Multi-photon nanosurgery in live brain

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.

Keywords: in vivo imaging; laser ablation; laser dissection; laser surgery; two-photon microscopy.

Figure 1

Schematic view of the optical window preparation for in vivo imaging. In the craniotomy technique a piece of bone (light brown) is removed and replaced by a thin cover glass (light blue). The chronically implanted glass window replaces the skull and gives optical access to the GFP-labeled cortical neurons. Imaging is performed using 20× long-working-distance water immersion dipping objective.

Figure 2

Custom-made two photon apparatus. The figure shows the laser beam [in red, coming from the bottom right side of the panel (1)] that is first scanned by two galvanometric mirrors (2), then expanded by a telescope (3), and finally focused by the objective (4) onto the specimen (5). The emitted light (yellow) is separated from the exciting beam by a first dichroic mirror (6) and then split by a second dichroic mirror (7) in the red and green components. Two photomultipliers detect the split fluorescence emissions (8a,b).

Figure 3

Neural nanosurgery in GFP-M transgenic mice. (A) Laser dissection of an interneuron cell body. Each image is a maximum-intensity projection of a set of 20 optical sections acquired at 2 μm z-step (at 150 μm depth). The tip of the red lightning symbol represents the laser irradiation point. The first panel was acquired just before the laser irradiation. The middle panel was acquired 24 h after the laser irradiation. The last panel shows an overlay of the neuron before (red) and after (green) laser irradiation. Scale bar, 25 μm. (B) Laser-induced lesion of a single dendrite. Maximum-intensity z projection (from 100 to 500 μm) of a pyramidal neuron before and 24 h after dendritic dissection. Scale bar, 60 μm. (C) Laser ablation of a single dendritic spine. Images of a portion of a dendrite before and 40 min after spine ablation. Scale bar, 15 μm. (B) and (C) are modified with permission from Sacconi et al. (2007).

Figure 4

Models of laser-induced vascular lesion. (A) Time lapse images of maximum intensity z-projections (from 20 to 60 μm) before (left) and after (right) the laser-induced ischemic hemorrhage. The figures show in green the GFP-labeled neuron in a GFP-M mouse and in red the vascular network labeled with Texas-red dextran dye. The tip of the yellow lightning symbol represents the laser irradiation point. The first image was acquired just before the laser irradiation. Scale bar, 20 μm. (B) Time lapse images of maximum intensity z-projections (from 20 to 100 μm) before (left) and after (right) the laser induction of an intravascular clot. Scale bar, 20 μm.

Figure 5

Effects of the nanosurgery on apical dendrites. (A) Laser-induced dendritic swelling. Time-lapse of maximum intensity z-projections (from 140 to 160 μm) of irradiated dendrite in GFP-M transgenic mouse. The tip of the red lightning symbol represents the laser irradiation point. The first panel was acquired just before the laser irradiation. Scale bar, 10 μm. (B) Laser-induced lesion of a single dendrite. Time-lapse of maximum intensity z-projections (from 90 to 110 μm) of irradiated dendrite. Scale bar, 25 μm. (B) is modified from Sacconi et al. (2007).