Laser Microsurgery in the GFP Era: A Cell Biologist's Perspective

Abstract

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 laboratory. 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.At last in an incredible manner he [Archimedes] burned up the whole Roman fleet. For by tilting a kind of mirror toward the sun he concentrated the sun's beam upon it; and owing to the thickness and smoothness of the mirror he ignited the air from this beam and kindled a great flame, the whole of which he directed upon the ships that lay at anchor in the path of the fire, until he consumed them all.1 I. History of the Field A. The Genesis of "Micro-Photo-Surgery"The origins of Cell Biology as a branch of "natural philosophy" can be traced to the English polymath Robert Hooke who, using a hand-crafted, leather and goldtooled compound light microscope (LM), published a book containing elaborate drawings of magnified objects which he called Micrographia. In this book, which became an immediate best-seller (and has been reprinted countless times), Hooke used the term "cell" to describe the repeating units seen in magnified slices of cork that resembled the monk cells of a monastery. Ironically, these repeating units were not actual cells but rather just the cellulose walls that surround cells in plant tissues. It took another 175 years of optical development and exploration, before Schleiden and Schwann (1839) convincingly asserted that cells are the fundamental building blocks of all life. In 1855, the Prussian physician and politician Virchow postulated that cells arise only from preexisting cells by reproduction and cannot be formed de novo from amorphous "living matter" or "protoplasma." This principle, which Virchow eloquently 1 Dio's Roman History, translated by Earnest Cary Loeb Classical Library, Harvard University Press, Cambridge, 1914, Vol. II, p. 171. NIH Public Access Author ManuscriptMethods Cell Biol. Author manuscript; availabl...

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