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Abstract
We demonstrate that the Stimulated Emission Depletion (STED) concept, which is usually invoked for fluorescence, can be extended to photoacoustic effects. When two-nanosecond pulses of exciting and stimulating light are synchronized, 80% of the acoustic signal generated through excited state absorption (ESA) can be quenched. Regarding the cross-sections for stimulated emission and ESA, a model gives a good order of magnitude in the depletion efficiency. The transient molecular orientation, usually measured via the fluorescence anisotropy, can be accessed in photoacoustic when STED is implemented.
© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Optical-resolution photoacoustic microscopy (OR-PAM) [1] is an emerging technique where ultrasound is produced locally upon light absorption by a thermoelastic effect. It cumulates the advantage of resolution due to optical excitation and sensitivity to absorption with the detection of ultrasonic waves. OR-PAM is a hybrid technique where the lateral resolution is given optically while its longitudinal resolution is limited acoustically. Recently, exploiting an Excited State Absorption (ESA) process, we have shown that the longitudinal resolution can become limited optically [2].
Until now, stimulation emission depletion (STED) has become a mature method for enhancing the spatial resolution in fluorescence microscopy [3], but it has never been envisaged in photoacoustic. However, the relaxation of an excited level population (Fig. 1(a)) is of common importance to both phonon emission and fluorescence. Hence, any phenomenon associated with depopulating excited levels is able to quench the fluorescence and the phonon emission. In general, all mechanisms associated with an excited state population could be affected by a STED effect. Along with the most popular example provided by STED fluorescence microscopy [3], photolithography [4] and Raman [5] have also shown an enhanced spatial resolution by STED. Recently, picosecond pump-probe experiments [6] and sound detection at laser threshold [7] have revealed that photoacoustic and stimulated emission can be combined. Nevertheless, the underlying mechanism has not been analyzed. This paper demonstrates that a depopulation by stimulated emission is suitable for photoacoustic signal quenching in a microscopic scale, and this technique, named Sound Stimulated Emission Depletion (S-STED), is comparable to Fluorescence Stimulated Emission Depletion (F-STED).
Fig. 1. (a) Jablonski diagram with excitation, vibronic/non-radiative relaxation, fluorescence, and stimulated emission. (b) Absorbance and fluorescence spectrum of Coumarin-490 in ethanol, where arrows indicate the two wavelengths of excitation (355 nm) and stimulated emission (532 nm).
2. Materials and methods
As shown in Fig. 1(b), we found that Coumarin 490 is a suitable dye to show S-STED when the absorption and fluorescence spectrum in an ethanol solution are compared with the two wavelengths (355 nm and 532 nm) of laser, respectively. Although the two laser wavelengths are slightly different from the maximum of the absorption and fluorescence spectrum, the large Stokes shift is beneficial to avoid any absorption at the wavelength of stimulation beam (532 nm). When 532 nm is used for excitation, Rhodamine 6G is suitable to observe optical resolution photoacoustic microscopy through ESA [2]. Therefore, Rhodamine 6G can be another candidate for S-STED. However, its small Stokes shift may limit the stimulation efficiency. In order to perform S-STED in Coumarin 490 and Rhodamine 6G, we used two different laser tandem: Tandem-1 and Tandem-2, respectively. Both have self aligned and self synchronized excitation and stimulation beams. In tandem-1 a nanosecond self Q-switched Nd:YAG laser (1064 nm, 10 to 130 KHz repetition rate, SNP models from TEEM- Photonics Inc) produces a 532 nm beam by second harmonic generation (SHG) in a potassium titanyl phosphate (KTP) crystal. Additionally a 355 nm beam is generated by sum frequency generation (SFG) of the residual 1064 nm and 532 nm in a beta barium borate (BBO). In Tandem-2 a more powerful 0.5 nanosecond self Q-switched Nd:YAG laser (1064 nm, 1 KHz repetition rate, PNP model from TEEM-Photonics) is frequency doubled to give a 532 nm beam. However, the 532 nm beam is used not only for excitation but also for generating a beam at 600 nm through a Rhodamine 101 dye laser. The dye laser cavity is obtained by the two parallel semi-reflective faces of a 1cm laser dye cuvette (model 119.000F-QS from Hellma Inc.) perfectly perpendicular to the pump beam. Therefore the partially transmitted 532 beam and the 600 nm beam are collinear. For Tandem-1 the two beams are perfectly overlapped