Time-Resolved Structured Illumination Microscopy for Phase

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B: Liquids, Chemical and Dynamical Processes in Solution, Spectroscopy in Solution

Time-Resolved Structured Illumination Microscopy for Phase Separation Dynamics of Water and 2-Butoxyethanol Mixtures: Interpretation of “Early Stage” Involving Micelle-Like Structures Shuichi Toyouchi, Shinji Kajimoto, Masatoshi Toda, Toshihiro Kawakatsu, Yohji Akama, Motoko Kotani, and Hiroshi Fukumura J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b10244 • Publication Date (Web): 03 Dec 2018 Downloaded from http://pubs.acs.org on December 3, 2018

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The Journal of Physical Chemistry

Time-Resolved Structured Illumination Microscopy for Phase Separation Dynamics of Water and 2-Butoxyethanol Mixtures: Interpretation of “Early Stage” Involving Micelle-Like Structures

Shuichi Toyouchi,†, Shinji Kajimoto,†,# Masatoshi Toda,†,※ Hiroshi Fukumura,*,† Toshihiro Kawakatsu,‡ Yohji Akama,§ and Motoko Kotani‖

†Department

of Chemistry, ‡Department of Physics, and §Mathematical Institute,

Graduate School of Science, Tohoku University, Sendai, 980-8578, Japan. ‖

Advanced Institute for Materials Research, Tohoku University, Sendai, 980-8577,

Japan.

Ⓢ Supporting Information

1 ACS Paragon Plus Environment

The Journal of Physical Chemistry 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

ABSTRACT: Phase separation dynamics of a water/2-butoxyethanol (2BE) mixture was studied with newly developed time-resolved structured illumination microscopy (SIM). Interestingly, an employed hydrophobic fluorescent probe for SIM showed spectral shifts up to 500 ns after a laser-induced temperature jump, which suggests 2BE micellar-like aggregates become more hydrophobic at the initial stage of phase separation. This hydrophobic environment in 2BE aggregates, probably due to the ejection of water molecules, continued up to at least 10 s. Time-resolved SIM and previously-reported light scattering data clearly showed that the size of a periodic structure remained constant (ca. 300 nm) from 3 to 10 s, and then the growth of periodic structures having the self-similarity started. We think that the former and the latter processes correspond to “early stage” (concentration growth) and “late stage” (size growth), respectively, in phase separation dynamics. Here we suggest that, in the early stage, the entity to bear 2BE phase be water-poor 2BE aggregates, and the number density of these aggregates would simply increase in time.


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INTRODUCTION Phase separation dynamics has attracted the attention of a wide-range of researchers because it affects resulting material structures such as metal alloys, composite polymers, minerals, biological systems, and so on. Thus, a numerous number of theoretical and experimental studies have been presented to date. Among them, the Cahn-Hilliard theory is known to describe time evolution of self-similar structures called spinodal decomposition (SD) during phase separation.1, 2 This theory is based on mass diffusion induced by free energy changes, and its validity has been experimentally confirmed in a variety of materials.3-10 According to the Cahn-Hilliard theory, SD occurs through two distinct stages as shown in Figure 1. During the “early stage”, only one periodic structure having a characteristic length grows in concentration caused by mass flow. After that, at the “late stage”, periodic structures grows in size keeping self-similarity due to merging of small structures without changing the component concentration in two phases. The late stage is rather macroscopic and the continuous model of matter can be applied. In contrast, the early stage, involving processes to gather alike particles, should be influenced by a variety of inter-molecular interactions when molecules are phase components. It is therefore interesting to study how molecular level dissociation/association is connected with the early stage of phase separation. In order to study ultrafast dynamics of phase separation, we have chosen a system having the lower critical solution temperature (LCST) because a temperature jump (T-jump) method using short-pulsed near infrared (NIR) irradiation is applicable. It is



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known that homogeneous mixtures of water and some organic solvents can split into two phases with a temperature rise.11-13 A phase diagram of water/2-butoxyethanol (2BE) mixtures is shown in Figure 2 as one such example. Following pioneering works of nanosecond laser-induced T-jump14,

