figure 2. Visible electronic absorption spectra of all dyes. For solubility considerations, spectra were obtained in various solvents (re. Table 1). The S0→S2 (Soret band) peak has been cut off to allow focus on some of the weaker dye absorption bands occurring at 532 nm.

Steady-state and time-resolved emission properties A collection of normalized emission spectra are presented in figure 3. Of the series of dyes investigated, BODIPY displays the highest energy fluorescence emission maximum (λem = 514 nm) consistent with it exhibiting a very small stokes shift of just 465 cm-1. Only the more red shifted methylene blue dye displays a smaller stokes shift of 402 cm-1 (λem = 678 nm), albeit with a lower fluorescence quantum yield (Φfl = 0.14) due to efficient intersystem crossing to its nonemissive T1 state. Similar to their absorption profiles, Cy3, merocyanine 540 and rhodamine 6G


15


display relatively narrow fluorescence emission peaks with small stokes shifts of 627 cm-1, 607 cm-1and 913 cm-1 respectively. In contrast, curcuminBF2 exhibits a broad emission band in THF with a larger stokes shift of 1735 cm-1 consistent with greater vibrational freedom in its S1 excited state, presumably due to the sterically unhindered vinyl arms of its quadrupolar donor-πacceptor-π−donor structure. Of note is the large stokes shift exhibited by [Ru(bpy)3]2+ of 5890 cm-1 which is due to quantitative ISC to its significantly lower energy T1 excited state, hence its broad emission spectrum being characterized as a phosphorescence emission.

ZnTPP Methylene blue [Ru(bpy)3]2+

1.0

BODIPY CurcuminBF2

Normalized Emission

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Cy3 Merocyanine 540 Nile red Rhodamine 6G

0.5

0.0 500

600

700

800

Wavelength (nm) Figure 3. Normalized emission spectra recorded at room temperature in various solvents (re. Table 1.1). Emission quantum yields range substantially throughout the series as is expected by the differing nature of emission spectra, multiplicity and lifetimes. Nile red displays the highest Φfl determined here to be 0.92 which is consistent with the longest observed fluorescence lifetime of 3.7 ns of the series of dyes.57 In contrast crystal violet and C60 are non-emissive and are


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potentially excellent linear absorbing PA emitters due to rapid non-radiative decay of their S1 and T1 excited states, respectively. The fluorescence quantum yield of Φfl = 0.16 for Cy3 is low consistent with its short fluorescence lifetime of 0.3 ns indicative of rapid non-radiative decay (knr = 2.80 x 109 s-1). ZnTPP and [Ru(bpy)3]2+ display weak fluorescence and phosphorescence emission, respectively, with Φfl = 0.03 and Φph = 0.06. Other molecules in the series show moderate Φfl values between 0.14-0.54 consistent with their observed 1τ and stokes shifts.

Optical and photoacoustic z-scan studies In order to effectively correlate the PA emission of each dye with its photophysical properties, optical and PA z-scan (PAZ-scan) experiments were performed on all samples.58 In this method all advantages offered by the conventional optical Z-scan technique and the remarkably sensitive PA detection are merged together. It should be emphasized that to unambiguously correlate all optical and PA signals each set of experiments was conducted on the same sample cuvette. The optical response of all dyes is presented in figure 4 illustrating the variation of optical absorbance of each dye with respect to the incident laser fluence ranging from 47 mJ cm-2 to 366 mJ cm-2. Similarly, the corresponding PA response of all dyes is presented in figure 5.


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Irradiance (107 W cm-2) 2

4

6

8

10

12

0.7

0.6

Optical absorbance (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.5

C60

ZnTPP

[Ru(bpy)3](PF6)2

Curcumin BF2

BODIPY Rhodamine 6G Merocyanine 540 Methylene Blue

Nile red Cy3 Crystal violet

0.4

0.3

0.2

0.1

0.0 40

80

120

160

200

240

280

320

360

Laser fluence (mJ cm-2)

Figure 4. Optical absorption as a function of laser fluence and irradiance for the complete series of commercial dyes absorbing at 0.3 OD in a 2 mm quartz cuvette at 532 nm.


