Electrospun Porous Nanofibers with Imprinted Patterns Induced by Phase Separation of Immiscible Polymer Blends.

Nanofibrous nonwoven fabrics have attracted attention as porous adsorbents with high specific surface areas for the safe and efficient treatment of spilled organic dyes and petroleum. For this purpose, a method of fabricating porous nanofibers with high specific surface areas would be highly beneficial. In this study, the phase separation in nanofibers electrospun from blended solutions of immiscible polymers [poly(styrene) (PS) and poly(vinylpyrrolidone) (PVP)] was investigated. The removal of PVP as a sacrificial polymer afforded the imprinting of mesopores (40-70 nm) in the PS nanofibers. The effects of solution composition (PS/PVP in N,N-dimethylformamide) on the structure formation in the fibers were investigated. The nanofibers thus obtained could selectively adsorb low-molecular-weight hydrophobic dyes, such as Nile Red and Oil Red O. Thus, it is expected that the combined approach of electrospinning of immiscible polymer blends and phase separation-induced patterning can be applied to the fabrication of functional nanofibers for diverse applications.

Introduction

Spillage and discharge of organic dyes and petroleum pose grave threats to the ecology and health of living organisms. Various techniques, such as oxidation,[1] coagulation,[2] biodegradation,[3] photocatalytic decomposition,[4] and adsorption,[5] have been developed and used to treat dyes and oils. In particular, porous adsorbents with large specific surface areas are highly desired owing to their reusability. Electrospun nanofiber-based nonwoven fabrics are promising porous materials that have been used in various biomedical applications, including scaffolds for tissue engineering,[6,7] wound dressings,[8] drug delivery system,[9] and affinity carriers.[10] Being more permeable than other porous materials, these fabrics are also suitable for separation applications.[10] Electrospinning is a well-known method for preparing nanofibers, in which a polymer solution is electrified and injected from a spinneret onto a collector.[11−15] Examples of electrospun nanofibers reported previously as adsorbent materials include poly(vinylidene fluoride)/graphene oxide,[16] poly(vinylidene fluoride)/SiO2,[17] poly(styrene) (PS)/superfine powdered activated carbon,[18] and PS/SiO2.[19] In all previous studies, the matrix polymer was blended with other compounds to improve the specific surface area and adsorption capacity. However, the porous structure of the matrix polymer inside remained unchanged.

Recently, molecular imprinting has garnered significant attention for the fabrication of surface-modified polymeric materials. Molecular imprinting is a technique in which a monomer is polymerized in the presence of a template molecule, followed by the removal of the template molecule to create voids that are complementary to the template molecule in shape, size, and chemical functionality.[20,21] Molecular imprinting has been investigated for a wide range of applications, including chromatography,[22] drug delivery,[23] and biosensing.[24] It is expected that nanofibrous adsorbent materials could be fabricated with molecular recognition sites on the surface by applying molecular imprinting technology.[25,26]

In this study, inspired by molecular imprinting, we focused on the effects of the polymer blend composition on the nanofiber structure and developed a novel method of patterning porous structures in nanofibers. Indeed, the phase separation occurs inside nanofibers electrospun from immiscible polymer blends as the solvent evaporates, forming various morphologies, such as core–shell and sea–island structures.[27−29] Using this process, melt-blown nonwoven fabrics with pores of approximately 100 nm have also been obtained.[30] However, electrospinning is a complicated process with several parameters that can affect the structure of the resulting fibers. Although numerous studies have been focused on this process, the mechanism of structure formation remains unrevealed. Therefore, it is difficult to control these phase-separated structures.

Herein, PS and poly(vinylpyrrolidone) (PVP), which are a hydrophobic matrix polymer and hydrophilic sacrificial polymer, respectively, were dissolved in N,N-dimethylformamide (DMF). They were then electrospun into nanofibers containing mesostructures through phase separation. The mesostructure formed by phase separation after electrospinning was imprinted onto the remaining hydrophobic polymer after the sacrificial polymer was washed away with water. The nanofibrous nonwoven fabric thus obtained had interconnected mesopores linked with the fiber surface and therefore could be used as an adsorbent with a large specific surface area. The adsorption potential of the obtained mesoporous fibers was demonstrated via adsorption experiments using hydrophobic dyes. These fibers have potential for use not only as filter materials but also as medical materials, including scaffolds for regenerative medicine and drug carriers loaded with biomolecules.

