In our neurophotonics laboratory, we develop and apply several methods and technologies for all-optical investigation of murine neurophysiology and neuropathology. The data acquired can complement and verify the computational brain models developed in our neuromodeling unit.
Mouse brains are large, interconnected structures of millions of neurons, and often the use of high resolution microscopy techniques implies focusing on just a small part of them, and forget about the big picture. Lightsheet microscopy doesn’t do that, allowing to image every small detail over the whole three dimensional volume of the brain.
In order to do so, the whole brain is extracted and chemically cleared, while structures of interest are stained with immunofluorescence. By illuminating the sample with a thin sheet of light from the side, fluorescence can be acquired by a high speed camera in a single plane at a time, completing the full three dimensional image acquisition extremely fast. A single brain can produce up to 2 Terabytes of storage, so big-data approaches to data analysis must be employed. A custom made 4-color lightsheet setup is available at our facility, capable of acquiring 3d datasets of up to 1.5×1.5×0.8 cm, with a resolution.of less than half a micrometer. In the video, autofluorescence from a iDISCO cleared full hemisphere of an adult mouse.
While lightsheet microscopy allows full imaging of whole brains, the need for chemical clearing implies the data is only morphological, and no physiological behaviour can be observed. Conversely, while operating on smaller fields of view, two-photon microscopy is capable of imaging deep through turbid and scattering media, such as live brain tissue, both in slice form and directly in-vivo. It does so by focusing a femtosecond laser in a sub micrometer spot inside the sample. Due to the non linearity of two-photon excitation, fluorescence light is only generated in a sub-micrometer three dimensional volume at the focus. By rapidly scanning the focus through the sample, a full three dimensional image can be acquired one pixel at a time.
A commercial twophoton microscope (Femtosmart2D, Femtonics) is available at our laboratory, with all the facilities enabling imaging both in brain slices and in-vivo.