Laser microsurgery in the GFP era : a cell biologist's perspective

Author Posting. © The Author(s), 2007. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Methods in Cell Biology 82 (2007): 237, 239-266, doi:10.1016/S0091-679X(06)82007-8.Modern biology is based largely on a reductionistic ‘dissection’ approach – most cell biologists try to determine how complex biological systems work by removing their individual parts and studying the effects of this removal on the system. A variety of enzymatic and mechanical methods have been developed to dissect large cell assemblies like tissues and organs. Further, individual proteins can be inactivated or removed within a cell by genetic manipulations (e.g., RNAi or gene knockouts). However, there is a growing demand for tools that allow intracellular manipulations at the level of individual organelles. Laser microsurgery is ideally suited for this purpose and the popularity of this approach is on the rise among cell biologists. In this chapter we review some of the applications for laser microsurgery at the subcellular level, and describe practical requirements for laser microsurgery instrumentation demanded in the field. We also outline a relatively inexpensive but versatile laser microsurgery workstation that is being used in our lab. Our major thesis is that the limitations of the technology are no longer at the level of the laser, microscope or software, but instead only in defining creative questions and in visualizing the target to be destroyed.Our work is sponsored by grants from the NIH (GM59363 to AK and GM40198 to CLR) and HFSP (RGP0064 to AK). Construction of the laser microsurgery workstation was supported in par by Summer Research Fellowship from Nikon/Marine Biological Laboratory (2003 to AK)