Results

Preparation of Polymer Blends

First, the phase separation behavior of the polymer blend was investigated. The blend solution was prepared by adding the PVP solution to the PS solution. The concentration of each polymer was adjusted between 5 and 15%. This range was selected because at concentrations ≤5%, fibers could not be electrospun, whereas the blend solution was too viscous to be injected at concentrations ≥15%. The states of the blend solution are shown in Figure a,b. The solution was clear (shown in orange) at a PVP concentration of 5% and PS concentrations of 5, 10, and 15% but appeared clouded (shown in blue and green) at higher concentrations of PVP. In particular, when the concentration was ≥10%, phase separation was evident after a couple of hours (shown in green). The results of the dynamic light scattering (DLS) measurements of the blend solutions are presented in Figure c. No colloidal particles were detected in the 1% PVP solution, suggesting that the PVP molecular chains were dispersed in the dilute solution. The colloidal particle size of the 1% PS solution was approximately 30 nm. Although the 1% PS/1% PVP blend was clear in appearance, particles with sizes of 30–300 nm were still observed. They were larger than those in the PS solution, indicating interactions with PVP chains. It is considered that the interactions are based on the entanglement of the molecular chains because the polarities of PS and PVP are different. In the clouded 10% PS/10% PVP blend, three size distributions were observed, which could be PVP, PS, and the agglomerate of PS and PVP. In particular, there was a broad size distribution beyond 300 nm, which suggests that high concentrations caused PS and PVP to agglomerate.

Figure 1

(a) Phase diagram of solution of PS, PVP, and DMF. (b) Cloudiness in PS/PVP/DMF solutions. (c) Particle size distributions of PS/PVP/DMF solutions as determined using DLS.

Fiber Morphology and Surface Property

The exposed hydrophilic PVP on the surface could be removed by washing with water. Morphological and hydrophilicity analyses of the electrospun fibers before and after washing were conducted using scanning electron microscopy (SEM) and water contact angle (WCA) measurement, respectively. For comparison, the WCAs of the PVP film, PS film, and PS fiber were also measured and were found to be 50, 89, and 145°, respectively.

The fiber morphology, average fiber diameter, and WCAs before washing are shown in Figure . The beads were ignored while measuring the diameter because it is difficult to define the bead diameter accurately. Finest fibers (diameter ∼ 371 nm) were obtained from the transparent solution with the lowest concentration (5% PS/5% PVP). However, there were many beads and the WCA was 80° (Figure g), indicating the presence of both PS and PVP on the fiber surface. The clouded solutions of 5% PS/10% PVP (Figure d) and 5% PS/15% PVP (Figure a) fibers and the clear solutions of 10% PS/5% PVP (Figure h) and 15% PS/5% PVP (Figure i) fibers had the same total polymer concentrations of 15 and 20%, respectively, and similar average fiber diameters. However, their morphologies were significantly different.

Figure 2

Surface characterization of PS/PVP electrospun nanofibrous fabrics before washing. SEM images: values are average diameter ± SD; scale bar = 10 μm. Insets: droplet images and WCAs. The polymer concentrations in the solution for electrospinning were (a–c) 15%, (d–f) 10%, and (g–i) 5% PVP and (a,d,g) 5%, (b,e,h) 10%, and (c,f,i) 15% PS.

The fibers with more PVP exhibited smoother surfaces, more variations in diameter, more beads, and greater adherence of the fibers with each other (Figure a,d). The corresponding WCA was also ≤80°, indicating the presence of PVP on the surface. The fibers with 10% PS/10% PVP showed similar characteristics. By contrast, the fibers with high PS contents were relatively straight and had numerous fine grooves on their surfaces. This feature was attributable to the condensation of water vapors in the air.[31] The WCA was ≥130°, indicating that the surface was primarily covered with PS. Thus, fibers with variable diameters and a hydrophilic surface were obtained from the PVP-rich 10% PS/15% PVP blend (Figure b), whereas the PS-rich 15% PS/10% PVP blend resulted in straight fibers with a hydrophobic surface (Figure f). The overall WCAs after washing were ≥130° (Figure ), indicating removal of PVP from the surface. The mass of 5% PS/5% PVP fibers decreased significantly after washing (Figure g), which could be due to the fragmentation of fibers during PVP removal. The numbers of beads in the fibers spun from the PVP-rich blends also decreased significantly after washing, as did the average fiber diameter (Figure a,b,d), suggesting that the outer surfaces of the fibers and beads were originally covered with PVP.

