Expansion super-resolution technology, in which resolution is improved by improving the corresponding sample, has emerged in recent years. Owing to its strong compatibility with other optical technologies and its high resolution, it has attracted an increasing amount of research attention. The combination of expansion super-resolution technology and other super-resolution techniques is one main development direction for expansion super-resolution technology. Expansion combined with optical fluctuation super-resolution technology (ExM-SOFI) is a widely used compound expansion technology with relatively few restrictions. To enhance the imaging of the existing ExM-SOFI, we applied an imaging buffer to enhance the anti-quenching ability of the expansion sample during shooting. The fluorescence intensity, fluorescence fluctuation amplitude, and on-time ratio of common dyes in ExM-SOFI were improved. Finally, the staining results of the microtubules and vesicles indicate that the use of this technique can make the sample more realistic, with fewer artifacts, and can improve the final resolution of expansion samples in high-order SOFI technology.

In this study, we derived an anti-quenching-enhanced ExM-SOFI technique by improving the existing ExM-SOFI technique using an imaging buffer. First, the samples were labeled with biotinylated antibodies. Biotins can retain recognition sites after expansion, for post-expansion staining, to reduce signal loss. An expanded hydrogel was then obtained using a common expansion protocol. Next, the expanded hydrogel was cut into a suitable size and re-embedded in a high-concentration solution to prevent it from shrinking. After re-embedding, the expanded hydrogel was incubated with a dye modified with streptavidin. During photography, the stained hydrogel was immersed in an imaging buffer for imaging. We used an imaging buffer with an oxygen-scavenging system as the main component. The fluorescence intensity, anti-quenching ability, and fluorescence fluctuation amplitude of the images before and after the buffer treatment were analyzed. In addition, the on-time ratio and artifacts of the SOFI images before and after buffer treatment were analyzed, and the changes in different orders of SOFI were compared.

The experimental design is illustrated in Fig. 1. According to the analysis results of the images before and after the imaging buffer treatment, the fluorescence intensity of the sample with the imaging buffer was approximately 60% higher than that without the imaging buffer [Fig. 2(c)]. The signal quenching speed of the sample with the imaging buffer was slower during the shooting process compared with that of the sample without the imaging buffer [Fig. 2(d)]. In the analysis of the fluorescence fluctuation amplitude [Fig. 2(e)], the fluorescence fluctuation amplitude of the image after the buffer was added was several times larger than that of the image before the buffer was added. Enhancements in the fluorescence intensity, anti-quenching ability, and fluorescence fluctuation amplitude are important to improve the quality and resolution of SOFI imaging. The on-time ratio is an important parameter that affects the imaging quality of SOFI; conventional dyes are often not conducive to SOFI analysis because of their high on-time ratios. We analyzed the on-time ratio of the images before and after the imaging buffer was added, and the results are shown in Fig. 3. Compared with the image without a buffer, the overall on-time ratio of the image with a buffer decreased from 80%-95% to 35%-40%. In a study by Wang et al. on SOFI, an on-time ratio in this interval was better for SOFI analysis. In addition, we analyzed the resolution-scaled Pearson (RSP) correlation values before and after the buffer was added; higher values indicate a better agreement. The RSP value after the buffer was added was higher than that before, indicating that the image with buffer was more authentic. In the comparison of different orders of SOFI (Figs. 4 and 5), in the imaging results of both the microtubule or vesicle, fewer image artifacts existed after the buffer treatment than before, and the real structure was better maintained in high-order SOFI.

ExM-SOFI technology is a composite expansion technology that has relatively few equipment limitations and can increase resolution. However, owing to the loss and dilution of fluorescence signals during the preparation of expanded samples, the signal of the expanded samples can be weak, making it difficult to achieve the best results during SOFI continuous shooting. In this study, we proposed an anti-quenching enhanced ExM-SOFI technology that combines imaging buffer technology with the original ExM-SOFI to reduce fluorescence quenching during shooting. We found that this technique enhanced both the intensity and fluctuation amplitude of the fluorescence. The on-time ratio was also reduced to a range that was more suitable for SOFI analysis, enabling ordinary dyes to perform better in ExM-SOFI. An increase in the RSP value also indicated that this technique increases the credibility of the image. Finally, a comparison of different order SOFI images showed that this method reduced artifacts and better maintained the real structure in high-order SOFI.