15

we have utilized nanosecond NIR pulses to

excite combination bands of water molecules. The T-jump method enables us to observe relaxation processes from unstable non-equilibrated state far from the equilibrated state with a deep quenching (more than several K) with nanosecond time resolution. The laser-induced phase separation has been investigated at molecular level and macroscopic phase growth by using picosecond/nanosecond time-resolved (TR) Raman scattering spectroscopy and nanosecond shadowgraph imaging, respectively.16-19 The molecular level understanding, namely hydrogen bond scission during phase separation, was validated with theoretical calculations of Raman spectra.20,

21

These

previous studies have shown that the hydrogen bond scission between water and organic molecules terminated within 1 s after T-jump, which has misled us to consider that the early stage of SD might be over in 1 s, because the early stage should involve local composition changes in phase separating media. The shadowgraph imaging showed the bicontinuous structures typical in the late stage of SD only after 20 s. Thus, there still remains a large temporal and spatial gap among these studies. This is because phases which are smaller than a few hundred nanometer have never been observed by using a conventional optical imaging technique due to the diffraction limit. In particular, the gap region (1 to 30 s in time, molecular scale to a few 100 nm in space), namely mesoscopic phase separation dynamics, is not yet understood well.


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One additional factor we should take into account is that water and organic solvent mixtures can have soft complex structures even in the one phase region.22-34 For example, 2BE molecules form micellar like aggregates in water/2BE mixtures due to the amphiphilic property of 2BE. D’Angelo et al. have reported critical micellar concentrations (CMCs) at various temperatures as shown in Figure 2.35 A study of depolarized light scattering by Micali et al. has suggested percolation structures which consist of the micellar aggregates.36 Recently, we also found that, by using the fluorescence correlation spectroscopy (FCS), water/2BE mixtures involve micelle like aggregates that mostly impeded the diffusion of a hydrophobic fluorescent probe just below LCST.37 This means the phase separation should start from such molecular-level inhomogeneous solution. There is a significant lack of knowledge about how these soft structures in solution play a role in phase separating media. It should be noted that we have also presented a new simplified molecular model showing LCST, which revealed string-like aggregate structures in the stable one phase region below LCST.38 In this report, we performed nanosecond TR fluorescence spectroscopy of a markedly hydrophobic fluorescent probe in water/2BE mixtures. Interestingly, fluorescence spectra of the probe changed prior to or at the beginning of the early stage, suggesting that the amount of water molecules inside 2BE aggregates decreased. This finding led us a new view to understand the early stage of phase separation. We have also developed nanosecond structured illumination microscopy (SIM) and light scattering technique to investigate the dynamics of mesoscopic phase separation. SIM is one of the super-resolution microscopy developed by Wilson et al.,39 Heintzmann et



al.,40 and Gustafsson et al.41-45 In general, SIM requires several different images to reconstruct one super-resolved image, and because of this, less temporal resolution has been recognized as a disadvantage. In our experiment, however, we need only periodicity lengths in objects, so that single-shot SIM is sufficient to extract super-resolved information when a sample has a single periodic structure. In this way, we successfully detected the mesoscopic region with a nanosecond temporal resolution during phase separation. We have clarified the time range of the early stage to be 1 to 10 s and that of the late stage to be after 10 s. Since molecular level hydrogen-bond scission was over at 1 s and only dry (less-water) 2BE aggregates existed throughout the early stage, the concentration increase keeping the spatial periodicity constant (Figure 1(b)) can be explained with an increase in the number density of dry 2BE aggregates. Thus, we present here an entirely new interpretation on the early stage in phase separation dynamics.

NANOSECOND SIM CONCEPT The spatial resolution limit of a microscope is determined by the “optical transfer function (OTF)” supporting region in reciprocal space, where the OTF has nonzero values, also known as the observable region (Figure 3). The Fourier transform (FT) of an obtained image is the pointwise product of the OTF and the original sample structure’s FT. The optical microscope can detect only information within the OTF 6 ACS Paragon Plus Environment

