Directed positioning of nuclei in living Paramecium tetraurelia: Use of the laser optical force trap for developmental biology

Abstract

AbstractWe have constructed a laser optical force trap (“laser tweezers”) by coupling an Nd:YAG laser to an optical microscope with a high numerical aperture objective. The laser beam (approximately 0.1 W power) is focused to a diffraction‐limited spot at the specimen plane of the objective: the wavelength chosen (1,064 nm) is not strongly absorbed by most biological materials and is thus not ablative. Because the intensity of the laser beam increases towards the center of the focal spot, small particles brought near the spot will be attracted to the center and held there. Movement of the laser beam will tend to move any trapped particles with it. The laser tweezers can permit precise, nondestructive repositioning of small structures inside a living cell, without recourse to micromanipulators. Initial work has involved the use of laser tweezers on cells of Paramecium tet‐raurelia held by a rotocompressor. We have been able to trap and reposition small organelles, especially the highly refractile structures known as crystals. Using a trapped crystal as a “tool”, we have been able to push micronuclei and other structures for many micrometers to virtually any desired location in a cell. In spite of extended exposure of specific structures and of individual cells to the laser beam, no damage has been detectible. Exposed cells, which were removed from the rotocompres‐sor and cultured, showed complete viabilty. The laser tweezers technique shows tremendous potential for applications to the study of many fundamental cellular and developmental phenomena in paramecia and other ciliates. For example, we intend to use this technique to investigate temporal and spatial characteristics of nuclear determining regions during sexual reorganization in Paramecium. © 1992 Wiley‐Liss, Inc.