Three-photon imaging of synthetic dyes in deep layers of the neocortex

Abstract

Multiphoton microscopy has emerged as the primary imaging tool for studying the structural and functional dynamics of neural circuits in brain tissue, which is highly scattering to light. Recently, three-photon microscopy has enabled high-resolution fluorescence imaging of neurons in deeper brain areas that lie beyond the reach of conventional two-photon microscopy, which is typically limited to ~450 µm. Three-photon imaging of neuronal calcium signals, through the genetically-encoded calcium indicator GCaMP6, has been used to successfully record neuronal activity in deeper neocortical layers and parts of the hippocampus. Bulk-loading cells in deeper cortical layers with synthetic calcium indicators could provide an alternative strategy for labelling that obviates dependence on viral tropism and promoter penetration. Here we report a strategy for visualized injection of a calcium dye, Oregon Green BAPTA-1 AM (OGB-1 AM), at 500-600 µm below the surface of the mouse visual cortex in vivo. We demonstrate successful OGB-1 AM loading of cells in cortical layers 5-6 and subsequent three-photon imaging of orientation-and direction-selective visual responses from these cells.

Characterization of red fluorescent reporters for dual-color in vivo three-photon microscopy

“…To maintain constant average power at the brain surface, the laser power after the objective was measured at each wavelength and adjusted prior to imaging. We found that the pulse energies required for imaging were similar to those recently published for structural and functional neuronal imaging in vivo, and importantly, below the limits for heating-and nonlinear absorption-induced damage 14,16,34 . To determine the in vivo 3P excitation properties of tdTomato, we measured the fluorescent signal, background, and signal-to-background ratio (SBR) in layer 6b of the PPC (760 µm depth) of tdTomato at a range of wavelengths (1225-1360 nm) and constant average power at the surface (Fig.…”

“…1a). With the ultimate goal of measuring the in vivo excitation properties of RFPs for simultaneous dual-color 3P microscopy, we first characterized our output pulses after the objective at a range of wavelengths using frequency resolved optical gating (FROG) 27 . Pulse characterization with FROG allows for the recovery of spectral intensity and phase using iterative spectrogram inversion algorithms and provides a ground-truth pulse width measurement 25,26 .…”

“…In the mouse brain, both 2P and 3P EAL are determined by a combination of scattering and water absorption. 2P and 3P EALs in different regions of the mouse brain have been characterized extensively, both in vivo and in silico 12,14,19,20,27 . Therefore, considerations such as the scattering and water absorption, as well as the output power from current NOPA configurations should be taken into account for dual-color 3P imaging with a single excitation source in the 1300 nm range.…”

Advanced high resolution three-dimensional imaging to visualize the cerebral neurovascular network in stroke

“…Gradient-index (GRIN) fiber probe implants can be used to go much deeper, distinguishing synapses in the hypothalamus that are 5mm deep [66]. MPM enabled high-resolution imaging in deeper brain areas (Figure 2) [67]. Chris Xu et al successfully used a three-photon microscope to image the vascular structure in the hippocampus of mice and neurons labeled with red fluorescent protein, with a resolution of about 4.4μm at a depth of about 900 μm [68].…”

Glycine and GABAA receptors suppressively regulate the inspiratory-related calcium rise in the thoracic inspiratory cells of the neonatal rat

“…The confocal microscope thus does not appear to be suitable for studying the neuronal networks existing in deep nerve tissue, even in neonatal rats. In studies using two-photon or multiphoton microscopes [15,16], cells in deep nerve tissue could be visualized more sharply even in the adult central nervous system. In the present experiments, although we tried to carefully remove the ventral funiculus using a fine insect pin, we were not able to record any respiratory activity from cells in the ventromedial region of the neonatal thoracic spinal cord in many preparations.…”