The optical monitoring of multiple single neuron activities requires high-throughput parallel acquisition of signals at millisecond temporal resolution. To this aim, holographic two-photon microscopy (2PM) based on spatial light modulators (SLMs) has been developed in combination with standard laser scanning microscopes. This requires complex coordinate transformations for the generation of holographic patterns illuminating the points of interest. We present a simpler and fully digital setup (SLM-2PM) which collects three-dimensional two-photon images by only exploiting the SLM. This configuration leads to an accurate placement of laser beamlets over small focal volumes, eliminating mechanically moving parts and making the system stable over long acquisition times. Fluorescence signals are diffraction limited and are acquired through a pixelated detector, setting the actual limit to the acquisition rate. High-resolution structural images were acquired by raster-scanning the sample with a regular grid of excitation focal volumes. These images allowed the selection of the structures to be further investigated through an interactive operator-guided selection process. Functional signals were collected by illuminating all the preselected points with a single hologram. This process is exemplified for high-speed (up to 1 kHz) two-photon calcium imaging on acute cerebellar slices.
In combination with fluorescent protein (XFP) expression techniques, two-photon microscopy has become an
indispensable tool to image cortical plasticity in living mice. In parallel to its application in imaging, multi-photon
absorption has also been used as a tool for the dissection of single neurites with submicrometric precision without
causing any visible collateral damage to the surrounding neuronal structures. In this work, multi-photon nanosurgery is
applied to dissect single climbing fibers expressing GFP in the cerebellar cortex. The morphological consequences are
then characterized with time lapse 3-dimensional two-photon imaging over a period of minutes to days after the
procedure. Preliminary investigations show that the laser induced fiber dissection recalls a regenerative process in the
fiber itself over a period of days. These results show the possibility of this innovative technique to investigate
regenerative processes in adult brain.
In parallel with imaging and manipulation technique, non-linear microscopy offers the opportunity to optically record
electrical activity in intact neuronal networks. In this work, we combined the advantages of second-harmonic generation
(SHG) with a random access (RA) excitation scheme to realize a new microscope (RASH) capable of optically recording
fast membrane potential events occurring in a wide-field of view. The RASH microscope, in combination with bulk
loading of tissue
with FM4-64 dye, was used to simultaneously record electrical activity from clusters of Purkinje cells in acute cerebellar
slices. Complex spikes, both synchronous and asynchronous, were optically recorded simultaneously across a given
population of neurons. Spontaneous electrical activity was also monitored simultaneously in pairs of neurons, where
action potentials were recorded without averaging across trials. These results show the strength of this technique in
describing the temporal dynamics of neuronal assemblies, opening promising perspectives in understanding the
computations of neuronal networks.
In this work, we combined the advantages of second-harmonic generation (SHG) with a random access (RA) excitation
scheme to realize a new microscope (RA-SHG) capable of optically recording fast membrane potential events occurring
in a wide-field configuration. The RA-SHG microscope in combination with a bulk staining method with FM4-64 was
used to simultaneously record electrical activity from clusters of Purkinje cells (PCs) in acute cerebellar slices.
Spontaneous electrical activity was also monitored simultaneously in pairs of neurons, where APs were recorded in a
single trial without averaging. These results show the strength of this technique to describe the temporal dynamics of