摘　要：激光扫描共聚焦显微镜（LSCM）是一种高分辨率的光学成像仪器，它利用“共轭成像”原理，获得的图片质量远超于普通荧光显微镜。LSCM有两组功能不同的针孔，即照明针孔和探测针孔，这是实现“共轭成像”的关键。由于照明针孔的大小和位置一般是固定的，LSCM主要通过调节探测针孔的大小来获得高质量的成像图片。然而，很多使用者对于LSCM中针孔大小与荧光成像质量的关联缺乏了解。因此，本文阐述了针孔在LSCM中的作用原理及其与显微镜分辨率的关系，并通过小鼠脊髓腹侧白质中的免疫荧光成像分析，发现针孔参数优化对提高LSCM荧光成像质量具有显著影响。这一发现将为LSCM成像分析提供重要参考。
关键词：激光扫描共聚焦显微镜；针孔；分辨率；荧光成像；成像分析
中图分类号：Q-334；Q-336                  文献标志码：A                    DOI：10.3969/j.issn.1007-7146.2023.01.004

Abstract: Laser scanning confocal microscopy (LSCM) is a high-resolution imaging device. It uses the principle of “conjugate imaging”, the image quality obtained is much higher than that of ordinary fluorescence microscopy. LSCM has two pairs of pinholes, namely illumination pinhole and detection pinhole, which have different function respectively and are the key to realize conjugate imaging. Although the size and position of the illumination pinhole are generally fixed, LSCM can obtain high-quality images by adjusting the size of the detection pinhole. However, there is a lack of knowledge about the link between parameter of pinhole and the higher quality picture for LSCM. Therefore, this paper reveals the principle of pinhole in LSCM and its relationship with resolution. Furthermore, based on immunofluorescence image analysis for specific proteins expression in white matter of mouse spinal cord, we found that optimization for pinhole play an important role in LSCM image quality improving. This study would provide important reference for LSCM imaging analysis.
Key words: laser scanning confocal microscopy; pinhole; resolution; fluorescence imaging; imaging analysis
（Acta Laser Biology Sinica, 2023, 32(1): 020-025）