Figure 3

Surface characterization of PS/PVP electrospun nanofibrous fabrics after washing. SEM images: values are average diameter ± SD; scale bar = 10 μm. Insets: droplet images and WCAs. The polymer concentrations in the solutions for electrospinning were (a–c) 15%, (d–f) 10%, and (g–i) 5% PVP and (a,d,g) 5%, (b,e,h) 10%, and (c,f,i) 15% PS.

Porosity in Fibers

Transmission electron microscopy (TEM) analysis of fibers was performed before (Figure ) and after (Figure ) washing to examine the internal structure. A characteristic speckled pattern was observed in the fibers from the blends with low PVP concentrations (Figures g,h and 5g,h). The contrast difference was more pronounced after washing, indicating the non-uniform localization of PS and PVP in the fibers. The patterns were approximately 40–70 nm (avg: 56 nm) in size and were elongated along the fiber direction, indicating the mesoporous structure in the fibers (Figure S1). In the fibers prepared from high-concentration blends, a core–shell structure with a clear contrast difference was observed (Figure b,c,e,f,i).

Figure 4

TEM images of the PS/PVP electrospun nanofibrous fabric before washing. Scale bar = 1 μm. The polymer concentrations of the solutions for electrospinning were (a–c) 15%, (d–f) 10%, and (g–i) 5% PVP and (a,d,g) 5%, (b,e,h) 10%, and (c,f,i) 15% PS.

Figure 5

TEM images of PS/PVP electrospun nanofibrous fabric after washing. Scale bar = 1 μm. The polymer concentrations of the solutions for electrospinning were (a–c) 15%, (d–f) 10%, and (g–i) 5% PVP and (a,d,g) 5%, (b,e,h) 10%, and (c,f,i) 15% PS.

After washing, these fibers exhibited a core–shell structure with a speckled pattern on the shell layer (Figure b,e,f), which was attributed to the non-uniform localization of PVP on the surface caused by the aggregation of PS in the core. In the washed fibers spun from the 15% PS/15% PVP blend, multiple contrast differences were observed in the axial direction, suggesting the existence of multiple core-like structures. One such fiber was frozen in liquid nitrogen, fractured, and washed with water to remove the PVP. The SEM analysis revealed a mesoporous structure (Figure S2). In contrast, the fibers spun from the 15% PS/5% PVP blend exhibited no significant speckled pattern, although a contrast difference was observed. Considering this aspect together with the previous results, it is speculated that excess PS formed the outer periphery of the fiber, preventing the removal of PVP trapped inside.

Residual PVP after Washing

Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy was performed to evaluate the residual PVP on the fiber surface after washing. The spectrum of PS has characteristic peaks corresponding to C–H (str.) from the phenyl group at 3026 cm–1, C–C (str.) from the phenyl group at 1601 and 1492 cm–1, and CH2 (bend.) at 1452 cm–1.[32] The spectrum of PVP has strong peaks due to C=O (str.) at 1652 cm–1 and C–N (str.) at 1286 cm–1.[33] The spectra of the washed fibers, however, exhibited only the peaks of PS (Figure ), confirming the removal of PVP. Interestingly, the peaks of PVP in the spectrum of the 15% PS/15% PVP fibers before washing were smaller than those in the spectrum of the 5% PS/5% PVP fibers even though the polymer ratio was the same (1:1). As ATR-FTIR spectroscopy analysis of nanofibrous sheets particularly emphasizes the information about the nanofiber shell,[34] it was assumed that PVP was encapsulated in the 15% PS/15% PVP fibers, which is consistent with the TEM observations.

Figure 6

ATR-FTIR spectra of electrospun PS/PVP fibers. Solid line: before washing. Dashed line: after washing. Controls: electrospun PS nanofibers (purple) and PVP nanofibers (red).

