Investigating relaxation processes in cells and developing organisms: from cell ablation to cytoskeleton nanosurgery.
Dynamic microscopy of living cells and organisms alone does not reveal the high level of complexity of cellular and subcellular organization. All observable processes rely on the activity of biochemical and biophysical processes and many occur at a physiological equilibrium. Experimentally, it is not trivial to apply a perturbation that targets a specific process without perturbing the overall equilibrium of a cell. Drugs and more recently RNAi certainly have general and undesired effects on cell physiology and metabolism. In particular, they affect the entire cell. Pulsed lasers allow to severe biological tissues with a precision in the range of hundreds of nanometers and to achieve ablation on the level of a single cell or a subcellular compartment. In this chapter, we present an efficient implementation of a picosecond UV-A pulsed laser-based nanosurgery system and review the different mechanisms of ablation that can be achieved at different levels of cellular organization. We discuss the performance of the ablation process in terms of the energy deposited onto the sample and compare our implementation to others recently employed for cellular and subcellular surgery. Above the energy threshold of ionization, we demonstrate how to achieve single-cell ablation through the induction of mechanical perturbation and cavitation in living organisms. Below this threshold, we induce cytoskeleton severing inside live cells. By combining nanosurgery with fast live-imaging fluorescence microscopy, we show how the apparent equilibrium of the cytoskeleton can be perturbed regionally inside a cell.
Colombelli
Julien
J
Light Microscopy Group, Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), D-69117 Heidelberg, Germany.
Reynaud
Emmanuel G
EG
Stelzer
Ernst H K
EH
D000818:Animals; D002477:Cells / Q000166:cytology* / Q000528:radiation effects; D003599:Cytoskeleton / Q000378:metabolism* / Q000528:radiation effects; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D007834:Lasers; D008866:Microsurgery / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*