BENCHMARKING <EM>IN VIVO</EM> FLUORESCENCE MONITORING VARIABILITY ACROSS ANIMAL STATE, CELLULAR SOURCE AND IMAGING MODALITY

Reliable measurement of fluorescence in vivo is essential for interpretation and planning of experiments in awake mice. While many methods for live, real-time readout is emerging, less is known about the variability and comparability of these approaches. Here, we provide a systemic characterization of common factors that influence fluorescence stability, addressing three questions relevant to everyday experimental design: does animal state modulated by the experimental context (newness, habituation and arousal) impacts signal stability; what is the level of signal consistency, depending on its source and how motion-driven variability manifests across two common imaging techniques (intensity-based vs image-based).
By using stable GFP signals expressed in two diverse cellular compartments of the brain (neurons and astrocytes) expressed in transgenic mouse lines (Thy1-GFP and Aldhl1l-CreER::EGFP), we investigate performance and characteristics of fiber photometry and miniscope imaging. The analyses focus on identifying ranges and patterns of variability, providing metrics for the reproducibility and robustness (e.g. signal-to-noise, coefficient of variation, drift). To evaluate modality-dependent effects, motion-sensitive analyses were performed using video-tracked behavior, which allow to compare signal stability during movement and inactivity. We also tested whether modulation of arousal state impacts signal variability.
This approach demonstrates how stable GFP serves as a controlled benchmark to systematically assess the impact of common experimental factors on in vivo fluorescence recordings. The framework provides practical guidance for experimenters and establishes a foundation for future studies employing dynamic fluorescent signals.