Improved Workflow for Analysis of Vascular Myocyte Time-Series and Line-Scan Ca²⁺ Imaging Datasets

Intracellular Ca²⁺ signals are key for the regulation of cellular processes ranging from myocyte contraction, hormonal secretion, neural transmission, cellular metabolism, transcriptional regulation, and cell proliferation. Measurement of cellular Ca²⁺ is routinely performed using fluorescence microscopy with biological indicators. Analysis of deterministic signals is reasonably straightforward as relevant data can be discriminated based on the timing of cellular responses. However, analysis of stochastic, slower oscillatory events, as well as rapid subcellular Ca²⁺ responses, takes considerable time and effort which often includes visual analysis by trained investigators, especially when studying signals arising from cells embedded in complex tissues. The purpose of the current study was to determine if full-frame time-series and line-scan image analysis workflow of Fluo-4 generated Ca²⁺ fluorescence data from vascular myocytes could be automated without introducing errors. This evaluation was addressed by re-analyzing a published "gold standard" full-frame time-series dataset through visual analysis of Ca²⁺ signals from recordings made in pulmonary arterial myocytes of en face arterial preparations. We applied a combination of data driven and statistical approaches with comparisons to our published data to assess the fidelity of the various approaches. Regions of interest with Ca²⁺ oscillations were detected automatically post hoc using the LCPro plug-in for ImageJ. Oscillatory signals were separated based on event durations between 4 and 40 s. These data were filtered based on cutoffs obtained from multiple methods and compared to the published manually curated "gold standard" dataset. Subcellular focal and rapid Ca²⁺ "spark" events from line-scan recordings were examined using SparkLab 5.8, which is a custom automated detection and analysis program. After filtering, the number of true positives, false positives, and false negatives were calculated through comparisons to visually derived "gold standard" datasets. Positive predictive value, sensitivity, and false discovery rates were calculated. There were very few significant differences between the automated and manually curated results with respect to quality of the oscillatory and Ca²⁺ spark events, and there were no systematic biases in the data curation or filtering techniques. The lack of statistical difference in event quality between manual data curation and statistically derived critical cutoff techniques leads us to believe that automated analysis techniques can be reliably used to analyze spatial and temporal aspects to Ca²⁺ imaging data, which will improve experiment workflow.