temporally. On the other hand, for tandem-2 the two pulses partially overlap, the maximum of the 600 nm pulse is delayed by ∼ 100 ps from the maximum of the 532 nm pulse. The experimental setups of S-STED in Fig. 2(a and b) are similar to the setup we have used for F-STED [8] except an addition of ultrasound detection. The two beams are focused simultaneously to the dye solution in a silica cuvette by an achromatic microscope objective lens (Mitutoyo Plan APO NUV) with an effective numerical aperture of 0.1, and the residual 1064 nm beam was removed by a filter. The nanosecond regime and the low repetition rate (several kHz) are important since they allow an efficient excitation and depletion of organic dyes without excessive average power and photo-damage. For the ultrasound detection a piezoelectric transducer (Olympus V2022) with 75 MHz bandwidth is attached directly to the bottom of a spectrometer cuvette. Two cascaded large band amplifiers (Mini-circuits ZFL-500+) are used before performing a fast signal averaging by a digital scope (Lecroy Wavesurfer). The laser pulse is also detected by a fast photodiode for triggering. Figure 2(c) shows a typical photoacoustic signal. In such a particular geometry of acoustic detection, the ringing signals after pulse excitation can be attributed to an echo at the interface between the transducer and the cuvette, and the peak-to-peak photoacoustic signal becomes enhanced by tight focusing unless the detector bandwidth is limited [9]. Although it is difficult to exclude the photo-bleaching effect in a dye solution, Coumarin 490 and Rhodamine 6G are known to be very stable against laser excitation. It is noticeable that we used a large volume of dye (the cuvette volume is 3.5 mL). In this case, a convection inside the liquid provides a renewal of fresh dye in the beam even if a photo-bleaching occurs. In spite of repeated experiments with the same sample, a signal fading effect has never been observed.
Fig. 2. (a) Experimental setup of S-STED in Coumarin 490. While two beams of 532 nm (SHG) and 352 nm (SFG) with a ns pulse duration are generated from the fundamental beam of 1064 nm, they are self-synchronized and self-aligned. The two beams are focused by an achromatic lens into a cuvette, which is filled with an alcoholic solution of Coumarin 490. While 355 nm is used for excitation, 532 nm induces a stimulated emission in Coumarin 490. Photoacoustic signal is collected by a 75 MHz bandwidth piezoelectric transducer attached directly to the cuvette, and the amplified signal is sent to a fast oscilloscope. A camera is also placed near the cuvette to image fluorescence simultaneously. (b) For S-STED in Rhodamine 6G, 532 nm (SHG) beam is used not only for excitation, but also for generating a stimulating pulse of 600 nm through Rhodamine 101. Note that the stimulating beam of 600 nm is delayed by ∼ 100 ps with a short pulse duration compared to 1 ns pulse duration of the excitation pulse (532 nm). (c) A typical photoacoustic signal collected by a 75 MHz bandwidth piezoelectric transducer.
3. Experimental results
3.1 Photoacoustic signal depletion
As shown in Fig. 3(a), photoacoustic signals in an alcoholic solution of Coumarin 490 under 355 nm excitation are compared in the presence (black) and absence (red) of a 532 nm stimulation beam. When the 532 nm stimulation beam is also involved together with the 355 nm excitation beam, a signal depletion appears significantly. However, the photoacoustic signal is not extinguished completely even if the stimulating power is increased. Because no photoacoustic signal is generated only by 532 nm beam excitation, the stimulation beam itself does not contribute to the background signal in Coumarin 490. Therefore, the background can be determined by the overlap condition at focus between excitation and stimulating beams but we have verified that a perfect overlap is achieved through a simultaneous observation of the STED effect on fluorescence. The best ratio of the peak-to-peak amplitude with and without STED is 0.18, this result implies that 82% of the photoacoustic signal generated only by 355 nm excitation becomes extinguished by the 532 nm stimulation beam. In Fig. 3(b), the depletion of photoacoustic signal is also observed in Rhodamine 6G dye, where the photoacoustic signal is generated by 532 nm beam and partially suppressed by 600 nm beam. Regarding the absorption spectrum of Coumarin 490 in Fig. 1(b), the large Stoke shift avoids any absorption at 532 nm wavelength. Therefore, no photoacoustic is generated when only the stimulation beam of 532 nm is used (Fig. 3(a)). However, it is difficult to avoid a small absorption in Rhodamine 6G when only the stimulation beam of 600 nm is used, whereby a minute photoacoustic signal appears (Fig. 3(b)).
