Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser

Abstract

In vivo two-photon microscopy has revealed vital information on neural activity for brain function, even in light of its limitation in imaging events at depths greater than several hundred micrometers from the brain surface. We developed a novel semiconductor-laser-based light source with a wavelength of 1030 nm that can generate pulses of 5-picosecond duration with 2-W output power, and a 20-MHz repetition rate. We also developed a system to secure the head of the mouse under an upright microscope stage that has a horizontal adjustment mechanism. We examined the penetration depth while imaging the H-Line mouse brain and demonstrated that our newly developed laser successfully images not only cortex pyramidal neurons spreading to all cortex layers at a superior signal-to-background ratio, but also images hippocampal CA1 neurons in a young adult mouse.

Figure 1. Schematic diagrams for in vivo two-photon microscopy using a novel picosecond pulse laser and a system to secure the head of mouse under standard upright microscope stage.

(a) Optical arrangement for the 1030-nm picosecond (newly developed) and 910-nm femto-second (Ti:Sapphire) lasers to be introduced into the microscope. The optical pathway from each laser is shown in green and red lines, respectively. The duration of an individual pulse of the 1030-nm laser beam was about 5 picoseconds (left). (b) The newly developed apparatus, with a horizontal adjustment mechanism, to secure the head of the mouse under an upright microscope stage. Three adjuster bolts are located on the adapter stage, and a 35-mm disposable dish was mounted on the head of the mouse. The mouse head was suspended from the adapter stage by the 35-mm dish, and the body was held under the stage by soft cloth. The stage angle could be manually controlled by adjuster bolt to ensure that the cover slip and objective lens were parallel, which would reduce optical aberrations.

Figure 2. Two-photon fluorescence imaging of cortical and hippocampal CA1 pyramidal neurons with 910-nm femto-second and 1030-nm picosecond excitation in a young adult H-line mouse brain.

(a) Maximum intensity projections of three-dimensional stacks were obtained with 910-nm and 1030-nm lasers (b). Four normalized xy frames from the z-stack at various depths are shown, including the dura matter, apical dendrite, soma, white matter, and hippocampus, respectively (left panel). The arrowhead shows a part of a basal dendritic process at cortex layer V.

Figure 3. Two-photon fluorescence imaging of cortical pyramidal neurons with 910-nm and 1030-nm excitation in an adult H-line mouse brain.

(a) Maximum intensity projections of three-dimensional stacks were obtained with 910-nm and 1030-nm lasers (b). Four normalized xy frames from the z-stack at various depths including the dura matter, apical dendrite, soma, and white matter, respectively (left panels). The arrowhead shows a part of a basal dendritic process at cortex layer V.

Figure 4. Quantitative analysis of signal-to-background ratios for in vivo imaging of neurons in the live mouse brain.

(a) A cross-sectional- (xy-) image obtained at 600-μm depth in a young adult mouse. The left white or left yellow box indicates the “soma” (upper right) or “apical dendrite” (lower right) region, respectively. In the upper and lower right panels, the averages of “signal” and “background” were calculated from fluorescence intensities in a circle (20 μm diameter) around an individual neural process (“signal”: red circle) and the background area (“background”: green circle), respectively. Ratio R at the each process was obtained from the average signal divided by the background (see Experimental procedure). Depth dependence of the average and SEM of signal-to-background ratios (R) obtained in the young adult (b) and the adult mouse (c). Red and black circles indicate averages with the 1030-nm picosecond and 910-nm femto-second lasers, respectively (n = 7 processes, ±: SEM).