Laser surgery and optical trapping in a laser scanning microscope.

Optical manipulation opens up many new possibilities for experiments in the field of microbiology and is a very powerful tool for investigating cellular structure. In this emerging field imaging retains an important role, and systems that combine advanced imaging techniques with optical manipulation tools, such as laser scalpels or optical tweezers, are an important starting point for researchers. We present a flexible experimental platform that contains both a laser scalpel and optical tweezers, in combination with confocal and multiphoton microscopy. A simple manipulation of the external optics is used to retain the three-dimensional imaging capabilities of the microscopes. Two applications of the system are presented. In the first, the laser scalpel is used to initiate diffusion of a fluorescent dye through Escherichia coli mutants, which exhibit abnormal cell division, forming filaments, or chains of bacteria. The diffusion assay is used to assess the potential for the exchange of cytoplasmic material between neighboring cells. The second application investigates the binding of endoplasmic reticulum (ER) to chloroplasts in Pisum sativum (garden pea). Individual plant protoplasts are ruptured using the laser scalpel, allowing individual chloroplasts to be trapped and manipulated. Strands of the ER which are attached to the chloroplast are identified. The magnitude and nature of the binding between the chloroplast and the ER are investigated.