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Irradiance (107 W cm-2) 2

Photoacoustic amplitude (a.u.)

0.25

4

6

8

10

12

(a)

ZnTPP C60 Methylene Blue [Ru(bpy)3]2+

0.20

Crystal violet

0.15

0.10

0.05

0.00 40

80

120

160

200

240

280

320

360

-2

Laser fluence (mJ cm ) Irradiance (107 W cm-2) 2

4

6

8

10

12

(b)

Crystal violet BODIPY Curcumin BF2

0.06

Photoacoustic amplitude (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cy3 Merocyanine 540 Nile Red Rhodamine 6G 0.04

0.02

0.00 40

80

120

160

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Figure 5. Photoacoustic response of all dyes plotted vs. laser fluence and irradiance. For clarity RSA dyes (a) and SA dyes (b, note reduced y-scale) are plotted independently with crystal violet (▲) included as a reference LA dye in both cases. All samples were recorded at 0.3 OD in a 2 mm quartz cuvette at 532 nm.


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Considering the optical signals in figure 4, it can be seen that crystal violet displays a characteristic LA optical response with incrementing laser fluence at least until 200 mJ cm-2 after which a slight depletion in absorption is observed due to sample saturation. The corresponding PA signal for crystal violet (Fig. 5) shows a linear increase with increasing laser fluence, again consistent with a linear absorbing material. The LA behavior of crystal violet can be explained by its relatively constant ground state optical absorption at 532 nm due to its ultrafast nonradiative decay. Beyond 200 mJ cm-2 laser fluence the PA emission of crystal violet does begin to deviate from linearity consistent with the onset of optical saturation as observed in figure 4. With its short singlet excited state lifetime of just 6 ps, and its lack of excited state absorption at 532 nm, crystal violet represents the most consistent LA reference dye investigated in our lab to date. This makes crystal violet an excellent PA reference material with respect to the contrasting SA and RSA behaviour exhibited by alternative dye samples. In fact, due to the absence of inner filter effects which so often hinder fluorescence, we have successfully utilized crystal violet to demonstrate the advantageous linear response of the PA technique up to an absorption value of 5.0 - this corresponds to a transmission of just 0.1% (Fig. 6). Similarly, the SA dye Cy3 also exhibits a linear PA response under identical conditions simply confirming the consistency of the PA technique even with such excessive absorbance. Cy3 exhibits a weaker PA response however in figure 6 due to its competing fluorescence excited state decay and SA behaviour.


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Figure 6. Optical transmission and photoacoustic emission of crystal violet and Cy3 recorded with a sample absorbance of up to 5 (0.1% transmission) at a laser fluence of 500 mJ cm-2 (1.70 x 108 W cm-2 irradiance).

Dyes with z-scan optical absorbance signals falling below that of crystal violet in figure 4 (i.e. nile red, merocyanine, BODIPY, Cy3, curcuminBF2, and rhodamine 6G) can be characterized as SA materials and exhibit a correspondingly weak/flat PA response with increasing laser fluence (Fig. 5b). For example, rhodamine 6G shows a weak PA emission. This is consistent with its negligible excited state absorption combined with the nature of its singlet excited-state (S1) which directs >90% of its absorbed photons to a S1→S0 fluorescence radiative decay (Φfl~ 0.9) leaving less than 10% of the excited state energy available for contribution to a non-radiative PA