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support. Improvement of the microscope resolution is equivalent to expanding the effective OTF support region, while SIM takes another strategy to convert high frequency information (outside of OTF support, opened star in Figure 3-A) into low frequency ones (inside of OTF support, closed star in Figure 3-A). Its concept can be easily understood in terms of the well-known Moire effect.42 If two fine patterns are superposed, an interference pattern, i.e. Moire fringes, will appear in their image product, which means the superposition of two patterns leads to frequency shift. Generally the temporal resolution of SIM is low because SIM requires multiple images taken with different phases and orientations of striped patterns for the reconstruction of one super-resolution image. However, a single structured illumination image contains sufficient information for the analysis of an isotropic periodic structure in a sample. The isotropic structure in 2D real space gives a ring pattern in the reciprocal space, and the ring radius corresponds to a characteristic length of the bicontinuous structure. When a characteristic length is larger than the diffraction limit, a whole ring pattern falls into the OTF supported region in reciprocal space (Figure 3-B, left). A vertical stripe-patterned illumination gives two horizontally separated dots in reciprocal space, (Figure3-B, middle), and with this structured 7 ACS Paragon Plus Environment


illumination, the origin of the ring pattern can be shifted to the two separated dots (Figure 3-B, right). If a characteristic length is smaller than the diffraction limit, the ring radius becomes larger than the OTF supported region, however, a part of the shifted large ring patterns would be transferred into the observable range. (Figure 3-C) Thus, we can obtain a characteristic length in solution structures in the range of super-resolution using only one-shot nanosecond illumination. We used hydrophobic fluorescent dyes to visualize organic phase. It should be noted that the lifetime of fluorescent dyes also affects temporal resolution of images.

MATERIALS AND METHODS Sample Preparation. 2BE (Wako) was mixed with water (Millipore, Simplicity UV) at various 2BE molar fractions. N,N’-Bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylic diimide (Sigma Aldrich, BPDI, dye content > 90 %) was dissolved in water/2BE mixtures as a fluorescent dye. BPDI is highly hydrophobic and much more soluble to 2BE than to water. BPDI also shows intense fluorescence in 2BE rich phase after phase separation, which is mainly due to a high fluorescence quantum yield.37 The concentration of BPDI was 5.1 x 10-5 M for transient fluorescence spectroscopy, 8 ACS Paragon Plus Environment

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fluorescence imaging, nanosecond SIM, and light scattering throughout the experiments. For steady state fluorescence spectroscopy, 1 x 10-6 – 1 x 10-5 M of BDPI solutions were used in various 2BE molar fractions. Temperature Jump Method. The nanosecond T-jump method has been described elsewhere.16-18 Briefly, phase separation of water/2BE mixtures was induced by nanosecond NIR laser pulses (wavelength 1.88 m) obtained by Raman shifting the fundamental Nd:YAG laser pulse (Spectra-Physics, 1064 nm, 8 ns, 10 Hz) in high pressure H2 gas. (Figure 4, left side) The incident angle of the NIR beam on a sample surface was 60 degree. NIR pulse energy was 1.07 J/cm2, resulting in a 33 K T-jump for the front surface of the sample (averaged within 10 m depth). This corresponds to a quench depth of 7 K, since the sample temperature was kept at 297 K. (LCST of water/2BE mixture, 323 K) Steady

State

and

Transient

Fluorescence

Spectroscopy.

Steady

state

fluorescence spectra of BPDI were measured at various 2BE molar fractions (0.005 1.000) with a fluorescence spectrometer (Hitachi, F-4500). Excitation wavelength was 490 nm. Transient fluorescence spectra of BPDI in water/2BE mixture with critical concentration were measured at various delay times after T-jump. A green laser 9 ACS Paragon Plus Environment


(Quantel, Nd:YAG second harmonic 532 nm, 6 ns, 10 Hz, 35 W/pulse) was used for excitation. The excitation pulse illuminated the sample through an objective lens (Nikon, 40x, N.A. 0.6). Fluorescence was collected through the same objective lens and guided into a spectrograph (Princeton Instruments, Acton, SP150, slit size 100 m) and a cooled CCD camera (Andor Technology, iDus, 223 K, 1024 x 256 pixels). The delay time between NIR and green pulses was controlled by using a digital delay generator (Stanford Research System, DG535). Sample solution was circulated through a 100 m-thick flow cell and its temperature was kept at 297 K by using a water chiller system. It is worth to mention that, with the circulation system, sample solution was replaced newly between T-jump events. (Figure S1) At various delay times, transient fluorescence spectra were integrated over ten events. Nanosecond SIM. The apparatus used for fluorescence imaging and nanosecond SIM is shown in Figure 4. The same green laser above mentioned with ten times of power (350 W/pulse) was used as excitation beam for BPDI. The excitation pulse illuminated the sample through an objective lens (Nikon, 40x, N.A. 0.6) with wide-field arrangement by focusing at the back aperture of the objective. The delay time between NIR and green pulses was controlled with the aforementioned method. Fluorescence 10 ACS Paragon Plus Environment

