Development of MEMS Mirror-Type Laser Microdissection
Development of MEMS Mirror-Type Laser Microdissection
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MEMS镜式激光显微切割的发展
DOI：
10.11239/jsmbe.59.95
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发表时间：
2021
影响因子：
--
通讯作者：
横田 浩章
中科院分区：
文献类型：
--
作者：
長谷川 正仁;工 藤 靖;平野 美奈子;横田 浩章
激光显微切割（LMD）是在显微镜下使用紫外（UV）激光束分离组织样品的特定区域或特定细胞的方法。可以从分离的生物样本中纯化和提取各种生物分子，例如核酸和蛋白质分子，其可以用于分子生物学分析。激光束在样品平面上扫描以切割出目标样品的任意区域，通常使用XY电动载物台或一组两个偏转棱镜来执行。除了这些传统激光扫描组件的可用性之外，还存在由UV激光照射引起的对样品的热损伤的担忧。本文报道了微机电系统（MEMS）镜型LMD仪器的发展，以减少热损伤的高速激光扫描。我们比较了两种主要的激光扫描方法：光栅扫描和矢量扫描中使用的激光偏转组件。然后，我们详细介绍了本研究中使用的静电型MEMS反射镜的工作原理和我们的仪器的发展。MEMS反射镜偏转（非谐振模式）由矢量扫描操作控制，并且不与激光脉冲照射同步。所开发的仪器演示了高速激光扫描，这是几十倍的速度比传统的LMD仪器。然后，我们展示了猪心肌切片的LMD，并确认较高的激光脉冲速率降低了完成LMD所需的扫描次数。此外，我们讨论了激光辐射引起的热损伤的目标组织与扫描速度和描述的模拟估计的激光扫描的数量，以完成显微切割通过改变激光扫描速度和重复率。模拟结果表明，我们的仪器提供LMD与样品的热损伤减少，并最大限度地减少激光扫描的次数和处理时间。仪器的特性，包括减少样品热损伤的能力，紧凑和简单的结构，单元化能力，与光学显微镜的高兼容性，和成本效益提供了一个有吸引力的替代传统的，市售的LMD仪器。
Laser microdissection (LMD) is a method for isolating a specific region of a tissue sample or a specific cell using an ultraviolet (UV) laser beam under a microscope. Various biomolecules, such as nucleic acids and protein molecules, can be purified and extracted from isolated biological specimens, which can be used for molecular biological analysis. The laser beam scan on the sample plane to cut out arbitrary areas of the target sample has been conventionally performed using an XY motorized stage or a set of two deflection prisms. In addition to the usability of these conventional laser scanning components, there is a concern about thermal damage to the sample caused by UV laser irradiation. This paper reports on the development of microelectromechanical system (MEMS) mirror-type LMD instrumentation for reducing thermal damage by high-speed laser scanning. We compare the laser deflection components used in the two major laser scanning methods: raster and vector scans. Then, we detail the operating principle of the electrostatic-type MEMS mirror used in this study and the development of our instrumentation. The MEMS mirror deflection (non-resonant mode) is controlled by a vector scan operation and is not synchronized with laser pulse irradiation. The developed instrumentation demonstrates a high-speed laser scan, which is several tens of times faster than that of conventional LMD instrumentation. We then demonstrate the LMD of a pig heart muscle slice and confirm that a higher laser pulse rate lowered the number of scans required to complete the LMD. Further, we discuss the laser irradiation-induced thermal damage to the target tissue in association with the scan speed and describe the simulation performed to estimate the number of laser scans to complete microdissection by varying the laser scan speed and repetition rate. The simulation results indicate that our instrumentation affords LMD with reduction in sample thermal damage and minimizes the number of laser scans and the process time. The characteristics of the instrumentation, including the ability to reduce sample thermal damage, a compact and simple structure, unitization capability, high compatibility with optical microscopy, and cost effectiveness offer an attractive alternative to conventional, commercially available LMD instrumentation.