6. Two color laser-induced phase transition of PNIPAM and VDP-PNIPAM
6.2 UV laser power dependence of particle expansion in VDP-PNIPAM
To clarify the reason of observed novel phase transition phenomenon with two color lasers, we examined further experiments. First, we check the excitation laser power dependence of phase transition expansion. There are two purposes for this experiment. First, to know how excitation light power affect to the phase transition
particle size change. Second, to know the laser fluence threshold of expansion effect because one of our interest is phase transition induced by “photon pressure”.
The results of laser power dependence experiments are shown in Figure 6.5 and 6.6 for H2O solution and Figure 6.7 and 6.8 for D2O solution, respectively. Trapping laser power was set to 200 and 600 mW in H2O and D2O, respectively, with following to the previous experimental results. The phase transition behavior in time and size under examined laser powers in two different solutions are very similar.
In the power dependence experiments, we found that particle size changed depending upon UV laser power. The particle size was enlarged by increasing the UV power and particle size expansion diminished by decreasing. We could see particle size expansion more than 1 mW of UV laser. Thus threshold laser power for expansion was considered as 1 mW.
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We also found that the phase transition expansion is more efficiently occur in D2O than in H2O solution under same excitation laser power. Here sample concentration was the same and excitation laser power was same. Just only trapping laser power was different. We observed very vigorous phase transition and large particle formation when very high power trapping laser irradiated to PNIPAM in H2O. At such high laser power we observed similar large particle and surrounding small particles. It implies quite strong heating effect affect to phase transition. However trapping laser power dependent heating effect has not examined yet and further detailed study should be necessary.
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Table 6.1 Mean size value of phase transition under each excitation power.
Power < 1 mW 1 mW 2 mW 3 mW 4 mW 5 mW 15 mW
Only trapping laser(μm) 6.2±0.9 10.2±0.3 10.6±2.6 8.2±0.9 8.6±1.2 8.4±0.9 9.4±0.5 Trapping + UV laser(μm) 6.2±0.9 10.8±0.5 12.1±3.5 17.6±8,5 27.4±5.5 27.0±9.9 30.2±8.7
Figure 6.5 Pictures of phase transition particles at each excitation laser power in H2O solution. (a) ~ (g) are the particles formed by trapping laser and their corresponding two colors laser expansion phase transition images are in (h) ~ (n). Trapping laser power= 200 mW.
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Figure 6.7 Pictures of phase transition particles at each excitation laser power in D2O solution. (a) ~ (f) are the particles formed by trapping laser and their corresponding two colors laser expansion phase transition images are in (g) ~ (l). Trapping laser power =600 mW.
Figure 6.6 Particle size change at each excitation laser power in H2O solution.
Trapping laser power =200 mW. (■) for trapping laser and (●) for two color lasers irradiation.
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6.3 The excitation wavelength dependence for two color effect
For the two color laser induced expansion effect, we need to irradiate sample with two different lasers simultaneously, and then the particle show the expansion. If just one laser, the expansion does not take place. As we show in Figure 6.9, the absorption and fluorescence spectra of VDP-PNIPAM, 325 nm laser light well corresponds to the
Figure 6.8 Particle size change at each excitation laser power in D2O solution. Trapping laser power = 600 mW. (■) for trapping laser and (●) for two color lasers irradiation.
Table 6.2 Mean size value of phase transition under each excitation power.
Power 1 mW 2 mW 3 mW 4 mW 5 mW 15 mW
Only trapping laser 6.7±1.0 11.5±2.9 6.9±1.4 9.5±3.0 10.4±3.4 9.7±0.5 Trapping + UV laser 7.0±1.0 21.2±2.9 37.3±21.8 50.1±20.0 >65 >65
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absorption band of VDP. It implies that the overlap of second excitation laser light wavelength and absorption wavelength of the molecule has some relationship. Here we will examine wavelength effect for particle expansion by using different wavelength laser light. We employ 375 nm light from mercury lamp obtained by using band-pass filter which bandwidth is 15 nm. A 405 and 488 nm diode laser output were also used as light source. For those experiments, the excitation light power is set as 15 mW for all light source. All of those experiments were done at wide-field illumination condition.
As shown in Figure 6.10 and 6.11, we could not see phase transition expansion except 325 nm light. In D2O solution with 488 nm laser, it seems that the particle enlarged after 488 nm, however the size did not change even after cut 488 nm irradiation. Slight change of the particle size was due to the long time NIR irradiation Figure 6.9 Absorption and emission spectrum of VDP-PNIPAM in H2O solution.
Concentration= 0.01 wt %. Excitation wavelength for emission spectrum: 325 nm.
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not due to second 488 nm light. Particle size change at each laser wavelength are summarized in Figure 6.12 and Table 6.3. Wavelength dependence results strongly suggest that the expansion phenomenon is induced by the light absorbed by the VDP molecule.
