The Impact of Optical Undersampling on the Ca2+ Signal Resolution in Ca2+ Imaging of Spontaneous Neuronal Activity.

Abstract

Background: In neuroscience, Ca²⁺ imaging is a prevalent technique used to infer neuronal electrical activity, often relying on optical signals recorded at low sampling rates (3 to 30 Hz) across multiple neurons simultaneously. This study investigated whether increasing the sampling rate preserves critical information that may be missed at slower acquisition speeds.

Methods: Primary neuronal cultures were prepared from the cortex of newborn pups. Neurons were loaded with Oregon Green BAPTA-1 AM (OGB1-AM) fluorescent indicator. Spontaneous neuronal activity was recorded at low (14 Hz) and high (500 Hz) sampling rates, and the same neurons (n = 269) were analyzed under both conditions. We compared optical signal amplitude, duration, and frequency.

Results: Although recurring Ca²⁺ transients appeared visually similar at 14 Hz and 500 Hz, quantitative analysis revealed significantly faster rise times and shorter durations (half-widths) at the higher sampling rate. Small-amplitude Ca²⁺ transients, undetectable at 14 Hz, became evident at 500 Hz, particularly in the neuropil (putative dendrites and axons), but not in nearby cell bodies. Large Ca²⁺ transients exhibited greater amplitudes and faster temporal dynamics in dendrites compared with somas, potentially due to the higher surface-to-volume ratio of dendrites. In neurons bulk-loaded with OGB1-AM, cell nucleus-mediated signal distortions were observed in every neuron examined (n = 57). Specifically, two regions of interest (ROIs) on different segments of the same cell body displayed significantly different signal amplitudes and durations due to dye accumulation in the nucleus.

Conclusions: Our findings reveal that Ca²⁺ signal undersampling leads to three types of information loss: (1) distortion of rise times and durations for large-amplitude transients, (2) failure to detect small-amplitude transients in cell bodies, and (3) omission of small-amplitude transients in the neuropil.