Botvinick Elliot L, Shah Jagesh V
Beckman Laser Institute, Department of Biomedical Engineering, University of California, Irvine, California 92612, USA.
Methods Cell Biol. 2007;82:81-109. doi: 10.1016/S0091-679X(06)82003-0.
In this chapter, we review the imaging techniques and methods of molecular interrogation made possible by integrating laser light sources with microscopy. We discuss the advantages of exciting fluorescence by laser illumination and review commonly used laser-based imaging techniques such as confocal, multiphoton, and total internal reflection microcopy. We also discuss emerging imaging modalities based on intrinsic properties of biological macromolecules such as second harmonic generation imaging and coherent anti-Raman resonance spectroscopy. Super resolution techniques are presented that exceed the theoretical diffraction-limited resolution of a microscope objective. This chapter also focuses on laser-based techniques that can report biophysical parameters of fluorescently labeled molecules within living cells. Photobleaching techniques, fluorescence lifetime imaging, and fluorescence correlation methods can measure kinetic rates, molecular diffusion, protein-protein interactions, and concentration of a fluorophore-bound molecule. This chapter provides an introduction to the field of laser-based microscopy enabling readers to determine how best to match their research questions to the current suite of techniques.
在本章中，我们回顾了通过将激光光源与显微镜相结合而实现的分子检测成像技术和方法。我们讨论了激光照明激发荧光的优势，并回顾了常用的基于激光的成像技术，如共聚焦、多光子和全内反射显微镜。我们还讨论了基于生物大分子固有特性的新兴成像模式，如二次谐波生成成像和相干反拉曼共振光谱。介绍了超越显微镜物镜理论衍射极限分辨率的超分辨率技术。本章还重点介绍了基于激光的技术，这些技术可以报告活细胞内荧光标记分子的生物物理参数。光漂白技术、荧光寿命成像和荧光相关方法可以测量动力学速率、分子扩散、蛋白质-蛋白质相互作用以及荧光团结合分子的浓度。本章介绍了基于激光的显微镜领域，使读者能够确定如何最好地将他们的研究问题与当前的技术套件相匹配。