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images were taken with the same objective lens and a cooled imaging CCD camera (Andor technology, DV435, 228 K, 1024 x 1024 pixels2). With this experimental setup, the original spatial resolution was determined to be 1.21 m by using 50 nm fluorescent polystyrene beads. For SIM measurements, a stripe pattern was generated with a spatial light modulator (SLM, Hamamatsu photonics, X-10468-01). The SLM worked as a grating and split the green laser beam into several diffracted light beams. A couple of the first order diffracted light beams were led onto a sample and the interference pattern between these two diffracted light beams at the focus plane was utilized (Figure 4, right circle). The period of the structured illumination was 2.15 m, which corresponds to ideal spatial resolution of 0.77 m by SIM. Solution (30 L) of water/2BE mixture with critical composition containing BPDI was sandwiched between two slide glasses (Matsunami Glass Ind., LTD) without any spacer. The solution thickness was estimated to be less than 12 m. The thin sample configuration allows us to obtain fine fluorescence images by reducing out-of-focus fluorescence signals. Note that the sample solution was not circulated with this configuration, unlike transient fluorescence spectroscopy described above. To avoid an 11 ACS Paragon Plus Environment


influence from the previous T-jump event, we obtained a single-shot image after confirming the recovery of sample solution. The temperature of a brass sample holder was kept at 297 K throughout the SIM measurement. All images were processed and analyzed with Matlab software (Mathworks, Inc.). Obtained fluorescence images were subtracted with a dark image and were multiplied with the Hann window function for minimization of an influence from image edges, then were converted to 2D FT (frequency domain) images. Power spectra of the 2D FT images were obtained with averaging more than 20 images for each delay time and with integration of FT intensities at various distances from the origin. The obtained power spectra were further subtracted with the power spectrum at 100 ns delay time. On this delay time, it is expected that sample temperature would have reached local thermal equilibrium but new phases have not yet appeared. For calculating power spectra of nanosecond SIM images, the right-shifted origin was regarded as a new origin. Light Scattering. The apparatus used for light scattering has been described elsewhere.46 The same green laser above mentioned generated a collimated probe beam. The spatial overlap between the probe beam and a NIR pulse was verified from a 12 ACS Paragon Plus Environment

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shadowgraph image. The probe beam size at the sample was approximately 200 m. The scattered light was then projected on a thin sheet of white paper placed above the sample at a specific distance ranging from 28 to 60 mm, and the projected scattering image was captured with a cooled imaging CCD camera (Andor Technology, iKon-M912, 223 K, 512 × 512 pixels2). The same sample with fluorescence imaging and nanosecond SIM was used, and the single-shot and single-imaging experiment was performed. Scattering images were averaged over more than 20 events for each delay time. Then power spectra of the averaged scattering images were obtained and further subtracted with the averaged power spectrum at 100 ns delay time.

RESULTS AND DISCUSSION Fluorescence spectroscopy. Figure 5(a) shows blue shift of steady state fluorescence spectra of BPDI in hydrophobic environment at room temperature. To avoid the light scattering effect in samples, we plotted the wavelength of the second spectral peak of BPDI fluorescence as a function of 2BE molar fraction in Figure 5(b). This clearly shows that the peak wavelength markedly shifts from 593 to 581 nm up to CMC (2BE molar fraction = 0.0175). Since BPDI is highly hydrophobic dye, it is 13 ACS Paragon Plus Environment


