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MBC in Press, published online ahead of print January 26, 2003
Mol. Biol. Cell 10.1091/mbc.E02-03-0163

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Submitted on March 25, 2002
Revised on December 9, 2002
Accepted on December 27, 2002

Controlled damage in thick specimens by multiphoton excitation

James A. Galbraith1 and Mark Terasaki2*

1 Laboratory of Neurobiology, NINDS, NIH, Bethesda, MD; and Marine Biological Laboratory, Woods Hole, MA
2 Department of Physiology, University of Connecticut Health Center, Farmington, CT; and Marine Biological Laboratory, Woods Hole, MA

* Corresponding author. E-mail address: terasaki{at}neuron.uchc.edu.

Controlled damage by light energy has been a valuable tool in studies of cell function. Here, we show that the Ti:Sapphire laser in a multiphoton microscope can be used to cause localized damage within unlabeled cells or tissues at greater depths than previously possible. We show that the damage is due to a multiphoton process, and made wounds as small as 1 µm in diameter 20 µm from the surface. A characteristic fluorescent scar allows monitoring of the damage and identifies the wound site in later observations. We were able to lesion a single axon within a bundle of nerves, locally interrupt organelle transport within one axon, cut dendrites in a zebrafish embryo, ablate a mitotic pole in a sea urchin egg, and wound the plasma membrane and nuclear envelope in starfish oocytes. The starfish nucleus collapsed ~1 hr after wounding, indicating that loss of compartmentation barrier makes the structure unstable; surprisingly, the oocyte still completed meiotic divisions when exposed to maturation hormone, indicating that the compartmentalization and translocation of cdk1 and its regulators is not required for this process. Multiphoton excitation provides a new means for producing controlled damage deep within tissues or living organisms.




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