It is difficult to evaluate accurately the PVP remaining inside the fiber using ATR-FTIR, which is a qualitative surface analysis technique. Therefore, proton nuclear magnetic resonance (1H NMR) spectroscopy analyses of the re-solubilized washed fibers were performed. The NMR spectra and peak attributions[35] are shown in Figure . The ratio of PS to PVP after washing was calculated from the proton intensities of PS and PVP and used to determine the percent of PVP removed (Table ). In the case of 5% PS/5% PVP fibers, 87% of PVP was removed. These findings together with the SEM and TEM results indicate that PS and PVP were well blended in the fiber and the internal PVP was almost removed, leaving behind an interconnected porous structure. The removal percentages of the 15% PS/5% PVP and 5% PS/15% PVP fibers were 41 and 98%, respectively. These results were attributed to the excess polymer distributed on the outside. For the 15% PS/15% PVP fibers, the core–shell structure was observed by TEM; however, as much as 88% PVP was removed, suggesting that the shell was an interconnected porous structure.

Figure 7

1H NMR spectra of PS/PVP fibers after washing.

Table 1

Amount of PVP (%) Removed as Estimated from 1H NMR Analyses

sample	area (a + b)	area (e + f + h + i)	PVP/PS % (before washinga)	PVP/PS % (after washing)	removed PVP %
5% PS/5% PVP	9.7	1.7	100	13	87
15% PS/5% PVP	16.1	4.5	33	20	41
5% PS/15% PVP	18.9	1.6	300	6	98
15% PS/15% PVP	12.8	2.1	100	12	88

Preparation ratio in electrospinning solution.

Dye Adsorption

The results presented thus far demonstrate that electrospinning of the clear solution of PS and PVP produced blended nanofibers and that washing the fibers with water resulted in porous PS nanofibers with interconnected pores ranging in size from 40 to 70 nm. Nanofibers with such a structure would be expected to adsorb dyes and oils via hydrophobic interactions. They could also exhibit different adsorption responses to dyes of different molecular sizes. Accordingly, adsorption tests were conducted using three dyes with different molecular weights.

Pieces of the washed nanofibrous fabric obtained from the 5% PS/5% PVP blend were evaluated. For comparison, pieces of native PS fibrous fabric (control) prepared by electrospinning of PS solution alone were also examined because the native PS fibers had a relatively smooth surface.

The dyes used were Nile Red (MW: 318.4 Da), Oil Red O (MW: 408.5 Da), and Rubrene (MW: 532.7 Da), all of which are hydrophobic in nature. Photographs of the fabrics after adsorption and the corresponding quantitative results are shown in Figure . The amounts of Nile Red and Oil Red O adsorbed on the washed 5% PS/5% PVP fibers were as much as five times higher than those on the control fibers, indicating a high adsorption performance toward hydrophobic molecules compared to that of native PS fibers. In the case of Rubrene, only a twofold increase in adsorption was observed because the Rubrene molecules were larger than the others and it was difficult for them to enter the pores in the fibers.

Figure 8

Adsorption test results for native PS fibers and washed 5% PS/5% PVP fibers. (a) Nile Red and (b) Oil Red O. Insets show pictures of the stained fabrics. (c) Rubrene. *p < 0.05, n = 3. The inset shows phase contrast and fluorescence microscopy images. The stained sheets were arranged in the same eye field. The intensity was measured with ImageJ software.

Discussion

Electrospinning of water/oil emulsions is known to result in a core–shell structure with the water-soluble polymer at the core.[36,37] The application of emulsion electrospinning for drug release systems has been investigated because it enables the encapsulation of low-molecular-weight compounds in nanofibers.[38] The mechanism of structuration has been ascribed to the elongation of the polymer jet during electrospinning and the rapid evaporation of the solvent, which causes the dispersed phase to localize as the core and extend into an elliptical shape, forming a core–shell structure.[39] However, this process is unstable and difficult to control. In this study, we systematically analyzed the effects of the compositions of PS/PVP/DMF solutions and emulsions on the phase-separated structures of the electrospun nanofibers.

The PS/PVP/DMF solution became clouded as the polymer concentration increased. With further increase, the solution became unstable and phase-separated. The clouded solution had particles with a size range of 300–10,000 nm as measured by DLS. The Hildebrand solubility parameters (SP values) of PS, PVP, and DMF are 17.4–19.0 MPa1/2,[40] 22.5–25 MPa1/2,[41] and 24.8 MPa1/2,[42] respectively. Thus, PVP is more soluble than PS in DMF. In addition, the molecular weight of the PVP was lower than that of PS. The molecules of PVP in DMF could be more extended than those of PS. As the PVP concentration increases, the space excluded by PVP would decrease and PS would agglomerate. Therefore, it was concluded that the large particles formed in the clouded solution were PS. Structural analysis of the fibers obtained from the clear solution revealed that PS and PVP were well blended within dozens of nanometers. The fibers formed from the clouded solutions contained similarly mixed polymers; however, the excess polymer was exposed on the shell surface.

