Abstract
In the last few years two-photon microscopy has been used to perform in vivo high spatial resolution imaging of neurons, glial cells and vascular structures in the intact neocortex. Recently, in parallel to its applications in imaging, multi-photon absorption has been used as a tool for the selective disruption of neural processes and blood vessels in living animals. In this review we present some basic features of multi-photon nanosurgery and we illustrate the advantages offered by this novel methodology in neuroscience research. We show how the spatial localization of multi-photon excitation can be exploited to perform selective lesions on cortical neurons in living mice expressing fluorescent proteins. This methodology is applied to disrupt a single neuron without causing any visible collateral damage to the surrounding structures. The spatial precision of this method allows to dissect single processes as well as individual dendritic spines, preserving the structural integrity of the main neuronal arbor. The same approach can be used to breach the blood-brain barrier through a targeted photo-disruption of blood vessels walls. We show how the vascular system can be perturbed through laser ablation leading toward two different models of stroke: intravascular clot and extravasation. Following the temporal evolution of the injured system (either a neuron or a blood vessel) through time lapse in vivo imaging, the physiological response of the target structure and the rearrangement of the surrounding area can be characterized. Multi-photon nanosurgery in live brain represents a useful tool to produce different models of neurodegenerative disease.
Highlights
The link between optics and biology has always been extremely strong
We show how the vascular system can be perturbed through laser ablation leading toward two different models of stroke: intravascular clot and extravasation
The first application developed for short-pulsed lasers in biology was multi-photon fluorescence microscopy (Denk et al, 1990)
Summary
The link between optics and biology has always been extremely strong. it was through the use of the first optical microscopes in the 17th century that fundamental concepts such as the existence of the cell itself were defined. In parallel to its application in imaging, multi-photon absorption has been used as a tool to manipulate the biological sample under investigation; in particular, this technique has been applied to the selective disruption of labeled cells and intracellular structures. The first published report of femtosecond laser subcellular nanosurgery was by König et al (1999), who demonstrated the potential of this technique by ablating nanometer-sized regions of the genome within the nucleus of living cells. In this work the spatial localization of multi-photon excitation was exploited to perform selective lesions on the processes of cortical neurons in living mice expressing fluorescent proteins. Non-linear laser ablation provides a method to induce vascular lesions one vessel at a time and to study cerebral microvascular diseases. In vivo time lapse imaging of cerebral fine structures in the days that follow a point disruption may represent an optimal tool to characterize the network response to injury and mimic a neurodegenerative pathology
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