In vivo multiphoton nanosurgery on cortical neurons.

Two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact neocortex of living mice. Multiphoton absorption has also been used as a tool for the selective disruption of cellular structures in living cells and simple organisms. In this work, we exploit the spatial localization of multiphoton excitation to perform selective lesions on the neuronal processes of cortical neurons in living mice expressing fluorescent proteins. Neurons are irradiated with a focused, controlled dose of femtosecond laser energy delivered through cranial optical windows. The morphological consequences are then characterized with time lapse 3-D two-photon imaging over a period of minutes to days after the procedure. This methodology is applied to dissect single dendrites with submicrometric precision without causing any visible collateral damage to the surrounding neuronal structures. The spatial precision of this method is demonstrated by ablating individual dendritic spines, while sparing the adjacent spines and the structural integrity of the dendrite. The combination of multiphoton nanosurgery and in vivo imaging in mammals represents a promising tool for neurobiology and neuropharmacology research.

[1]  A. Vogel,et al.  Mechanisms of femtosecond laser nanosurgery of cells and tissues , 2005 .

[2]  W. Webb,et al.  Nonlinear magic: multiphoton microscopy in the biosciences , 2003, Nature Biotechnology.

[3]  K. Svoboda,et al.  Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex , 2002, Nature.

[4]  D. Kleinfeld,et al.  Targeted insult to subsurface cortical blood vessels using ultrashort laser pulses: three models of stroke , 2006, Nature Methods.

[5]  G. Feng,et al.  Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP , 2000, Neuron.

[6]  Mehmet Fatih Yanik,et al.  Neurosurgery: Functional regeneration after laser axotomy , 2004, Nature.

[7]  Renzo Antolini,et al.  Combined intracellular three-dimensional imaging and selective nanosurgery by a nonlinear microscope. , 2005, Journal of biomedical optics.

[8]  James A Galbraith,et al.  Controlled damage in thick specimens by multiphoton excitation. , 2003, Molecular biology of the cell.

[9]  K J Halbhuber,et al.  Intracellular nanosurgery with near infrared femtosecond laser pulses. , 1999, Cellular and molecular biology.

[10]  F. Helmchen,et al.  Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo , 2004, Nature Methods.

[11]  W. Denk,et al.  Deep tissue two-photon microscopy , 2005, Nature Methods.

[12]  Aravinthan D. T. Samuel,et al.  The role of the AFD neuron in C. elegans thermotaxis analyzed using femtosecond laser ablation , 2006, BMC Neuroscience.