Scheme shows the possible mechanism of structure formation. In the cases of clear solutions, both PS and PVP were uniformly dispersed. Electrospinning enabled the solvent to evaporate rapidly, resulting in fibers with miscible PS and PVP domains. When PVP was present in excess, fibers covered with PVP were obtained. Likewise, in the cases of clouded solutions, when the PS concentration was low, the PS aggregates localized in the PVP matrix were elongated into fibers. This mechanism resembles the mechanism proposed for the electrospinning of block copolymers, where the island phases of the core component aggregate to form a core–shell structure.[43,44]

Scheme 1

Schematic Illustration of the Formation Mechanism of the Mesoporous Structure via Electrospinning of Polymer Blend Solutions with Different Polymer Concentrations

At high concentrations, PS tended to form large particles. Even at the tip of the Taylor cone during electrospinning, the PS particles aggregated to form a core–shell-like structure with PS localized to the outside, which could be explained by the fact that functional groups and molecular species with low surface free energies localize to the fiber surface during electrospinning in air.[45] The structure formation during electrospinning of polymer blends depends on whether phase separation occurs before the solvent evaporates. In the present case, it was assumed that a mesoscale structure was formed because macroscale phase separation did not proceed due to the large molecular weight of PS and the close solubilities of PS and PVP. A combination of immiscible polymers would be expected to form fibers with distinct core–shell interfaces.

In the adsorption tests, compared to Rubrene, Nile Red and Oil Red O were easily adsorbed on the porous PS nanofibers obtained after removing PVP. The difference in the degree of adsorption could be due to the difference in molecular weight, which affects the entrapment in the fiber pores. The pores on the nanofibers formed using PVP as a template were approximately 40–70 nm in diameter according to the TEM analysis. Mesoporous materials with selectivity are desired as adsorbents for oils and dyes. Zeolites, for instance, are well-known porous materials with pores 1 nm or less in size; however, mesoporous zeolites with >10 nm pores have been reported to exhibit larger molecular sieving effects.[46,47] Many porous polymers also have been reported, but the fabrication of porous nanofibers remains challenging.[48,49]

The fabrication of PS nanofibers with porous surfaces via electrospinning has been reported. Changing the humidity during electrospinning of the PS/DMF system[50,51] and changing the mixing ratio of the DMF/THF solvent[52,53] affect the solvent volatilization rate to induce the microstructure of the PS fiber surface. These methods are simple and effective, but they require strict control of the spinning environment, making it difficult to control the structure precisely. Although the resulting porous structure improves the surface area, it does not necessarily form interconnective pores inside the fibers. In contrast, our method successfully formed a mesoporous structure using a hydrophilic polymer, in this case, PVP, as a template. As our method provided the porous structure by washing PVP, the surface pores are considered to be interconnected. Thus, dye adsorption would have occurred not only by simple adsorption on the rough surface but also by entrapment inside the mesopores following the diffusion of molecules into the pores. The mobility of dye molecules inside mesopores decreases as their molecular weight decreases and as the molecules become bulkier, which makes it more difficult for them to pass through the mesopores. Therefore, even for molecules with slightly different molecular weights, such as Nile Red and Rubrene, large differences can be observed in the adsorption properties. This characteristic is expected to be effective for the selective recovery of low-molecular-weight molecules, which is particularly difficult in oil spills. The desorption of dye was difficult because the adsorption was based on hydrophobic interactions. It is difficult to reuse the PS fibers as an adsorbent for oil and dye, but it could be employed as a disposable material exclusively for adsorption. The difficulty in desorbing would also lead to a procedure to stain polystyrene, which is a difficult material to stain.