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conceivable that BPDI molecules are surrounded with aggregates of 2BE.37 Interestingly, the blue shift of fluorescence spectra continued even at the concentration higher than CMC, particularly after 0.02. This implies that micelle- like aggregates of 2BE contains a little amount of water molecules inside at concentrations around CMC. The result is consistent with studies of small-angle neutron scattering and neutron spin echo by Yoshida et al., which showed that the 2BE micellar aggregates are unstable

and

incomplete

micellar

type.47,

48

Thus,

we

can

monitor

hydrophobic/hydrophilic environments surrounding BPDI molecules during phase separation dynamics. The transient fluorescence spectra of BPDI in water/2BE mixture with critical composition at -100 ns (gray solid line) and 1 s (dotted line) after T-jump are shown in Figure 6 (a) together with a steady state fluorescence spectrum (black solid line) before T-jump. A blue shift after T-jump was observed from 580 to 578 nm. Based on the aforementioned steady state result, this transient blue shift can be interpreted as dehydration process of 2BE micellar aggregates. Fluorescence peak wavelengths are plotted as a function of delay time after T-jump in Figure 6 (b). The peak shift reached a plateau within 500 ns, implying that the composition of micellar aggregates is 14 ACS Paragon Plus Environment

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equilibrated in this time range. It should be noted that the peak shift in this time range is not due to temperature changes because the laser-induced T-jump can rise water temperature within 10 ns and its decay is negligible small within 400 ns.49 Interestingly, the observed time scale by fluorescence is found to be faster than that by the TR Raman scattering experiments.16-18 Raman spectral changes showed a slower kinetic region indicating that hydrogen bond scission continued up to 1 s after the T-jump. We currently consider that fluorescence spectra observed the microenvironment of BPDI, while Raman spectra observed the total amount of hydrogen bonds between water and 2BE. This means water may be ejected from micelle-like aggregates earlier, and then overall hydrogen bond scission seems to continue. This suggests that hydrogen bond scission from 500 ns to 1000 ns observed by Raman spectroscopy may occur mainly at the outside of micelle-like aggregates. Thus, local equilibration would take place depending on the observation scale. Nanosecond SIM. First, we compared previous and present methods to visualize morphology of phase separation in solution. Typical observed images at 100 s are shown in Figure S2 upper line. In the shadowgraph image, bright worm-like structures in dark background were observed. Although the worm-like structure width didn’t 15 ACS Paragon Plus Environment


grow, the dark region became larger as phase separation proceeded. The results suggest that the worm-like structures reflect the shape of interface between neighbor phases, and the dark region would correspond to both water and 2BE rich phases. Although fluorescence images gave similar worm-like structures, the structure width grew as phase separation proceeded. Because of marked hydrophobicity of fluorescent BPDI, bright fluorescent region should represent the spatial distribution of 2BE rich phase during phase separation. The power spectra of shadowgraph, fluorescence imaging, and light scattering at various delay time are shown in Figure S2 lower line. Power spectra of fluorescence imaging and light scattering gave clear single peaks, being the hallmark of the SD process.6 On the other hand, several peaks were observed in the power spectra of shadowgraph imaging. The multiple peaks might result from that the shape of interfaces is observed in shadowgraph imaging. Since it is difficult to assign each peak, we assumed a single Gaussian function for fitting multiple peaks. The characteristic lengths of phase separating media can be determined from the peak position of power spectra. The determined characteristic lengths are plotted as functions of delay time. (Figure S3) The results of fluorescence imaging and light scattering methods clearly showed that the domain sizes follow power law, 𝐿(𝑡) ~ 𝑡𝛼 ( 16 ACS Paragon Plus Environment

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= 0.61 by fluorescence imaging and 0.64 by light scattering). Obviously, the result of shadowgraph imaging is different from the other methods ( = 0.36) due to aforementioned multiple peaks. This comparison indicates that the fluorescence imaging technique is highly reliable for monitoring phase separation dynamics. The exponent obtained with the present shadowgraph imaging is different from those in the previous studies.16-18 We believe this difference is due to the difference in the optical thickness of samples. We estimated the thickness to be 12 m in this study, while the one in the previous studies was 1 mm. The images taken with thick samples showed marked growth with time even in bright regions together with dark area, which is in contrast to a constant thickness of bright-worm like structures in the present images. Moreover, the power spectra of shadowgraph imaging with 1 mm thick samples always gave single Gaussian-like peaks. Thus, the shadowgraph imaging is markedly influenced by optical thickness. Fluorescence images taken with uniform and structured illumination at various delay time are shown in Figure 7. In the both illumination cases, growing phase structures became obvious at later than 50 s delay time. In structured illumination images, stripe patterns were superimposed in spatial domain images, while the stripe 17 ACS Paragon Plus Environment


