Fig. 6: Optical layout of CRASH2p microscope.
From: Closed-loop two-photon functional imaging in a freely moving animal
The dual-beam hyperscope (shaded gray area in the figure) has two independent scan heads, combined internally with a polarizing beam splitter. One ("tracking") carries a 1070 nm pulsed laser that excites mCherry but not GFP or GCaMP variants; the other ("imaging") carries both 1070 nm and 920 nm pulsed lasers, combined with a dichroic beamsplitter. The 1070 nm laser is split between the two paths, with a delay arm on the tracking path to enable temporal demultiplexing of the photons excited by the independent scan heads. The power of each laser is modulated using a Pockels cell; the ratio of power in the 1070 nm power delivered to the imaging and tracking paths is controlled using a 1/2 wave (λ/2) plate and polarizing beamsplitter (PBS). Following the pockels cell, the beams are expanded by a factor of 3× to fill the effective aperture of the TAG lenses when operating at 190 kHz. Each path contains a TAG lens in a double-pass configuration. A 1:1.5 demagnifying relay between each TAG lens and the hyperscope conjugates the TAG lens onto an initial blank mirror (place-holder for unused resonant galvo, not shown), the x-galvo, and the y-galvo, which are all placed in the same conjugate plane by means of internal 1:1 relays. The two scan paths are recombined internally by a polarizing beamsplitter and then expanded 6.85× to fill the back aperture of the 8 mm focal length 0.95 NA objective lens. The objective was mounted on a piezo actuator attached to the Hyperscope MDU, which contained filters and optics to separate the IR excitation light from the visible fluorescence emission and direct the latter onto red-tuned and green-tuned PMTs. A 3-axis stage with piezo-electric immobilizer was attached to the table. The larva’s behavior was monitored using an infrared (IR) camera mounted below.