Conclusions

In this study, we focused on the relationship between the inner structures of fibers comprising two polymers and their miscibility. To the best of our knowledge, this is the first report of the successful fabrication of mesoporous PS nanofibers by imprinting the phase separation pattern of one polymer as a sacrificial template. In addition, the effects of the PS/PVP blending ratio on the structure of electrospun fibers were studied. The fibers obtained from the clear solution exhibited nanoscale blending of PS and PVP, and mesoporous PS fibers were realized after PVP removal. The fibers obtained from the phase-separated clouded solutions showed a core–shell-like structure. The adsorption of hydrophobic dyes onto the porous PS fibers was noticeably higher than that on the native PS fibers owing to the very large specific surface area imparted by the porous structure. The average size of the mesopores throughout the fiber was approximately 56 nm, and a difference in the amount of adsorption was observed depending on the size of the dye molecules, suggesting selectivity toward the adsorbed dyes. It is anticipated that the presented methodology inspired by molecular imprinting technology will lead to the electrospinning of various functional nanofibers for not only adsorbents but also for medical materials such as drug delivery materials.

Experimental Section

Electrospinning

The polymer solutions were prepared by dissolving PS (MW: 316,000 Da; Sigma-Aldrich, USA) and PVP (MW: 40,000 Da; Nacalai Tesque, Japan) separately in DMF, and the PVP solution was added to the PS solution under gentle stirring. The resulting PS/PVP/DMF mixture was vortexed, taken into a syringe, and electrospun (NANON, MECC, Japan) through a 27G nozzle at a flow rate of 1.0 mL h–1 and linear velocity of 3.14 m s–1 under an electric field of 1.25 kV cm–1. The fabricated fibers were washed with water for 10 min (six times) to remove PVP. The solution was vortexed every 20 min during electrospinning.

Dynamic Light Scattering

Each mixture was vortexed immediately before measurement, and a 2 mL aliquot was prepared in a quartz cell (optical path length: 10 mm) and analyzed (SZ-100, Horiba, Japan). The measurement range was 0.1–10,000 nm with a backscatter angle of 173°, the wavelength was 532 nm, the measurement time was 2 min, and the temperature was 25 °C. The measurements were repeated three times.

Electron Microscopy

The specimens were sputter-coated with Pt/Pd (120 s, 15 mA, 6 Pa) prior to SEM (JSM-6390, JEOL, Japan) analysis at an applied voltage of 15 kV. The fiber diameter and orientation were measured from SEM images using ImageJ (ver. 1.52e). For TEM (H-7650, Hitachi, Japan) analysis, and the fibers were spun on a carbon support membrane (ELS-C10, Oken Shoji Co. Ltd., Japan) for approximately 10 s and observed at a voltage of 200 kV.

Water Contact Angle

A water droplet (2 μL) was dropped on a specimen electrospun onto a glass slip, and the WCA was measured using a drop shape analyzer (DSA-25, KRUSS, Germany) via a tangential method at 2 s after dropping. The Young–Laplace method was applied for hydrophobic specimens with contact angles greater than 130°. The measurement was repeated five times for each specimen.

Attenuated Total Reflectance Fourier-Transform Infrared

The ATR-FTIR (Nicolet 6700, Thermo Scientific, USA) spectra were obtained in the range of 400–4000 cm–1 with a resolution of 4 cm–1 and an average of 64 scans for each data point.

Proton Nuclear Magnetic Resonance

The 1H NMR (JNM-ECX500II, JEOL, Japan) spectra were recorded to determine the detailed structures of the polymers. To set an accurate chemical shift, a coaxial insert (Wilmad-LabGlass) was used with the NMR tube for external locking (reference: CDCl3 with 0.1% tetramethylsilane).

Adsorption Tests

Fabrics of native PS fibers and washed 5% PS/5% PVP fibers were cut into 6 × 6 cm2 pieces, immersed in dye solutions for 3 h, and then washed with 60% propanol until no color appeared. Propanol solutions of 0.02% Nile Red (FUJIFILM Wako, Japan), 1% Oil Red O (Sigma-Aldrich, USA), and 0.04% Rubrene (FUJIFILM Wako, Japan) were used as the dye solutions. The amounts of adsorbed Nile Red and Oil Red O were determined by measuring the absorbance (520 nm) of the fiber/tetrahydrofuran solutions using a spectrophotometer (U-2900, Hitachi, Japan). Rubrene was quantified via fluorescence microscopy, where the fluorescence intensity was normalized by the intensity in the bright field.