In vivo two-photon imaging of mouse hippocampal neurons in dentate gyrus using a light source based on a high-peak power gain-switched laser diode

Abstract

Abstract:In vivo two-photon microscopy is an advantageous technique for observing the mouse brain at high resolution. In this study, we developed a two-photon microscopy method that uses a 1064-nm gain-switched laser diode-based light source with average power above 4 W, pulse width of 7.5-picosecond, repetition rate of 10-MHz, and a high-sensitivity photomultiplier tube. Using this newly developed two-photon microscope for in vivo imaging, we were able to successfully image hippocampal neurons in the dentate gyrus and obtain panoramic views of CA1 pyramidal neurons and cerebral cortex, regardless of age of the mouse. Fine dendrites in hippocampal CA1 could be imaged with a high peak-signal-tobackground ratio that could not be achieved by titanium sapphire laser excitation. Finally, our system achieved multicolor imaging with neurons and blood vessels in the hippocampal region in vivo. These results indicate that our two-photon microscopy system is suitable for investigations of various neural functions, including the morphological changes undergone by neurons during physiological phenomena.

Functional Imaging of Dentate Granule Cells in the Adult Mouse Hippocampus

“…Be that as it may, the substantial rate of remapping of spatial representations (Bonnevie et al, 2013;Danielson et al, 2016) and the flexibility of granule cell activity in relation to behavioral-state preference (i.e., running vs resting) shown here both suggest that granule cell activation patterns in the adult DG change across days, presumably undergoing context-and experience-dependent plasticity. Together with previously described approaches allowing for structural imaging of the DG (Gu et al, 2014;Kawakami et al, 2015), the method described here will substantially expand the available toolbox to analyze granule cell activity in an intact hippocampal circuitry to study the mechanisms underlying functional (e.g., experienceinduced) and structural (e.g., neurogenesis-associated) plasticity of the adult DG.…”

“…In awake experiments, the average laser power was 234 Ϯ 75 mW and 133 Ϯ 50 mW, respectively. We estimate that the laser power in the focal volume (600 -800 m deep in the tissue) was Ͼ10 -40 times lower, assuming a scattering length of 200 -250 m at 920 -1040 nm (Kobat et al, 2009). Laser intensity presumably was also attenuated by scattering in the white matter tract of the corpus callosum and potentially by clipping of the beam at the window implant edges.…”

“…Two-photon calcium imaging. We used a custom-built, two-photon microscope of the Sutter Instrument Movable Objective Microscope type, equipped with a 16ϫ long-working-distance, water-immersion objective (0.8 numerical aperture, model CFI75 LWD 16ϫW, Nikon), a Pockels cell (model 350/80 with controller model 302RM, Conoptics), and galvanometric scan mirrors (model 6210, Cambridge Technology), controlled by HelioScan software (Langer et al, 2013). For excitation of R-CaMP1.07, we used a ytterbium-doped potassium gadolinium tungstate (Yb:KGW) laser (1040 nm, Ͼ2 W average power, ϳ230 fs pulses at 80 MHz; model Ybix, Time Bandwidth Products).…”

“…This is partly due to the lack of optical imaging methods for resolving granule cell activity within the intact hippocampal circuit in vivo, given that the DG is located several hundred microns deep below the ventral borders of the neocortex, which makes optical access to the DG difficult without interfering with or damaging overlying hippocampal structures. Consequently, DG granule cells have hardly been resolved structurally in vivo and have been imaged functionally in only one study so far (Gu et al, 2014;Kawakami et al, 2015;Danielson et al, 2016). Functional imaging of granule cell ensembles is, however, a prerequisite to further reveal fundamental principles of DG computation during encoding and expression of hippocampal engrams.…”

Deep tissue imaging with multiphoton fluorescence microscopy

“…Calcium transients due to spontaneous neural activity were clearly observed. CA1 hippocampal neurons have also been observed with a high-peak power gain-switched laser diode at 1,064 nm with a 7.5 picosecond pulse duration [52]. Taken together, these results demonstrate the capability of current technology to acquire structural and functional neurovascular information with high spatial and temporal resolution deep within scattering brain tissue.…”