Fig. 3. (a) When Coumarin 490 in ethanol (∼millimolar concentration) is excited by only 355 nm pulse with 0.4 µJ (black curve), strong photoacoustic signals appear. However, the signal becomes suppressed significantly when a stimulating 532 nm beam is added (red curve). When only the stimulation beam of 532 nm is used (blue), no photoacoustic signal is observed. The laser beam waist at the entrance of the dye is 1.5 µm. (b) With 532 nm excitation, photoacoustic signal of Rhodamine 6G dye in ethanol (500 µmol/L) is observed with (red) and without (black) 600 nm stimulating beam, where other experimental conditions are similar to those for Coumarin 490. In both the cases, the polarizations of excitation and stimulation beams are parallel. The time difference between Fig. 3(a) and 3(b) is irrelevant to sample properties but due to different delay line settings of ultrasonic transducers.
3.2 Effect of excitation and stimulation energy
As shown in Fig. 4(a), the ratio (R) of the photoacoustic signal with and without STED shows an exponential decrease for increased stimulating laser energy at 532 nm. It eventually levels off at a value of 0.18 corresponding to the remaining background. Regarding the range of laser fluence used in this experiment, two remarks can be made. First, the fluence used for stimulation is comparable to those used in conventional F-STED microscopy where a saturation of the stimulated emission is mandatory to achieve super resolution [10]. Secondly the range of fluence used for excitation is one commonly found in ORPAM experiments [11], but far above the fluence needed for fluorescence microscopy. Therefore, various nonlinearities may be present. Figure 4(b) shows the photoacoustic intensity signal as a function of excitation energy without any stimulation. In this experiment, a broad range of excitation energy was used, but the laser beam was defocused to avoid damaging the dye. The photoacoustic signal does not show a linear excitation dependence (see inset), but a quadratic dependence is observed up to 0.7 µJ.
Fig. 4. (a) Ratio of the photoacoustic signal of Coumarin 490 in ethanol (millimolar concentration) with and without STED is plotted as a function of stimulating energy at 532 nm. The excitation energy at 355 nm is 0.4 µJ, and the laser beam waist at the entrance of the dye solution is 1.5 µm. (b) Excitation (355 nm) laser energy dependence of photoacoustic signal for Coumarin 490 in ethanol is plotted as a function of the square of excitation laser energy. The linear scale excitation energy is also shown in inset. The beam waist at the entrance of the dye solution is 2.2 µm. The polarizations of excitation and stimulation beams are parallel
3.3 Polarization dependence
Using a dichroic wave plate (half-wave retardation at 532 nm and full-wave retardation at 355 nm), it is possible to adjust the polarization angle between the excitation and the stimulation beams. This technique enables to maintain the same conditions of alignment, pulse simultaneity, and beam intensity. As shown schematically in Fig. 5(a), the photoacoustic signal depletions in Coumarin 490 were measured separately with two different polarization configurations. Concerned with a photo-bleaching effect, we repeated experiments in different orders, but a signal fading was not observed. When excited only by 355 nm (①), a large photoacoustic signal is observed in Fig. 5(b). However, the signal becomes suppressed with 65% extinction when the two polarizations of excitation and stimulation are parallel (②). If the polarizations of excitation and stimulation beams are perpendicular to each other (③), the signal extinction becomes decreased to 47%.
Fig. 5. Given three optical configurations for the linear polarization of excitation (355 nm) and stimulation (532 nm) beams (a), photoacoustic signals are observed from Coumarin 490 in ethanol. The energy of the stimulation pulse is 0.9 µJ and the beam waist at the cuvette entrance is 1.5 µm.