pattern frequency was indicated as two horizontally separated points in frequency domain images. As explained in the CONCEPT section, what we need in this study is the peak values of power spectra that appear as ring patterns in frequency domain images. At the delay time of 500 s, it is clear that a ring structure observed with uniform illumination was duplicated and shifted to the right and left sides centered at the shifted origins in the frequency domain image. The analyzed power spectra taken with uniform and structured illumination are shown in Figure 8 (a). The dotted and dash lines, respectively, show diffraction and SIM limits of the current experimental setup. With the uniform illumination, the apparent peak in the power spectrum of 18 s looked similar to the peak position of 20 s. This means that the peak positions are influenced by the diffraction limit. Although there is no significant peak in the power spectra before 18 s under uniform illumination, single peaks appeared under structured illumination at the delay times of 15 and 16 s. Calculated characteristic lengths for both illumination cases are plotted as functions of delay time in Figure 8 (b). This result shows that one-shot time-resolved structured illumination can break the optical diffraction limit if we limit the necessary information to the periodicity of spatial patterns. The characteristic length obtained 18 ACS Paragon Plus Environment

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from the SIM experiment followed a power law with the exponent () of 0.66. As shown in Figure S3, this exponent value is very close to the value obtained by light scattering ( = 0.64 for 20 – 100 s). Now we can safely state that SIM results are consistent with light scattering results and the late stage would start earlier than 20 s. Light Scattering. The light scattering method directly delivers frequency domain images of phase separating media. The power spectra obtained from the images at earlier delay times are shown in Figure 9 (a). Peaks of the power spectra, namely characteristic lengths of phase separating domains, were recognized after 3 s, and the peak values stayed almost constant up to 10 s. Then after 10 s, the peak shifted toward smaller wavenumbers, meaning the growth of characteristic lengths started. The calculated characteristic lengths are plotted as a function of delay time together with the results of nanosecond SIM in Figure 9 (b). When the earlier time range from 10 to 20 s is involved, the power law exponent by the light scattering method becomes 0.72 instead of 0.64. The values around 0.63 are obtained for the same system in a previous shadowgraph study, and its reason is attributed to a high Reynolds number and a large surface tension.18 The constant characteristic length from 3 to 10 s corresponds to the domain size 19 ACS Paragon Plus Environment


of 300 nm. Similar results by light scattering experiments for water/2BE mixture with no fluorescent molecules have been reported elsewhere.37 During this time range, the light scattering intensities at wavenumber Q = 10 m-1 are plotted as a function of delay time in Figure 9 (c). This clearly shows the scattering intensities grow exponentially. According to the linearized Cahn-Hilliard theory, the characteristic length remains constant while the density fluctuation increases exponentially with time in the early stage of SD.1,

2

Thus, we can phenomenologically assign this time

range to the early stage of SD, and the late stage would start after 10 s. What is the entity to bear SD in “early stage”? During the early stage of SD, concentration fluctuation should exponentially grow in time, while the fluctuation periodicity remains constant. Simply the local concentration growth might correspond to gathering of 2BE organic molecules in solution. However, this view looks inconsistent with the previous results by TR Raman spectroscopy, which showed that molecular level changes (hydrogen bond scission) terminated within 1 s after the T-jump.16-17 In the present work, based on TR fluorescence spectra, we revealed that the dehydration of the 2BE micellar aggregates started just after T-jump and continued up to 0.5 s. (Figure 6)

These experimental results suggest that 20

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molecular-level hydrogen bond scission is completed and the binary system reaches a local equilibrated state within 1 s. Namely, only dry 2BE micellar aggregates should be floating in water-rich phase. To explain the exponential growth of local concentration fluctuation, we have to invoke an increase in the number density of dry 2BE micellar aggregates in solution. Simple merging of micellar-like aggregates to form soft structures having the characteristic length of 300nm can not explain the exponential growth keeping the same periodicity. Figure 10 shows conceptual description of our model for the early stage of SD. In the early stage (1 -10 s), the amplitude of number-density fluctuation of micellar aggregates would increase while the ratio between 2BE and water molecules would stay constant at the inside of the aggregates. Thus, we consider that the entity baring the early stage is not the concentration of 2BE molecules, but the number density of micellar-like aggregates. It is known that micellar-like aggregates take an important role in such kind of binary mixtures. Corti et al. have measured light scattering from n-dodecyl hexaoxyethylene glycolol monoether aqueous solution and found a large increase of the correlation range of concentration fluctuation when the system approaches the critical temperature.50 This long range correlation over a few 100 nm is ascribed to large 21 ACS Paragon Plus Environment


clusters including micelles and water rather than the size of micelles. Telgmann and Kaaze have also explained their results of broad band ultrasonic absorption spectra of water/2BE mixture along the discussion of micelle’s density fluctuation.32

