Material below is adapted from the SfN Short Course Simultaneous Holographic Imaging of Neuronal Circuits in Three Dimensions, by Sean Quirin, PhD, et al. Short Courses are daylong scientific trainings on emerging neuroscience topics and research techniques held the day before SfN’s annual meeting.
Traditional microscopy techniques are limiting in that most only reflect two dimensions of a three-dimensional biological system. Optical imaging, such as two-photon microscopy to measure voltage-sensitive or calcium-sensitive dyes, is minimally invasive and has the resolution to study the individual cells that make up large neuronal networks. However, images from these experiments are captured in two dimensions; to collect data spanning a chunk of tissue, sequential images are scanned one at a time, losing any activity that occurs at the same time in different planes.
A new technique seeks to solve this problem by simultaneously collecting data in three dimensions. To achieve this, a conventional two-photon microscope first collects structural data (such as the location of cells), and then a hologram is generated to target regions of interest (such as active cells) in three-dimensional space. A spatial light modulator illuminates the chosen cells while a phase mask system simultaneously picks up their fluorescence. With the new method, two neurons firing together can be imaged together, even if they are spread apart.
The capabilities of this system have been demonstrated both in vitro and in vivo. Using this technique in slices from the mouse dentate gyrus, individual cells were identified and imaged — a feat in and of itself in an area so densely packed with cells. No matter the cell’s location in three-dimensional space, calcium signals could be clearly separated and quantified. Thus, the technique could be used to visualize patterns of activity responsible for memory with unprecedented detail and precision.
This technique was also demonstrated in vivo in larval zebrafish. Using the two-photon, holographic approach, calcium dynamics were measured across the brain with enough precision and speed to make a three-dimensional map of synaptic activity, a first in these animals. However, this was possible in larval zebrafish because they are transparent, a convenience not true of most animals. This new imaging system is not yet optimized to image through layers of tissue because the light scatters too much. Imaging techniques are continuing to improve, though, and applying new strategies may remedy this problem soon.