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response. This weak/flat PA response for SA dyes is consistent with ground-state photobleaching, negligible or zero excited state absorption and an increase in incident photon transmission over the laser pulse width at increasing laser fluence. In contrast, ZnTPP, C60, and methylene blue each show RSA behavior in the optical z-scan experiment (Fig. 4). The PA profile of these three dyes (Fig. 5a) correlates well with their RSA optical behavior with a nonlinear enhancement of the PA response observed to a greater or lesser degree for each of these dyes.27 Relative to the linear PA emission profile of crystal violet, a 3.8-fold enhancement is observed for the PA emission of ZnTPP at the highest laser fluence of 366 mJ cm-2. Similarly, C60 and methylene blue exhibit nonlinear enhancements of 2.15-fold and 1.38-fold, respectively. Finally, the single inorganic dye here studied [Ru(bpy)3]2+ exhibits a weak RSA optical response and its PA emission is correspondingly weak. However, at high laser fluence (> 240 mJ cm-2) a nonlinear PA enhancement is observed possibly due to a weak excited state absorption cross section for the T1 state of [Ru(bpy)3]2+ at 532 nm. Dye concentration dependence – low laser fluence Due to the wide range of εg displayed by the molecules at the 532 nm excitation wavelength here utilized, an evaluation of the PA signal produced as a function of optical density/molar concentration has also been conducted at fixed low and high laser fluences. This approach effectively examines the potential of each dye as a PA contrast agent with respect to both the dye and light dosage. The optical z-scan absorption and corresponding PA emission at low laser fluence (13 mJ cm-2) is presented in figure 7. It is noteworthy that the low laser power of 13 mJ cm-2 was chosen as it lies below the maximum permissible exposure limits of 20 mJ cm−2 at 532 nm according to the American National Standards Institute (ANSI).59


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Figure 7. Optical z-scan absorption (top) and photoacoustic emission (bottom) of all dyes as a function of optical density and concentration, respectively, measured at 13 mJ cm-2 laser fluence (4.39 x 106 W cm-2 irradiance). A narrow concentration scale is utilized on the left side of the optical z-scan plot to resolve the similar optical response of rhodamine 6G, merocyanine 540, Cy3 and crystal violet. The same crystal violet data is also plotted for reference with the broader concentration scale on the right side of the optical z-scan plot.

Due to the relatively low density of photons at 13 mJ cm-2 laser fluence, RSA and SA behavior is not observed and all dyes behave as LA materials. Thus the plot of optical z-scan absorption vs dye concentration is effectively a standard Beer plot. While this observation is unremarkable it warrants inclusion if not just for clarification but also as it is in stark contrast to the optical zscan data recorded at high laser fluence (vide infra). All dyes exhibit the expected linear correlation between optical absorption and dye concentration with the observed slopes equivalent to the corresponding molar absorption coefficients at 532 nm (re. Table 1). The corresponding PA emission exhibited by each dye at low laser fluence (Fig. 7, bottom) is also linear in accordance with Eq. 1. The PA response for each dye plotted versus dye concentration effectively illustrates the dosage vs PA response profile for each dye across the series. The PA emission varies considerably across the series of dyes with respect to concentration even at the low laser fluence of 13 mJ cm-2. The trend in observed PA amplitudes with respect to dye concentration is strongly dictated by the magnitude of the S0→S1 molar extinction coefficient at the excitation wavelength. For example, even though rhodamine 6G exhibits a very weak PA emission when comparing all dyes at equal optical density (Fig. 5), this weak PA response is effectively compensated by its excellent ability at absorbing photons at 532 nm (εg @ 532 nm =


