The multiphoton system (two-photon/2p and three-photon/3p) enables 3 types of innovative experimental paradigms:
- transcranial imaging
- deep multiphoton imaging
- high-speed volumetric imaging with Bessel beams
Transcranial imaging enables the recording of brain activity and imaging of cell-cell interactions at cellular and sub-cellular resolution in awake behaving head-fixed animals without the need for invasive cranial window surgery. The native state of the cells is not disrupted. It is essential to the study of neuro-immune interactions, as any disruption of the skull triggers cellular signalling that would alter the cell-cell interactions of the neuro-immune response.
Employing a typical cranial window implantation, deep multiphoton imaging enables recording from previously inaccessible sub-cortical structures (hippocampus, striatum) at cellular and sub-cellular resolution while leaving the overlying brain regions (usually cortex) within a live mouse intact.
Bessel beam volumetric imaging allows high speed functional imaging to be performed with a multiphoton microscope allowing dendritic Ca2+, voltage and neurotransmitter imaging at significantly higher speeds than conventional point-scanning.
Two-photon vs. Three-photon, deeper imaging and structures resolved: (a) Sagittal view of a mouse brain with overlay of imaging depth of 2p (green) and 3p (red) using a cranial window. 3p reaches multiple brain regions while 2p is restricted to superficial cortex. (b) Adapted from Takasaki et al. (2020); neurons are labeled with genetically encoded calcium sensor. 2p signals begin to give unreliable activity recordings after 350-400μm while 3p maintains signal fidelity and contrast. (c) Transcranial 2p imaging provides no useful contrast of brain cells (MacVicar lab), while 3p produces useful images deep in the cortex through the intact skull (adapted from Wang et al. (2018)).
Ouzounov et al. “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain” Nat. Methods. 14 (2017) 388.
Wang et al. “Three-photon imaging of mouse brain structure and function through the testing skull” Nat. Methods. 15 (2018) 789.
Lu et al. “Video-rate volumetric functional imaging of the brain at synaptic resolution” Nat. Neuroscience 20 (2017) 620.