1. Introduction

Biomaterials, since the dawn of human society, have been widely used as tissue engineering scaffolds and drug delivery carriers. For example, nacre, also known as mother-of-pearl, was used as dental implants by ancient Mayans [1, 2 ]. Modern biomaterials date back to 1940s. Sir Harold Ridley pioneered in achieving first implantation of an intraocular lens made of poly(methyl methacrylate) [1]. Later on, various types of biomaterials have been studied. These biomaterials can be grouped into four categories: metallic, ceramic, polymeric and hybrid materials, based on their types; or two categories: synthetic and natural materials, based on their origins. Commercially available biodevices, such as stainless steel-based bone implants and silicone-based breast implants, are celebrating their clinical success for decades. Not only choosing the type of biomaterials is crucial, the design of biomaterial-based implants or scaffolds is equally essential to sustain their success even longer. A recent study done by Robert Langer and Daniel Anderson’s group showed that by simply tuning the size and shape of a porous scaffold, its overall performance in terms of biocompatibility can be significantly improved [3]. Other than size and shape, other nano-features, such as nano-pillars or nanogrooves, have shown to be important parameters. One study showed that synthetic nanostructures were able to guide mesenchymal stem cells to neuronal differentiation [4]. Therefore, tuning the morphological and structural parameters of a biomaterial has a great impact on the fate of a biomaterial-based biodevice in vivo.

Traditionally, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are used to characterize the morphology and microstructure of biomaterials [5]. Although these techniques can provide nanometer-scale resolution, they have their limitations. For example, conventional SEM and TEM require high vacuum, which is not a biological relevant environment. This also makes them difficult for real-time imaging. They can only provide a “snap-shot” of how a biomaterial interacts with cells or tissues. Compared to these techniques, optical microscopy is superior as it can monitor cell-biomaterial interaction in situ and provide three-dimensional information with optical section [6, 7 ].

Optical microscopies, since their invention at 1660s, have been developed to a very much more sophisticated level. Presently, biologists are heavily depending on confocal laser scanning microscopy. Super-resolution microscopy, which overcomes light diffraction limit, has emerged as an alternative to SEM and TEM. It is also known as nanoscopy which perfectly describes its capability with nanometer accuracy [8]. Besides these recent achievements in the field of super-resolution microscopy, nonlinear optical microscopy (NLOM) has also gained widespread use in the field of biomedical optics [9]. NLOM include multiphoton excitation fluorescence (MPEF), second-harmonic generation (SHG), third-harmonic generation (THG), coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering (SRS) and many others. Among all the NLOMs, SHG microscopy receives much attention with advantages of being label-free, inherent optical sectioning, minimal out-of-plane absorption, near-infrared excitation for superior optical penetration, and capable of imaging live tissues [10]. Recently, endogenous signals and exogenous probes that can be used by SHG microscopy have been reviewed [9, 11, 12 ]. However, in comparison to the number of fluorescent probes available for fluorescence microscopy, the probe inventory for SHG microscopy is limited: only a small number of biopolymers are SHG-active, including collagen, silk, cellulose and poly(hydroxybutyrate-co-hydroxyhexanoate) [10, 13–15 ]. Here, we investigate a biomaterial, polysulfone (PSF) as a new SHG-active biopolymer.

PSF is an amorphous thermoplastic with high thermal, mechanical and chemical resistance. It has been widely used as ultrafiltration [16], hemodialysis [17], gas-separation [18], and fuel-cell membranes [19]. Besides industrial applications, PSF has been used as a biomaterial for decades. It has been used as orthopaedic implants since 1970s [20]. Efforts have also been made to improve its blood compatibility [21]. PSF-based hollow fiber cartridges have also been applied in artificial kidney. It out-performs other membrane materials such as cellulose acetate, polyacrylonitrile, and polycarbonate [22]. One study showed that PSF-based membrane material with double coating of collagen IV and 3,4-dihydroxy-L-phenylalanine (DOPA) can support renal tubular epithelial cells’ growth and differentiation, which could be a suitable scaffolding material for bioartificial kidney [23]. Another study showed that PSF membranes coated with DOPA can be used as synthetic cell culture substrates to support human pluripotent stem cells growth under chemically defined condition [24]. Over the years, researchers were focusing on surface modification of PSF or blending PSF with other materials such as grapheme [23]. Other material properties of PSF, such as its nonlinear optical (NLO) property, were rarely investigated [25]. Therefore we investigated the NLO properties of PSF so that we can widen its applications in biomedical field.

2. Materials and methods

2.1 Materials

PSF (average MW ~22 kDa) was purchased from Sigma-Aldrich. PSF pellets were used without further treatment.

2.2 Digital microscopy

The surface of PSF was examined by digit microscope (VHX-500, Keyence).

2.3 SEM

The samples were sputtered with gold, and SEM was conducted with a JSM-7400F field emission scanning electron microscope (JEOL, Tokyo, Japan).

2.4 SHG microscopy

SHG measurements were performed as previously described by Zhuo et al [26]. In short, SHG images were recorded by the use of a commercial laser scanning microscopic imaging system (Zeiss LSM 510 META, Jena, Germany) coupled to a mode-locked femtosecond Ti: sapphire laser (110 fs, 76MHz), tunable from 700 nm to 980 nm (Coherent Mira 900-F). The polarization direction of the laser light is the horizontal polarization. A LD Plan-Neofluar 20x/0.40 Corr objective was employed to focus the excitation beam into the sample and collect the backscattered SHG signals. To avoid material damage caused by other processes such as two-photon absorption, we controlled the average laser power of sample at <5 mW and there was no photobleaching at this low power level [26].