CONCLUSION We experimentally studied IR-laser-induced phase separation dynamics of water/2BE mixtures using time-resolved spectroscopy and microscopy. The transient fluorescence spectra showed that hydrophobicity of micellar aggregates increased up to 1 s, which suggests the ejection of water from the aggregates. Newly developed nanosecond SIM successfully demonstrated time-resolved super resolution. Together with the light scattering results, we found that the onset of the late stage of SD would be around 10 s. The light scattering method also showed the early stage of SD as described by Cahn-Hilliard Model occurred in the time range of 1 to 10 s. This means that the hydrogen bond scission and water ejection up to 1 s should be distinguished from the early stage. We proposed a new model for the early stage of SD of water/2BE mixtures, in which the number density of 2BE micellar aggregates plays a role in exponential intensity growth keeping the same domain size of 300 nm. Therefore, the entities to 22 ACS Paragon Plus Environment

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bear the phase separation in the continuous model would not be continuous in space. This is the first comprehensive study to understand the whole SD process in binary liquid mixtures.

ASSOCIATED CONTENT Ⓢ

Supporting Information

The Supporting Information is available free of charge on the ACS Publication website at DOI: Effect of solution flow, comparison of power spectra obtained with different methods, and domain sizes obtained with different methods (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail:

[email protected]

ORCID Hiroshi Fukumura: 0000-0003-2392-935X 23 ACS Paragon Plus Environment


Present Addresses 

Shuichi Toyouchi, Department of Chemistry, University of Leuven, B-3001 Leuven,

Belgium #

Shinji Kajimoto, Graduate School of Pharmaceutical Sciences, Tohoku University,

Sendai, 980-8578, Japan ※ Masatoshi

Toda, National Institute of Advanced Industrial Science and Technology,

Tsukuba, 305-8568, Japan Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest

ACKNOWLEDGMENTS HF thanks Dr Motohiro Kasuya at IMRAM Tohoku Univ. for his generous support in fluorescence measurement. This work was partially supported by the JST Mathematics Program (CREST, A Mathematical Challenge to a New Phase of Material Science) 24 ACS Paragon Plus Environment

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Figure captions

Figure 1.

(a) Schematic illustration of spinodal decomposition from one phase at

low temperature (left) into two phases at high temperature (right), through a transient interconnecting network structure (middle). (b, c) Spatial fluctuations of 33 ACS Paragon Plus Environment


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concentration in the early and late stage of the spinodal decomposition. Ci, Cw, and Co denote initial concentration, concentration of water phase, and of organic phase, respectively. The fluctuations grow with time (from solid line to dash line) with keeping a characteristic length in the early stage, and keeping self-similarity in the late stage.

Figure 2.

Phase diagram of water and 2BE mixture. Solid and dotted lines

represent the coexistence curve and critical micellar concentration (CMC) line, respectively. 2BE molecules in water form micellar aggregates above the CMC. The mixture splits from one phase into two phases across the coexistence curve with a temperature rise. (Ref. 35)

Figure 3.

Conceptual

explanation

of

nanosecond

structured

illumination

microscopy. (A) The resolution of a conventional optical microscope is limited by diffraction. A set of high frequency information such as the opened star is not detectable in reciprocal (k) space. Only optical transfer function (OTF) supported information such as a closed star can be detected. (B) An isotropic spinodal 34 ACS Paragon Plus Environment

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decomposition structure in real space (top) is converted to a ring pattern in reciprocal space (bottom), the radius of which corresponds to the structure’s periodicity (characteristic length). When the illumination pattern is vertical stripes in real space, it gives two dots horizontally separating from the origin in reciprocal space. Here the distance of each dot from the origin represents the stripe interval inverse. With this stripe pattern illumination, the origin of the ring pattern is also shifted to both two dots in reciprocal space. (C) When a ring size is larger than the OTF supported region (namely a characteristic length is smaller than the optical resolution limit), a part of the shifted rings still falls into the OTF region. In this way, we can obtain characteristic lengths of transient structures by using one-shot nanosecond illumination.

