The application of the pulsed ruby laser to experimental microsurgery at the cellular and intracellular level

Abstract

Conventional microsurgery using microneedles is a very useful technique for the developmental biologist. However, there are some limitations imposed on this technique because of both practical physical considerations and the time required for an operation. Very elaborate equipment is necessary for the most delicate work and the nature of the equipment is such that the variety of usable experimental material is somewhat limited. Work with microneedles at the tissue level is often a difficult proposition and the necrosis of cells adjacent to a puncture can be a difficult problem to overcome with certain types of material. At the levels requiring greatest precision even the micromanipulator has one serious limitation in plant micrurgy. In order to perform an intracellular operation, one must first alter the integrity of the plant cell wall.

With the advent of the laser beam a new approach to micro­surgery was suggested. Laser microsurgery showed great promise as a supplement to conventional techniques. The possibility of using focused laser light as a tissue, cell or organelle disrupting agent suggested immediate application to studies in cytology and developmental biology. Theoretical considera­tions showed that one could expect to have a range of destruc­tive capability from altered volumes of material at the cubic micron level up past altered volumes of the order of cubic centimeters. The unusual properties of the laser light might even allow one to perform an operation on material surrounded by other material without affecting the intervening material.

Before such research could be attempted it was necessary to design a system whereby one might align target material for a predetermined operation and observe the effects. This necessitated an extensive study of the properties of laser light and the application of these with respect to a given physical system designed for a specific range of operations. Thus, an instrumentation had to be developed and a technique evolved for practical use of the laser in studies of a cytol­ogical nature. It was very important that the overall limit­ations of the technique be defined. Prior to this, the nature of the laser effects had to be determined and the relative contributions of the controllable variables to producing an effect had to be ascertained. A description of all the com­ponents of the system was required as well as a description of the reproducible effects obtained with living material.

With the instrumentation and techniques established, it was also desired to demonstrate the applicability of laser microsurgery to the solution of a purely biological problem with a view to demonstrating the ease and versatility of this new technique. A "model tissue" used to determine the relative contributions of energy input, objective focal length and degree of defocusing In producing a laser effect was found to be of great utility In this work. Thus, three distinct re­searches were undertaken: the development of the instrument­ation, the study of the laser effects and the solution of a biological problem using the laser technique. Because of the interdisciplinary nature of the work, an elaboration of laser action and the properties of laser light as related to their application is introduced at this point.