2.5 Quantification of fibril organization

PSF fibril organization was determined by analysis of SHG images using the Fast Fourier Transform (FFT) module of ImageJ software (NIH). The details of quantitative analysis was described by Zhuo et al [27]. Briefly, FFT can represent all frequencies present in an image by using of the power plot. This makes the FFT of an image a good method to calculate the fibril bundles orientation. In detail, for an image containing a set of aligned fibril bundles, FFT image will show higher values along the direction orthogonal to the direction of the fibril bundles and its intensity plot will have an elliptic behavior; for an image with randomly oriented collagen-fibril bundles, the intensity plot of FFT image will show a circular behavior. In this work, we fitted the intensity plot of FFT image with an ellipse, and the ratio between its short and long axes is defined as the fibril bundles orientation. According to this definition, the fibril bundles orientation approaches the maximum value of 1 when only fibril bundles are not organized and randomly oriented; and the fibril bundles orientation approaches 0 when only the fibril bundles are organized in an aligned fashion.

3. Results and discussion

We first examine the PSF pellets under traditional digital microscope and SEM. As it can be seen in Fig. 1(a) , all pellets are transparent, and cubic shaped (~3 mm). When we examine it under digital microscope and SEM, the fibrillar structure can be seen (Figs. 1(b) and 1(c)).

 

Fig. 1 Morphology and microstructure of polysulfone (PSF) pellets. (a) Digital image of PSF pellets reveals their macroscopic structure as cubic-shaped and transparent; (b) Digital microscopic image of PSF pellets reveals their fibrillar mesostructure; (c) Scanning electron microscopic image of PSF pellets reveals their fibrillar microstructure.

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We next examine these samples under SHG microscope. As expected, PSF is a second order nonlinear optical material, which can generate SHG signal. It can be seen in Fig. 2(a) that the polar and fibrillar-like structure is revealed. The structure seen in these SHG images strongly resembles that obtained from traditional digital microscope and SEM. Quantitatively, the fibril-bundle orientation ratio was 0.66 ± 0.05 and the diameter of fibril-bundle was 19.3 ± 1.2 μm.

 

Fig. 2 PSF is SHG active. a) SHG image of PSF pellets. PSF fibrils (green) are aligned in parallel; scale bar = 50 μm; (b) SHG spectral peaks at various excitation wavelengths (810-890 nm, coded with different colors); (c) Log-log plot of the above SHG signal measurements demonstrating a log[I₄₁₅] = 0.32 + 2.08 × log [I₈₃₀] dependence, quadratic to a good approximation, consistent with nonlinear second order optical upconversion; (d) Emission λ-scan of the SHG signal (λ_ex = 830 nm) acquired from SHG imaging of PSF. The solid spheres (D) represent back scattering SHG data and the solid line represents a Gaussian fit (red). The full width at half-maximum of the fitted curve bears a 1/√2 relation to the spectral profile of the corresponding beam.

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To further confirm PSF is SHG-active, we varied the excitation wavelength from 810 to 890 nm. The peaks of emission wavelength were exactly one half of the excitation wavelength (Fig. 2(b)), which indicates PSF is indeed SHG-active. The dependence of the output signal on irradiation intensity was measured by varying the 830 nm excitation intensity. The linear regression was applied to the log-log plots revealed a quadratic power dependence of SHG intensity to the power intensity, as shown in the Fig. 2(c).

The intensity of SHG signal is proportional to the square of the incident laser intensity. This confirmed the two-photon nature of the emission from PSF pellets. Figure 2(d) showed the emission wavelength data of the SHG signal (λ_ex = 830 nm). The data in the graph were fitted to a Gaussian curve exhibiting a maximum at 415 nm, which is exactly half the excitation wavelength of 830 nm. The full width at half-maximum of Gaussian distribution (~11 nm) was narrow and it obeys a relationship to the corresponding wavelength profile of the fundamental beam (~15 nm).

From above, we demonstrated that PSF is SHG-active. It is bright under SHG microscope. Furthermore, we observed the mesostructure of PSF is fibrillar-like. The fibril-bundle orientation and the diameter of fibril-bundle were determined. It is worth noting that the mesostructure of PSF is polar and fibrillar-like, which makes it SHG-active. This interesting nonlinear optical property makes PSF a more interesting scaffolding material. SHG uses near-infrared light which penetrates deeper into biological tissues and provides an optical window with low auto-fluorescence signals from background. As such, PSF implants can be easily traced under SHG. With the new development, PSF-based medical devices can be visualized via SHG endoscope. This widens the application of PSF as a biomaterial. PSF can also be fabricated into nanoparticles [28]. It thus can be used as nanocarriers for drugs, genes and many others. We could also expect PSF as a therasonic nanoagent for drug delivery and disease diagnostics. So far, only collagen [10], silk [14], cellulose [13], poly(hydroxybutyrate-co-hydroxyhexanoate) [15] and cellular DNA [26] have been investigated by SHG microscopy. Our work of PSF could add PSF as the sixth SHG biopolymer to enlarge the very short existing list of such kind of biomaterials.

4. Conclusion

In summary, we have showed that PSF is a second order nonlinear optical material, which can generate SHG signals. We found that SHG imaging can quantitatively reveal the mesostructure of PSF as polar and fibrillar-like. With the advent of the SHG-based endoscopy [29], PSF will be a more interesting scaffolding biomaterial for implants and therasonic nanoagents.