As a nonlinear optical imaging technique that offers high spatial resolution and high penetration depth, second harmonic imaging holds great promise for clinical diagnosis and various applications in the biomedical field, because it overcomes photobleaching and saturation absorption owing to energy absorption, which are commonly encountered in fluorescence imaging. Second-harmonic generation (SHG) is a nonlinear optical process in which two identical photons interact with a nonlinear material and are effectively converted into a single photon with precisely twice the frequency of the incident beam. In biologically relevant SHG imaging, the predominant approach has traditionally relied on the use of exogenous dye markers or endogenous proteins with a relatively low SHG efficiency. In fact, numerous studies have demonstrated strong and photostable SHG signals generated by inorganic crystalline materials. However, most of these inorganic crystalline materials contain heavy metals and have relatively large sizes (~100 nm in diameter). Recently, silicon quantum dots (SiQDs) were developed and have attracted growing interest owing to their remarkable properties, such as aqueous solubility, low cytotoxicity, high quantum yield, and exceptional stability against photobleaching. However, only few studies have investigated the generation of SHG signals from SiQDs without structural reconstitution, which has great potential for advanced optical applications, particularly in the field of SHG imaging. In this study, we developed polyethylene glycol (PEG)-coated SiQDs, an asymmetric material with a high nonlinear optical effect, as second-harmonic probes. To enhance the biological affinity and reduce the surface oxidation of the SiQDs, we modified their surface with PEG and investigated the imaging effect of PEG-coated SiQDs as a biological probe for second-harmonic wave imaging in HepG2 cells. Compared to two-photon fluorescence imaging, the second-harmonic imaging technique based on PEG-coated SiQDs provides more reliable and stable results. This finding can promote the future applications of SiQDs in molecular imaging, drug delivery, and stem cell therapy. By combining the advantages of the SHG dye, which has good biocompatibility and extremely low cytotoxicity, and the SHG inorganic crystalline materials with the photostability of the crystal structure, our SiQDs are expected to become the primary choice among many probes. We labeled hepatocellular carcinoma (HepG2) cells with non-functionalized SiQDs for cell imaging using SHG.

First, the nonlinear material used in this study, PEG-coated SiQDs, was synthesized by directly reducing the precursor with silicon-oxygen bonds and then modifying with organic ligands. The morphology and chemical composition of the SiQDs were characterized through transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDS). Furthermore, the physical mechanism behind the strong SHG of SiQDs was examined using finite-difference time-domain (FDTD) numerical simulations. The second harmonic characteristics of the SiQDs were then evaluated experimentally using a custom-built setup. Finally, to verify the feasibility of using the PEG-coated SiQDs in cell labeling and imaging, SHG imaging studies on HepG2 cells were conducted using confocal microscopy.

The TEM image of the PEG-coated SiQDs [Fig.1(a)] reveals that they are approximately spherical and have an average diameter of (2±0.5) nm. The excellent second-order nonlinear effects of these SiQDs were verified both theoretically and experimentally. By scanning the SHG signals of the SiQDs [see Fig.4(b)], we confirmed that they exhibit strong and stable SHG signals. Furthermore, we used these SiQDs to perform nonlinear optical imaging of HepG2 cells. Confocal microscopic visualization of the HepG2 cells treated with PEG-coated SiQDs confirmed the excellent tracking and imaging ability of the SiQDs (Fig.5). Furthermore, a TPL scan of the cells incubated with SiQDs demonstrated the advantages of SHG imaging over TPL imaging (Fig.6). Overall, the PEG-coated SiQDs serve as stable and reliable biological probes, significantly improving the image contrast compared with that of two-photon fluorescence imaging. The advantages of SHG imaging, including the absence of photobleaching, blinking, and saturation absorption, are highlighted. In addition, the intensity of the SHG signal produced by the PEG-coated SiQDs is 100 times higher than that obtained in two-photon fluorescence imaging [Fig.6(m)]. These results indicate that SHG imaging based on PEG-coated SiQDs has great potential for a wide range of applications in biomedical imaging and other related fields.

This paper presents a method for preparing PEG-coated SiQDs and their application in cell imaging. The PEG-coated SiQDs have a dynamic fluid diameter of only (2±0.5) nm, and the PEG molecules on their surface enhance their biocompatibility and show no apparent toxicity. The main innovation lies in the exceptionally strong and stable SHG signals exhibited by SiQDs. The SiQDs were employed as biological probes for SHG imaging of human liver cancer cells (HepG2). The advantages of SHG imaging, including the absence of photobleaching, blinking, and saturation absorption, were highlighted by comparing the results with those of two-photon fluorescence imaging. Thus, SiQDs can serve as highly biocompatible photosensitizers without causing toxic side effects and thus have promising prospects in biomedical applications.