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65,700 M-1 cm-1). Whilst εg at the excitation wavelength does dominate the concentration dependence of the PA response under a LA regime it is still influenced by the nonradiative rate constant of each dye; hence the trend in PA emission observed across the series of dyes is a balance of both variables. This is clarified upon taking a second glance at Eq. 1 where the slope of the PA vs plot equals Γ I Φ, the laser fluence I is constant and the Grunesien coefficient Γ is only a minor contributing factor upon changing the solvent, e.g. C60 and [Ru(bpy)3]2+. It follows that crystal violet exhibits the highest gradient in its PA amplitude with respect to concentration. Although its molar extinction coefficient at 532 nm (45,600 M-1 cm-1) is slightly lesser in comparison to rhodamine 6G, crystal violet is non-emissive and thus effectively possesses a PA quantum yield of unity. Cy3 and merocyanine 540 also exhibit excellent ‘PA amplitude vs concentration’ behavior, almost comparable to crystal violet, followed closely by rhodamine 6G and curcuminBF2. ZnTPP and nile red exhibit a weaker PA vs concentration response due to an order of magnitude decrease in molar extinction coefficient at 532 nm relative to the former dyes. This deficiency is even more exasperated for the remainder of the series which only exhibit weak absorption at 532 nm with the poorest performing being BODIPY with an εg of just 700 M-1 cm-1 at 532 nm. Dye concentration dependence – high laser fluence Plots of optical absorption and PA amplitude vs concentration are presented in figure 8 for the same series of dyes irradiated at a high laser fluence of 360 mJ cm-2. All SA dyes (BODIPY, curcuminBF2, Cy3, merocyanie 540, nile red, rhodamine 6G) and the LA crystal violet dye exhibit a linear optical and PA response with increasing dye concentration. The optical response of all SA dyes underperform relative to the LA crystal violet reference as anticipated due to ground state bleaching and an absence of excited state absorption. Although at higher laser


25


fluence, all SA dyes show a similar trend in optical absorbance vs dye concentration as observed under low laser fluence conditions. This is due to the sole contribution of their S0→S1 transition and lack of nonlinear absorption contributions consistent with SA behavior (Figs. 7 & 8 top). If anything the differences in slope for each dye are slightly more pronounced at high laser fluence.

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Figure 8. Plots of optical z-scan absorption vs concentration recorded at 360 mJ cm-2 laser fluence (1.22 x 108 W cm-2 irradiance) for SA dyes (top) and RSA dyes (bottom) each plotted alongside the LA crystal violet reference dye. The RSA dyes of C60, methylene blue, and [Ru(bpy)3]2+ each exhibit an almost identical optical absorbance vs concentration response at high laser fluence (Fig. 8, bottom), which is consistent with their comparable S0→S1 molar extinction coefficients and weak RSA behavior. Relative to the majority of the SA dyes (apart from BODIPY) the latter RSA dyes exhibit a weak optical response vs dye concentration slightly lesser than that of the nile red dye. In contrast, ZnTPP exhibits a strong optical absorbance vs concentration response consistent with a significant excited state absorption for this dye at 532 nm (Fig. 8, bottom).60 At low concentrations, the nonlinear enhanced optical absorption of ZnTPP is comparable to the linear optical absorption of


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crystal violet. This is remarkable due to their order of magnitude difference in S0→S1 molar extinction coefficients (4,200 M-1 cm-1 and 45,600 M-1 cm-1, respectively) which confirms the nonlinear contribution to the ZnTPP response as confirmed earlier (Fig. 4). Although the optical absorbance of ZnTPP increases with dye concentration, a decreasing nonlinear curve of its optical response is also observed. This is explained by a reduced nonlinear absorption contribution at increased dye concentration. At a fixed laser fluence upon increasing the dye concentration the number of ground state dye molecules competing for the same incident density of photons for excitation is increased; hence the lower probability of two photon absorption as more photons are participating in the initial S0→S1 excitation process. The amplitude of PA emission vs dye concentrations recorded at high laser fluence is plotted in Figure 9. A linear enhancement of PA signal amplitude is observed with the increase in concentration for all studied dyes. The PA amplitude soars sharply with concentration for crystal violet, closely followed by ZnTPP, curcuminBF2 and merocyanine 540 with remaining SA and RSA dyes exhibiting a weak dependence of PA emission on dye concentration. Except for ZnTPP, the substantial growth of PA signal in these dyes is conforming to their extinction coefficients. This confirms that the nonlinear contribution of the RSA dyes C60, methylene blue, and [Ru(bpy)3]2+ is negligible under the experimental conditions. At high laser fluence, ZnTPP featuring strong RSA behavior shows superior characteristics in the conversion of absorbed photons into acoustic signal despite its relatively low S0→S1 extinction coefficient at 532 nm. Despite the strong nonlinear response of ZnTPP at high laser fluence, the PA emission of the LA crystal violet dye is still slightly superior at 532 nm irradiation due to its order of magnitude greater molar extinction coefficient.


