抗淬灭增强的ExM‑SOFI技术

Anti⁃Quenching Enhanced ExM⁃SOFI Technology

膨胀超分辨技术是近几年出现的一种对样品制备进行改进实现分辨率提升的超分辨技术,由于其与其他光学技术的兼容性强,可以进一步提高分辨率,引起了越来越多研究人员的关注。复合膨胀技术是膨胀超分辨技术改进的一个主要发展方向之一,膨胀结合光学波动超分辨技术(ExM‐SOFI)在复合膨胀技术中是一种受限较小且使用较为广泛的技术。为了增强现有ExM‐SOFI技术的成像效果,本课题组将成像缓冲液技术应用于ExM‐SOFI技术,以增强膨胀样品在拍摄过程中的抗淬灭能力,从而使普通染料在ExM‐SOFI中的荧光强度、荧光波动幅度和闪烁比等均有增强。微管和囊泡的染色成像结果表明,使用这种技术可以使样品在高阶SOFI中保持真实结构,伪影更少,因而高阶SOFI技术可以提升膨胀样品的最终分辨率。

Objective Expansion superresolution 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 superresolution technology and other superresolution techniques is one main development direction for expansion superresolution technology.Expansion combined with optical fluctuation superresolution technology(ExMSOFI)is a widely used compound expansion technology with relatively few restrictions.To enhance the imaging of the existing ExMSOFI,we applied an imaging buffer to enhance the antiquenching ability of the expansion sample during shooting.The fluorescence intensity,fluorescence fluctuation amplitude,and ontime ratio of common dyes in ExMSOFI 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 highorder SOFI technology.Methods In this study,we derived an antiquenchingenhanced ExMSOFI technique by improving the existing ExMSOFI technique using an imaging buffer.First,the samples were labeled with biotinylated antibodies.Biotins can retain recognition sites after expansion,for postexpansion 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 reembedded in a highconcentration solution to prevent it from shrinking.After reembedding,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 oxygenscavenging system as the main component.The fluorescence intensity,antiquenching ability,and fluorescence fluctuation amplitude of the images before and after the buffer treatment were analyzed.In addition,the ontime ratio and artifacts of the SOFI images before and after buffer treatment were analyzed,and the changes in different orders of SOFI were compared.Results and Discussions 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,antiquenching ability,and fluorescence fluctuation amplitude are important to improve the quality and resolution of SOFI imaging.The ontime 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 ontime ratios.We analyzed the ontime 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 ontime ratio of the image with a buffer decreased from 80%‒95%to 35%‒40%.In a study by Wang et al.on SOFI,an ontime ratio in this interval was better for SOFI analysis.In addition,we analyzed the resolutionscaled 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 highorder SOFI.Conclusions ExMSOFI 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 antiquenching enhanced ExMSOFI technology that combines imaging buffer technology with the original ExMSOFI to reduce fluorescence quenching during shooting.We found that this technique enhanced both the intensity and fluctuation amplitude of the fluorescence.The ontime ratio was also reduced to a range that was more suitable for SOFI analysis,enabling ordinary dyes to perform better in ExMSOFI.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 highorder SOFI.