4. Discussion
4.1 Excited state absorption
A first attempt to interpret our experimental results is to follow the usual scheme valid for F-STED. Figure 1 shows the energy diagram, restricted to the S0 ground state and S1 first excited state. In addition to the usual transitions involved in a laser excitation ($h{\nu _{ex}}$) and fluorescence cycle, we consider two additional paths of relaxation. One is stimulated emission, induced by a second laser with energy chosen to match the maximum of the fluorescence energy ($h{\nu _{fl}}$). Another one is the phonon emission since it is at the origin of the photoacoustic signal. Figure 1 also shows a typical example given by Coumarin 490 in ethanol. The energy difference between the maxima of the absorption and fluorescence spectra (the pseudo-Stokes shift) is not only due to phonon relaxation between the vibronic levels (the Stokes shift). A part of it goes into solvent relaxation. This part can be important for polar solvents such as ethanol [12]. Finally, the entire pseudo-Stokes shift energy is transformed into phonons. Practically this corresponds to the energy difference between the excitation and the fluorescence energy ($\Delta = h{\nu _{ex}} - h{\nu _{fl}}$). For a fluorophore having a fluorescence quantum efficiency (QE), non-radiative relaxation produces phonons with an average energy per molecule $\delta = (1 - QE)h{\nu _{fl}}$. Therefore, in absence of stimulation, the total phonon energy is $\delta + \Delta = h{\nu _{ex}} - h{\nu _{fl}}.QE$. Even if stimulation is efficient enough to completely deplete (radiatively) the excited level, a phonon background with an energy Δ is still present. Therefore, the minimum ratio R between the phonon energy with and without stimulation is given by
Obviously, this simple model is not satisfactory for experimental photoacoustic signal quenching in Coumarin 490 dye (R = 0.18) and Rhodamine 6G dye (R∼0.5). The two-level model predicts a linear dependence of the photoacoustic signal on the excitation fluence. However, when increasing the excitation laser fluence, the photoacoustic signal shows a quadratic dependence (Fig. 4(b)). A superlinear dependence has already been observed in other systems [14]. Particularly, this effect becomes pronounced when the final highly excited level becomes non-radiative through excited state absorption (ESA). In fact, a quadratic dependence is common for dyes with a good quantum efficiency, in presence of ESA.
As shown in Fig. 6 the absorption bands of Coumarin 490 have positions and amplitudes that favour the occurrence of ESA. The dynamic of the ESA in Coumarin 153 has been analysed carefully [15], and Coumarin 490 is known to have a high ESA cross-section (S1→Sn) at the third-harmonic of the Nd:YAG laser (3hν, 355 nm). In addition, as already measured, an efficient stimulated emission (S1→S0) occurs for the second harmonic (2hν, 532 nm). Therefore, the following mechanism is suggested:
Fig. 6. Absorption (black) and fluorescence spectra of Coumarin 490 in ethanol. The length of arrows for absorption and ESA are the same.
In absence of stimulation, the S1 level is excited and its population is simultaneously transferred to Sn. A fast non-radiative relaxation then occurs, which is at the origin of the photoacoustic signal. Considering the multi-phonon energy gap law [16] and the large energy gap between Sn and S1, the non-radiative de-excitation of Sn may follow two different routes: i) a direct Sn→S0 phonon emission with a total relaxed phonon energy of ΔE = 6hν and/or ii) a two-step relaxation Sn→S1→ S0 with a lower total released phonon energy since a part of the S1→ S0 relaxation is radiative ΔE= (6-2QE)hν.
To the contrary, the radiative depopulation of S1 by an efficient simulation forbids ESA. As a result, the photoacoustic signal is suppressed. Only a faint phonon emission remains, which is generated through the relaxation within the vibronic levels of S1 and S0 with cumulated energy equal to the difference between the excitation and the stimulation (δE = hν). For Coumarin 490 dye (QE = 0.82), the expected ratio between the phonon energy with and without stimulation R= δE/ ΔE ranges between R = 1/6 and R = 1/4.4, which depends on the Sn→S0 non-radiative relaxation process. These values are close to the experimental value.
4.2 The saturation level of stimulation
For F-STED, the saturation STED flux (Φsat) is defined as the value for which the rates of the stimulated depletion and the spontaneous emission (W) become equal: $W = {\sigma _{st}}{\Phi _{sat}}$, where σst is the stimulated emission cross section. In the present model for S-STED the stimulated depletion rate is now opposed to the ESA rate and the saturation is reached when ${\sigma _{st}}{\Phi _{sat}} = {\sigma _{ESA}}{\Phi _{exc}}$ where σESA and Φexc are respectively the cross section for the excited state absorption and the flux of the excitation. On Fig. 4 we can observe that a 0.5 µJ value for Φsat is attained for a Φexc equal to 0.4 µJ. This Φsat value is roughly twice the one that can be calculated from the cross sections given in Fig. 6. This result is satisfactory since it results from some approximations, Fig. 6 shows for example that the stimulating wavelength can also be slightly absorbed through an additional ESA.
4.3 Simultaneity of excitation and depletion
This model can explain our efficient S-STED effect. It is based on the population depletion as in F-STED but differs fundamentally. While the stimulation has to compete with a relaxation process for F-STED, it is opposed to an absorption (an ESA more precisely) for S-STED. Therefore, the simultaneity of the excitation and the stimulation is mandatory unless the ESA occurs. Our laser tandem based on harmonic generation provides the simultaneity by principle. As a confirmation of this requirement, the case of Rhodamine 6G provides a counterexample, where the stimulation pulse does not temporally overlap the excitation. As shown in Fig. 3(b), the S-STED efficiency in Rhodamine 6G, also known for its particularly efficient ESA and its excellent stimulation capability [17], is low. We were not able to obtain a good contrast between the photoacoustic signal without and with stimulation. For this experiment, the laser tandem was based on a 532 nm-excited dye lasing at 600 nm. The dye laser pulse is shorter and slightly delayed from the pump as usual for nanosecond pumped dye laser. We can assume that it is the reduced overlap between the two pulses which is at the origin of the lack of efficiency of the S-STED effect in the Rhodamine 6G.