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Figure 9. Photoacoustic emission of all dyes as a function of concentration measured at 360 mJ cm-2 laser fluence (1.22 x 108 W cm-2 irradiance).

Photoacoustic tomography Finally, to compare the capability of each dye as a PA imaging contrast agent, a beneficial study is to perform photoacoustic tomography (PAT). The same series of LA, SA and RSA dyes were recorded side by side along with a THF solvent blank (Fig. 10) using the apparatus detailed in the experimental section. The same experiment was conducted at 13 mJ cm-2 and 360 mJ cm-2 to allow correlation of each dye’s PA tomography performance with the corresponding PA zscan experiments discussed above.


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Figure 10. (top) PAT image recorded at the low laser fluence of 13 mJ cm−2 (4.39 x 106 W cm-2 irradiance) and high laser fluence of 360 mJ cm-2 (1.22 x 108 W cm-2 irradiance) at λexc = 532 nm; dimension = 32 mm×5 mm. All samples had 0.15 O.D. at 532 nm in the 1 mm path length borosilicate capillary tubes. The x-axis color scale represents the normalized acoustic intensity. (bottom) A plot of the normalized PA emission extracted from both low and high fluence PAT images illustrating the nonlinear PA amplification for RSA dyes.

At low laser fluence PAT establishes the LA crystal violet dye as the superior PA contrast agent when exposed to low laser fluence, benefitting from its high molar absorptivity at 532 nm


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and short lived excited state. At high laser fluence, consistent with the PA z-scan experiments presented above, ZnTPP exhibits the strongest PA contrast at high laser fluence followed by C60, methylene blue and crystal violet. All of the SA dyes show weak contrast features as a result of their ground state bleaching at high laser fluence.

CONCLUSIONS A selection of well characterized and commercially available dyes have been evaluated in order to establish a relationship between their photophysical and PA properties. Optical and PA z-scan experiments have revealed four of the select dyes, namely ZnTPP, C60, methylene blue, and [Ru(bpy)3]2+, possess RSA behavior upon excitation with a 3 ns pulse width of 532 nm laser photons. The impact of RSA behavior on the production of a PA signal is seen in comparing samples of equal optical density. For example, ZnTPP displays a 3.8-fold enhancement in PA emission relative to the linear absorbing and linear PA emitting crystal violet reference. Due to the wide range of S0→S1 molar extinction coefficients across the series of dyes at the 532 nm excitation wavelength, an evaluation of the PA signal produced as a function of molar concentration was also conducted at fixed low (13 mJ cm-2) and high (366 mJ cm-2) laser fluences. While the PA response under a LA regime is still influenced by the nonradiative rate constant of each dye, overall the trend in observed PA amplitudes with respect to dye concentration was found to be more strongly dictated by the magnitude of the S0→S1 molar extinction coefficient at the excitation wavelength. Remarkably, at high laser fluence, nonlinear optical absorption amplification of the PA emission for ZnTPP generates a PA response comparable to crystal violet thus compensating for an order of magnitude difference in its S0→S1 molar extinction coefficient. This result suggests that a dye which possesses strong RSA


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behaviour, similar to ZnTPP, at the excitation wavelength but which also exhibits a high S0→S1 molar extinction coefficient (εg) and good optical limiting properties (a high ratio of excited state vs ground state absorption) could be highly advantageous for in vitro PA microscopy applications.61

ACKNOWLEDGMENTS Funding for this research was provided by NIH grant U54CA156734. EA thanks the Oracle Education Foundation for financial support. ASSOCIATED CONTENT Open aperture optical z-scan data is provided in the electronic supporting information for all dyes.

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Correlation of Photophysical Properties with the Photoacoustic

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