No abstract

The vestibular system, which detects gravity and motion, is crucial to survival, but the neural circuits processing vestibular information remain incompletely characterised. In part, this is because the movement needed to stimulate the vestibular system hampers traditional neuroscientific methods. Optical trapping uses focussed light to apply forces to targeted objects, typically ranging from nanometres to a few microns across. In principle, optical trapping of the otoliths (ear stones) could produce fictive vestibular stimuli in a stationary animal. Here we use optical trapping in vivo to manipulate 55-micron otoliths in larval zebrafish. Medial and lateral forces on the otoliths result in complementary corrective tail movements, and lateral forces on either otolith are sufficient to cause a rolling correction in both eyes. This confirms that optical trapping is sufficiently powerful and precise to move large objects in vivo, and sets the stage for the functional mapping of the resulting vestibular processing.

Looming visual stimuli result in escape responses that are conserved from insects to humans. Despite their importance for survival, the circuits mediating visual startle have only recently been explored in vertebrates. Here we show that the zebrafish thalamus is a luminance detector critical to visual escape. Thalamic projection neurons deliver dim-specific information to the optic tectum, and ablations of these projections disrupt normal tectal responses to looms. Without this information, larvae are less likely to escape from dark looming stimuli and lose the ability to escape away from the source of the loom. Remarkably, when paired with an isoluminant loom stimulus to the opposite eye, dimming is sufficient to increase startle probability and to reverse the direction of the escape so that it is toward the loom. We suggest that bilateral comparisons of luminance, relayed from the thalamus to the tectum, facilitate escape responses and are essential for their directionality.

Since the time of their introduction, optical tweezers (OTs) have grown to be a powerful tool in the hands of biologists. OTs use highly focused laser light to guide, manipulate, or sort target objects, typically in the nanoscale to microscale range. OTs have been particularly useful in making quantitative measurements of forces acting in cellular systems; they can reach inside living cells and be used to study the mechanical properties of the fluids and structures that they contain. As all the measurements are conducted without physically contacting the system under study, they also avoid complications related to contamination and tissue damage. From the manipulation of fluorescent nanodiamonds to chromosomes, cells, and free-swimming bacteria, OTs have now been extended to challenging biological systems such as the vestibular system in zebrafish. Here, we will give an overview of OTs, the complications that arise in carrying out OTs in vivo, and specific OT methods that have been used to address a range of otherwise inaccessible biological questions.

Light-sheet microscopy is used extensively in developmental biology and neuroscience. One limitation of this approach is that absorption and scattering produces shadows in the illuminating light sheet, resulting in stripe artifacts. Here, we introduce diffuse light-sheet microscopes that use a line diffuser to randomize the light propagation within the image plane, allowing the light sheets to reform after obstacles. We incorporate diffuse light sheets in two existing configurations: selective plane illumination microscopy in which the sample is illuminated with a static sheet of light, and digitally scanned light sheet (DSLS) in which a thin Gaussian beam is scanned across the image plane during each acquisition. We compare diffuse light-sheet microscopes to their conventional counterparts for calcium imaging of neural activity in larval zebrafish. We show that stripe artifacts can cast deep shadows that conceal some neurons, and that the stripes can flicker, producing spurious signals that could be interpreted as biological activity. Diffuse light-sheets mitigate these problems, illuminating the blind spots produced by stripes and removing artifacts produced by the stripes' movements. The upgrade to diffuse light sheets is simple and inexpensive, especially in the case of DSLS, where it requires the addition of one optical element.