The Cousa objective: a long-working distance air objective for multiphoton imaging in vivo.

Multiphoton microscopy can resolve fluorescent structures and dynamics deep in scattering tissue and has transformed neural imaging, but applying this technique in vivo can be limited by the mechanical and optical constraints of conventional objectives. Short working distance objectives can collide with compact surgical windows or other instrumentation and preclude imaging. Here we present an ultra-long working distance (20 mm) air objective called the Cousa objective. It is optimized for performance across multiphoton imaging wavelengths, offers a more than 4 mm2 field of view with submicrometer lateral resolution and is compatible with commonly used multiphoton imaging systems. A novel mechanical design, wider than typical microscope objectives, enabled this combination of specifications. We share the full optical prescription, and report performance including in vivo two-photon and three-photon imaging in an array of species and preparations, including nonhuman primates. The Cousa objective can enable a range of experiments in neuroscience and beyond.

Stimulus Contrast Information Modulates Sensorimotor Decision Making in Goldfish.

Animal survival relies on environmental information gathered by their sensory systems. We found that contrast information of a looming stimulus biases the type of defensive behavior that goldfish (Carassius auratus) perform. Low-contrast looms only evoke subtle alarm reactions whose probability is independent of contrast. As looming contrast increases, the probability of eliciting a fast escape maneuver, the C-start response, increases dramatically. Contrast information also modulates the decision of when to escape. Although response latency is known to depend on looming retinal size, we found that contrast acts as an additional parameter influencing this decision. When presenting progressively higher contrast stimuli, animals need shorter periods of stimulus processing to initiate the response. Our results comply with the notion that the decision to escape is a flexible process initiated with stimulus detection and followed by assessment of the perceived risk posed by the stimulus. Highly disruptive behaviors as the C-start are only observed when a multifactorial threshold that includes stimulus contrast is surpassed.