Figure 4.

Experimental setup for nanosecond time-resolved SIM. DM1 = 1064

nm dichroic mirror. HWP = half-wave plate. L = lens, f = 50 mm (L1), f = 120 mm (L2), f = 300 mm (L3), f = 400 mm (L4), f = 300 mm (L5). PolBSC = polarizing beam splitter cube. SLM = spatial light modulator. EX = excitation filter, EX540/25. DM2 = 532 nm dichroic mirror, DM565. BA = Band pass filter, BA605/55. CCD = 35 ACS Paragon Plus Environment


charge-coupled device.

Figure 5.

(a) Steady state fluorescence spectra of BPDI in water and 2BE

mixture with various 2BE molar fractions. These spectra were normalized at the second peak which ranges from 578 to 594 nm. A vertical dotted line represents the second peak position of critical concentration (580.6 nm). (b) Wavelength shifts of the second peak from critical concentration plotted as a function of 2BE molar fraction. Dotted and dash vertical lines represent the CMC at room temperature (2BE molar fraction 0.0175) and the critical concentration (2BE molar fraction 0.052), respectively. Dotted and dash horizontal lines indicate  = 0 and -3.0 nm, respectively, for eye guide.

Figure 6.

(a) Transient fluorescence spectra of BPDI in phase separating water

and 2BE mixture at the critical concentration (2BE molar fraction 0.052). Gray solid and black dotted lines represent spectra taken at -100 ns and 1 s of delay time, respectively. The black solid line shows a steady state spectrum below the critical temperature for comparison. (b) Fluorescence wavelength shifts of the 36 ACS Paragon Plus Environment

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second peak from the value of -1 s plotted as a function of delay time. A dotted horizontal line represents the peak shift of pure 2BE ( = -2.8 nm). Inset is an expanded figure up to 1 s. Gray solid line is a fitting curve with a single exponential function (t = 115 ns).

Figure 7.

Nanosecond fluorescence images of water and 2BE mixture during

laser-induced phase separation taken at various delay times. Left and right columns’ images were taken with uniform and structured illumination, respectively. Insets are frequency domain images of respective spatial domain images.

Figure 8.

(a) Power spectra obtained from fluorescence images taken with

uniform (left) and structured illumination (right). Dotted and dash lines represent diffraction limit of the optical system and expected SIM detection limit, respectively. Solid lines in the structured illumination spectra are fitting curves obtained with Gaussian functions. (b) Domain sizes plotted as a function of delay time. Closed and opened circles show the domain size by uniform and structured



illumination, respectively. Solid line is a fitting curve of structured illumination obtained with a power function, t,  = 0.66. Dotted and dash lines also represent diffraction limit of the optical system and expected SIM detection limit, respectively.

Figure 9.

(a) Scattering spectra of phase separating water and 2BE mixture

taken at various delay times. (b) Domain sizes plotted as a function of delay time. Closed square and opened circle represent light scattering and fluorescence imaging with structured illumination result, respectively. Solid line is a fitting curve of light scattering obtained with a power function, t,  = 0.72. Dotted and dash lines represent diffraction limit of the optical system and expected SIM detection limit, respectively. (c) Scattering intensity at wavenumber, Q = 10 m-1, plotted as a function of delay time.

Solid line represents a fitting curve obtained

with an exponential function.

Figure 10.

Conceptual description of a proposed model for spinodal decomposition

in early stage. A, B, C, and D describe delay time 0 s, up to 1 s, from 1 to 10 s, 38 ACS Paragon Plus Environment

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and after 10 s, respectively. Left illustrations display spatial distribution of 2BE micellar aggregates. Right graphs show local 2BE concentration in a phase separating mixture.

Figures



FIGURE 1


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FIGURE 2



FIGURE 3


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FIGURE 4



FIGURE 5


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FIGURE 6



FIGURE 7


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FIGURE 8



FIGURE 9


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FIGURE 10



TOC Graphic


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Time-Resolved Structured Illumination Microscopy for Phase

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