4.4 Polarization sensitivity
Molecules such as Coumarins are not spherically symmetrical, excitation by a polarized electrical field will create an excited state population of molecules, and their dipolar moment are preferentially aligned. Depending on solvent viscosity, the molecules in their excited state can rotate during relaxation. This interesting phenomenon has been used to measure the interaction of dye molecules with various substrates. Particularly, it is useful for biological species [18]. Fluorescence anisotropy measurement is the usual method to access the distribution of the excited fluorophore dipoles [19]. The photoacoustic signal cannot carry a molecular orientation information. The anisotropy can also be measured by F-STED [20,21]. Since the S-STED effect also probes the excited state population, it is interesting to verify whether this effect can be used to measure an anisotropy.
Figure 5 shows the anisotropy of the photoacoustic signal under S-STED, the photoacoustic signal quenching by STED is more important when the exciting and stimulating beam polarizations are parallel. The STED effect, for a given stimulating energy, is minimal when the two beams are cross polarized. Quantitatively the anisotropy is defined as:
where ${D_\parallel }$ and ${D_ \bot }$ are respectively the sound depletion (D = 1-R) for the stimulating polarization parallel and perpendicular to the excitation polarization. Using the signals of Fig. 5 and subtracting the background we can compute an anisotropy r = 0.18. This value has to be compared with the anisotropy r’ = 0.08 measured by the conventional method using a polarized excitation at the 355 nm and detecting the polarized fluorescence. It is not the purpose of this paper to make a quantitative analysis of S-STED anisotropy which would need to measure its dependence on the solvent viscosity and on the delay between the excitation and the stimulation. We can just mention that Coumarin 490 is a small molecule and ethanol has a low viscosity. A rotational depolarization time in the range of 0.15 ns is expected [21] and a depolarization can erase part of the initial anisotropy. But, since the excitation and stimulation pulses are short (0.7 ns for 1064 nm, less for 532 and 355 nm) and simultaneous, the loss of polarization is certainly reduced compared to the depolarization during the ∼2 ns fluorescence lifetime. To the best of our knowledge this is a first demonstration of an anisotropy measurement on a homogeneous dye solution via a simple photoacoustic setup. Our S-STED method differs strongly to the PPAM method based on the vectorial optical absorption method recently published [22]5. Conclusion
The main purpose of this paper was to provide experimental evidence that the stimulated emission depletion (STED) concept commonly used for fluorescence (F-STED) can be extended to photoacoustic (S-STED). While both the fundamental mechanism of S-STED and F-STED rely on the depletion of an excited state, an efficient S-STED involves an excited state absorption (ESA). This gives an interesting non-linear behavior to S-STED. For this process, the chromophores do not have necessarily a low quantum efficiency. The phonon emission is not generated from the first excited state, but from the non- radiative relaxation of upper excited states. Therefore, the stimulating laser does not have to compete with a high non-radiative rate but with the ESA probability. This can be obtained at moderate power. We believe this is the principal originality of S-STED. In addition, the information on the molecular orientation induced by the excitation was lacking in photoacoustic methods. S-STED provides an original way to access to the anisotropy. The demonstration of the efficiency of S-STED opens a way towards an extension of various optical methods to photoacoustic as soon as an optical probe can be found. In this frame, Föster Resonance Energy Transfer (FRET) [23,24] and Fluorescence Lifetime Imaging (FLIM) can also be envisaged. The S-STED concept can be applied to super-resolved microscopy, using a dichroïc phase mask as designed and validated for F-STED microscopy [8]. In addition, the nonlinear dependence of the photoacoustic signal upon the excitation fluence provides optical sectioning capability [2]. Combining these effects, 3D super-resolved photoacoustic scanning microscopy could be achieved.
Funding
National Research Foundation of Korea (2018R1A6A3A01013285); Centre National de la Recherche Scientifique; Ministry of Education.
Acknowledgement
We would like to thank Irene Wang for the careful reading of the manuscript and for the pertinent corrections she suggests.
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