Abstract
The application of 2-photon laser scanning microscopy (TPLSM) techniques to measure the dynamics of cellular calcium signals in populations of neurons is an extremely powerful technique for characterizing neural activity within the central nervous system. The use of TPLSM on awake and behaving subjects promises new insights into how neural circuit elements cooperatively interact to form sensory perceptions and generate behavior. A major challenge in imaging such preparations is unavoidable animal and tissue movement, which leads to shifts in the imaging location (jitter). The presence of image motion can lead to artifacts, especially since quantification of TPLSM images involves analysis of fluctuations in fluorescence intensities for each neuron, determined from small regions of interest (ROIs). Here, we validate a new motion correction approach to compensate for motion of TPLSM images in the superficial layers of auditory cortex of awake mice. We use a nominally uniform fluorescent signal as a secondary signal to complement the dynamic signals from genetically encoded calcium indicators. We tested motion correction for single plane time lapse imaging as well as multiplane (i.e., volume) time lapse imaging of cortical tissue. Our procedure of motion correction relies on locating the brightest neurons and tracking their positions over time using established techniques of particle finding and tracking. We show that our tracking based approach provides subpixel resolution without compromising speed. Unlike most established methods, our algorithm also captures deformations of the field of view and thus can compensate e.g., for rotations. Object tracking based motion correction thus offers an alternative approach for motion correction, one that is well suited for real time spike inference analysis and feedback control, and for correcting for tissue distortions.
Highlights
AND INTRODUCTIONBehaviorally relevant information in the brain does not reside in the firing events of individual neurons, but instead in the collective activity of groups of neurons
Our approach works best with a signal that is independent of neuronal activity and under ideal circumstances would appear at uniform brightness
If not slightly higher, signal to noise ratio (SNR) for each neuron with our tracking-based motion correction approach when compared to TurboReg
Summary
Relevant information in the brain does not reside in the firing events of individual neurons, but instead in the collective activity of groups of neurons. Understanding the collective behavior of neurons is essential for understanding how the brain processes information and encodes memory. Real-Time Capable Tracking-Based Motion Correction neuronal activity using Ca2+ indicators is a powerful approach that allows for analysis of the inner workings of the brain at the level of single cells for large groups of neurons within an awake behaving organism (O’Connor et al, 2009; Peron S. et al, 2015). While electrophysiology approaches allow for very accurate measurements of the neuronal circuit a few neurons at a time, TPLSM allows for the simultaneous observation of hundreds to thousands of neurons P. et al, 2015) and yields information on the collective behavior of groups of neurons. To detect synchronous activity of 100 neurons with more than 95% confidence, the detection of single neuron events must be made with close to 100% accuracy
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