Figure 6.10 Wavelength dependence of phase transition expansion in H2O
solution. (a)~ (d) are phase transition induced by only NIR trapping laser. (e) ~ (f) are phase transition irradiated by second light with 325, 375, 405 and 488 nm, respectively. Graph (d) and (f) are recorded in D2O solution. Trapping laser power was 200 mW in H2O and 600 mW in D2O. All the excitation laser power was 15 mW.
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6.4 Proposed mechanism of two color laser induced phase transition expansion
Comparisons of two color phase transition of non-labeled PNIPAM and VDP-PNIPAM strongly suggest that the phase transition expansion is related to the
Table 6.3 Size of particles induced by trapping and two color laser irradiation.
wavelength(nm) 325 nm 375 nm 405 nm 488
nm(D2O) Trapping size(μm) 9.4±0.5 6.7±2.6 7.6±0.3 4.7±1.4 Two color laser phase
transition (μm) 30.2±8.7 6.72.6 7.6±0.3 6.1±2.1 Figure 6.11 Excitation wavelength dependence of VDP-PNIPAM phase
transition expansion. The excitation light power at each wavelength is 15 mW.
(■) for trapping laser and (●) for two color lasers irradiation.
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existence of VDP. Wavelength dependence indicates importance to use the light which is overlapped with absorption band of chromophore.
The phase transition expansion of VDP-PNIPAM is dramatically larger than PNIPAM phase transition when irradiated with trapping laser and blue laser. Here, we also did the experiment without trapping laser to be reference. It did not show any change by irradiate blue laser even for a long time. That means the phase transition expansion need both two color laser irradiate simultaneously.
Here we can propose two possible reasons for two color laser induced phase transition expansion. One is a resonance effect and another is a photothermal heating effect. An enhanced mechanical interaction between an electromagnetic field and a nanoscopic object near electronic resonance has predicted by theoretical calculations.[51, 52] It suggests that if the wavelength of trapping laser is overlapped with an electronic absorption of the object which will be trapped, stronger mechanical force will be exerted to that object and trapping force will be enhanced. It has been experimentally observed by under optical trapping conditions. Hosokawa et al. has reported resonance laser enhanced biased diffusion of dye-doped polymer nanoparticle which size is 40 nm.[53] They introduced a 1064-nm NIR trapping laser and weak 532 nm laser light for fluorescence excitation to dye-doped nanoparticle dispersed solution and measured diffusion constant of the particles with or without
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resonant light by using FCS. It should be noted that 532 nm fluorescence excitation laser is overlapped with the absorption band of doped dye. They observed clear difference in diffusion time when they introduced resonant second laser although the power of resonant light was very low which was in the range of W. We can notice that an interesting coincidence between their result and ours in terms of the laser condition. They used ~MW/cm2 trapping laser and much weaker second resonance laser which power density was ~kW/cm2 to sub ~ kW/cm2. We observed similar enhancement-like phenomena even we used much lower power density of second UV laser. Another example of resonance enhanced trapping has reported for the trapping study on protein in solution.[54] They examined laser trapping of several protein molecules such as lysozyme, cytochrome c and myoglobin. They observed rapid molecular assembly formation in cytochrome c and myoglobin, but not in lysozyme.
They attributed observed enhancement of trapping of cytochrome c and myoglobin to the resonance enhancement of trapping of molecules by two-photon absorption of these proteins. These proteins possess heme in their structure which has electronic absorption band around 500-600 nm region. In the experiment they used ~MW/cm2 level of power density trapping laser which is sufficiently high fluence that we can expect two-photon absorption of the molecules. Thus they attributed observed enhancement of trapping and formation of assembly due to the resonance effect.
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In our experiment we used intense NIR trapping laser and weak UV laser as well as first example as depicted in Figure 6.12. The wavelength of UV laser is overlapped with electronic transition band of labeled fluorophore, VDP, in the polymer molecule.
Wavelength effect of second laser by changing its wavelength clearly showed no particle enlargement at 375, 405 and 488 nm at these wavelengths VDP’s absorption coefficient is extremely low as we can see in the absorption spectrum. Based on these results we can propose resonance enhancement of trapping of VDP-PNIPAM as one of the reason of observed two color laser enhanced phase transition and particle enlargement.
Another possible reason, photothermal heating, is also related to the absorption of labeled fluorophore VDP. When we introduce 325 nm excitation light, VDP-PNIPAM
Figure 6.12 Absorption spectrum of VDP-PNIPAM. An arrow in the figure indicates the wavelength of UV laser used in our experiments which has large overlap with electronic transition band of labeled fluorophore.
