Calculating DF/F

This table summarizes Calcium Activity (ΔF/F) detection methods reviewed by Paudel et al. (2024).

Method	How It’s Performed	Pros	Cons
Manual Spike Counting	Visual inspection of raw fluorescence or dF/F traces to count events.	Simple; no code needed.	Subjective; non-scalable; low reproducibility.
dF/F Thresholding	Compute (F − F₀)/F₀ and define a fixed or adaptive threshold for events.	Widely used; compatible with GCaMP, Fluo dyes.	Sensitive to F₀ definition; arbitrary thresholds can bias results.
Z-Score Thresholding	Normalize trace by mean/SD, define events above N standard deviations.	Removes baseline drift; good for noisy data.	Sensitive to noise if SD is low; assumes Gaussian distribution.
Percentile Baseline Subtraction	Use a moving window to define F₀ as low percentile (e.g., 10–20th) of the trace.	Adaptive baseline; handles long-term drift.	Choice of window size/percentile affects sensitivity.
Ratiometric Imaging (Fura, YC2)	Compute ratio of Ca²⁺-sensitive and insensitive fluorophores per ROI.	Controls for volume/motion artifacts; yields [Ca²⁺].	Requires dual excitation/emission; reduced spatial/temporal res.
Photon Counting (Aequorin)	Count emitted photons per ROI/pixel over time; map to Ca²⁺ levels via calibration.	Quantitative; good dynamic range.	Low spatial resolution; complex calibration.
Image Subtraction (Frame-to-Frame)	Compute ΔF = Fₙ − Fₙ₋₁ or F − background to detect sudden changes.	Simple, fast; used for wave detection.	Sensitive to noise; misses gradual changes.
Savitzky-Golay Filtering	Smooth trace to reduce noise while preserving spikes.	Good for noisy signals.	Requires tuning; can mask small or fast events.
Tensor Voting / Cluster Detection	Identify spatially coordinated activity from 2D/3D image stacks.	Detects population events (e.g., waves).	Not standard; needs spatially dense data.
Standard Deviation (SD) Masking	Define active frames/regions where ΔF exceeds N×SD of baseline.	Objective thresholding for event detection.	Threshold choice heavily affects results.

Example of ΔF/F trace showing different baseline choices. Adapted from Fig. 1 of .