Fig. 1: CRASH2p microscopy.
From: Closed-loop two-photon functional imaging in a freely moving animal
Top (a): Principle of operation. Tracking path: a pulsed 1070 nm laser was directed through a resonant TAG lens and relayed on to one scan head’s galvanometers. Imaging path: a pulsed 920 nm laser was combined with the 1070 nm laser, directed through a separate tag lens, and relayed on to a second scan head’s galvanometers. The 1070 nm pulses were displaced by 1/2 cycle using an adjustable delay line (Fig. 6). The two scan paths were combined and directed through a tube lens onto a 25× objective mounted on a piezo scanner. Fluorescent red and green photons were separately collected by PMTs operated in photon counting mode. The signal from the “red" PMT was temporally demultiplexed; all three (tracking red, imaging red, and imaging green) PMT signals were sent to an FPGA card. Tracking was achieved by rastering the tracking beam in a cylinder about a target neuron; the position of the beam and the record of emitted photons were combined to form an estimate of the neuron’s position. Each new estimate was combined with previous estimates using a Kalman filter and used to direct the next cylindrical scan. The tracked neuron’s location was added to the imaging path target location, so the scan was always referenced to the tracked neuron. The piezo positioner provided feedback in z, while a slower motorized stage adjusted all three axes to bring the tracked neuron to the center of the field of view. Bottom: Red (b, d) and green (c, e) XY and XZ projections from a 200 ms interval without (b, c) and with (d, e) tracker correction, during forward crawling. Note that although the tracker output was not used in the assembly of the left images (b, c), the tracker was still required for their acquisition; otherwise, the labeled cells would have moved outside the imaged volume during scanning. In (b, c) the path of the tracked neuron is shown with circles indicating the starting (left) and ending (right) positions. In 200 ms, the neuron traveled 97 µm at a top speed of 0.8 mm/s. Genotype: R36G02 > mCherry,GCaMP6f.