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will be excited to lowest singlet excited state, i.e. S1 state, and then excited
VDP-PNIPAM transfer to ground state with emitting fluorescence, without emitting by vibronically or through triplet state, and so on, “without” trapping laser. However
if NIR trapping light coexist with 325 nm light, NIR light can be absorbed by excited VDP-PNIPAM and it will be excited to higher singlet excited state, Sn state. Typically an excited state lifetime of Sn state is quite short and Sn S1 relaxation occurs at the time scale of picoseconds with vibronically in which process photoexcitatin energy will be converted to thermal energy due to the vibronic relaxation of the molecule;
namely it generates a heat. Molecules returned from Sn to S1 state are still exposed to the intense NIR light even after relaxation from higher excited singlet state, and these molecules will be excited and may repeat the same excitation/relaxation process and, most importantly, they generate heats on this cyclic SnS1 excitation/relaxation process as depicted Figure 6.13. Similar photothermal energy conversion with two laser has reported for anthracene-doped polystyrene films. Fukumura et al. suggested cyclic multiphoton absorption in excited state as well as we mentioned above.[55]
Thus once excited states are formed, it works as an efficient photothermal converter with raising local temperature and it can induce further phase transition in a wider region. Again, by changing the excitation laser power, we observed obvious change of the phase transition enhancement. Enhancement of the phase transition efficiency
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strongly depends on whether second light has overlap with absorption band of VDP.
Additionally, we observed clear difference of the particle size change rate in H2O and D2O. VDP-PNIPM/D2O showed larger change of the particle size under the same UV light power. We used different laser power in H2O and D2O since a heat generation ration depends on the solvent and we need much intense NIR laser in D2O case. As we described above, how much heat will be generated will strongly depend on how efficiently multiphoton absorption will occur in excited state. Therefore we assume that higher power density trapping laser irradiation with resonant UV laser can enhance more heat generation. Of course higher intensity of UV laser also enhance heat generation since we can increase the number of excited VDP molecule by using more excitation photons. It can concisely explain observed two color laser induced enhancement of phase transition.
At the moment we cannot declare which process is correct or not since both possibility can work together in examined conditions. It should be necessary to examine some more experiments for example by changing excitation UV laser wavelength within absorption band, change the chromophore or use non-covalently bonded chromophric system and so on. Further investigation is going on.
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Figure 6.13 Schematic drawing of photothermal effect induced by multiphoton absorption in excited state. Here we omit relaxation path through triplet state in this scheme.
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7. Summary
In this study, we have studied the phase transition dynamics of PNIPAM and fluorescently labeled PNIPAM, VDP-PNIPAM, with time- and space-resolved fluorescence microscopy combining with laser trapping technique. Phase transition of PNIPAM molecule will be induced by focusing NIR trapping laser into the PNIPAM solution. Laser irradiation induced micrometer-sized particle formation which can be interpreted due to mainly temperature elevation in H2O and both temperature and photon pressure of focused trapping laser in D2O.
We combined the wide-field imaging, confocal spectrum measurement and fluorescence lifetime measurement with TCSPC at the trapping laser spot to elucidate phase transition dynamics. Time- and space-resolved fluorescence microspectroscopy with VDP-PNIPM gave us quantitative information of phase transition dynamics which we cannot obtain just only using conventional imaging methods.
We also found novel phase transition behavior. By combining the trapping laser with additional UV excitation laser, we observed unconventional enhancement of phase transition. This phenomenon can be induced only by simultaneous introduction of two lasers. We experimentally studied this phenomenon in detail and proposed the mechanism of it.
In chapter three, we discuss the thermal effect of solvent and substrate. For
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trapping experiment, we also need to take care about the absorption of solvent and substrate because heat is one issue in our study. We check the thermal effect which comes from the absorption of solvent by changing solvent to D2O. In D2O solution, the phase transition will not appear by using low power laser which can induce phase transition in H2O. That means the phase transition in H2O is mainly due to heating effect. For the heating effect from the absorption of substrate, it can be neglected by changing the substrate to quartz. The difference caused by heating effect from substrate is like change the system temperature.
In chapter four we introduced our FLIM system. It enables us to measure fluorescence dynamics by measuring fluorescence lifetime with TCSPC. By using FLIM we can observe phase transition dynamics by probing the excited state fluorescence dynamics of probe chromophore, VDP. Spatial resolution of our system is determined to 280 nm for lateral plane. We can measure fluorescence spectra and images simultaneously in addition to fluorescence decay curves. It will give us more clear insight for phase transition dynamics of PNIPAM.
In chapter five, we studied phase transition dynamics by using VDP-PNIPAM as molecular probe and by probing fluorescence image, spectrum, and lifetime.
Simultaneous observation of image, spectrum and decay curve is quite powerful method. We can visualize the phase transition dynamics with higher sensitivity and
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time resolution. We observed that molecular environment did not returned to the original coil state even after disappearing of phase transition particle. It could not observed just only conventional bright field imaging but the combination multimodal detection enabled it.
In chapter six, we discussed two color laser induced unusual phase transition behavior. The expansion of VDP-PNIPAM phase transition particle is explained with the interaction between electronic transition band of laveled fluorophore and irradiated two lights, UV excitation laser and NIR trapping laser. We proposed two possible pathway for the particle expansion by resonance effect and photothemal effect. Both mechanisms are acceptable to explain observed particle expansion and we could not conclude which mechanism induced observed enhanced phase transition behavior. However Experimental results gave us many clues to solve this puzzle and we are doing further experiments to clarify the mechanism of this interesting phenomenon.
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