EZcalcium: Open-Source Toolbox for Analysis of Calcium Imaging Data

Abstract

Fluorescence calcium imaging using a range of microscopy approaches, such as two-photon excitation or head-mounted "miniscopes," is one of the preferred methods to record neuronal activity and glial signals in various experimental settings, including acute brain slices, brain organoids, and behaving animals. Because changes in the fluorescence intensity of genetically encoded or chemical calcium indicators correlate with action potential firing in neurons, data analysis is based on inferring such spiking from changes in pixel intensity values across time within different regions of interest. However, the algorithms necessary to extract biologically relevant information from these fluorescent signals are complex and require significant expertise in programming to develop robust analysis pipelines. For decades, the only way to perform these analyses was for individual laboratories to write their custom code. These routines were typically not well annotated and lacked intuitive graphical user interfaces (GUIs), which made it difficult for scientists in other laboratories to adopt them. Although the panorama is changing with recent tools like CaImAn, Suite2P, and others, there is still a barrier for many laboratories to adopt these packages, especially for potential users without sophisticated programming skills. As two-photon microscopes are becoming increasingly affordable, the bottleneck is no longer the hardware, but the software used to analyze the calcium data optimally and consistently across different groups. We addressed this unmet need by incorporating recent software solutions, namely NoRMCorre and CaImAn, for motion correction, segmentation, signal extraction, and deconvolution of calcium imaging data into an open-source, easy to use, GUI-based, intuitive and automated data analysis software package, which we named EZcalcium.

Plasticity after cortical stroke involves potentiating responses of pre-existing circuits but not functional remapping to new circuits

“…Fluorescence traces ( ΔF/F ) of neuronal calcium transients were extracted using custom-written semi-automated MATLAB routines as previously described (He et al, 2017). The vast majority of movies (>90%) did not require motion correction, but for those that did, X–Y drift was corrected using either a frame-by-frame, hidden Markov model–based registration routine (Dombeck et al, 2007) or the motion correction module from EZcalcium (Cantu et al, 2020) based on NoRMCorre nonrigid template matching (Pnevmatikakis and Giovannucci, 2017). To determine whether individual neurons showed time-locked responses to whisker stimulations, we first calculated a modified Z score vector for each neuron and then used a probabilistic bootstrapping method to correlate calcium transients with epochs of stimulation, as described (He et al, 2017).…”

Software for Non-Parametric Image Registration of 2-Photon Imaging Data

“…We can report state-of-the-art registration results in terms of reduced temporal standard deviation and MSE ratios, when comparing our method with NoRMCorre [5] and rigid image registration, see Figure 2. NoRMCorre has been adopted in recent toolboxes for 2P and calcium imaging such as CaImAn (2019) [15], EZcalcium (2020) [16] or Begonia (2021) [17] and can be considered the state of the art for the alignment of 2-photon image sequences.…”

“…with respect to the maximum value 2 16 , experiment specific properties as well as the applied low-pass filtering (see section 5.5) as well as SNR of the raw data (e.g. 30.9 Hz vs 6.2 Hz) have an impact on the PSNR differences between the datasets.…”

Neuronal hyperactivity in a LRRK2-G2019S cellular model of Parkinson’s Disease

“…Giovannucci et al [19] developed automatic and scalable analysis methods to address problems common to preprocessing, including motion correction, neural activity identification, and registration across different sessions of data collection. Cantu et al [8] developed a graphical user interface-based and automated analysis software package for motion correction, segmentation, signal extraction, and deconvolution of calcium imaging data.…”