Leybaert L, de Meyer A, Mabilde C, Sanderson M J
Department Physiology and Pathophysiology, Faculty of Medicine and Health Sciences, Ghent University, De Pintelaan 185, B-9000 Ghent, Belgium.
J Microsc. 2005 Sep;219(Pt 3):133-40. doi: 10.1111/j.1365-2818.2005.01502.x.
Most currently available confocal or two-photon laser scanning microscopes (LSMs) allow acquisition rates of the order of 1-5 images s(-1), which is too slow to fully resolve dynamic changes in intracellular messenger concentration in living cells or tissues. Several technologies exist to obtain faster imaging rates, either in the video-rate range (30 images s(-1)) or beyond, but the most versatile technology available today is based on resonant scanners for horizontal line scanning. These scanning devices have several advantages over designs based on acousto-optical deflectors or Nipkow discs, but a drawback is that the scanning pattern is not a linear but rather a sinusoidal function of time. This puts additional constraints on the hardware necessary to read-in the image data flow, one of which is the generation of a pixel clock that varies in frequency with the position of the pixel on the scanned line. We describe a practical solution to obtain a variable pixel clock add-on that is easy to build and is easy to integrate into a custom-built LSM based on resonant scanning technology. In addition, we discuss some important hardware and software design aspects that simplify the construction of a resonant scanning-based LSM for high-speed, high-resolution imaging. Finally, we demonstrate that the microscope can be used to resolve calcium puffs triggered by photolytically increasing the intracellular concentration of inositol trisphosphate.
目前大多数可用的共聚焦或双光子激光扫描显微镜（LSM）的采集速率约为每秒1 - 5幅图像，这对于充分解析活细胞或组织中细胞内信使浓度的动态变化来说太慢了。有几种技术可以实现更快的成像速率，要么处于视频速率范围（每秒30幅图像），要么更快，但目前最通用的技术是基于用于水平行扫描的共振扫描仪。与基于声光偏转器或尼普科夫圆盘的设计相比，这些扫描设备有几个优点，但一个缺点是扫描模式不是时间的线性函数，而是正弦函数。这对读取图像数据流所需的硬件提出了额外的限制，其中之一是生成一个频率随扫描线上像素位置变化的像素时钟。我们描述了一种实用的解决方案，即获得一个易于构建且易于集成到基于共振扫描技术的定制LSM中的可变像素时钟附加装置。此外，我们讨论了一些重要的硬件和软件设计方面，这些方面简化了用于高速、高分辨率成像的基于共振扫描的LSM的构建。最后，我们证明该显微镜可用于解析通过光解增加细胞内三磷酸肌醇浓度而触发的钙瞬变。
