This digital resource is a scientific article published in the Journal of Cell Biology, focusing on 'Accuracy and precision in quantitative fluorescence microscopy.' With the increasing use of fluorescence microscopy for quantitative measurements in cell biology, understanding and mitigating sources of error are paramount. This article provides a detailed examination of factors affecting the quantitative reliability of fluorescence imaging. The article systematically discusses parameters of digital image acquisition that influence the accuracy and precision of quantitative fluorescence microscopy measurements. It covers critical aspects such as focus, signal-to-noise ratio (SNR), detector settings, and the inherent variability in biological specimens. It explains how factors like imprecise focus or low SNR can lead to inaccurate or imprecise intensity and spatial measurements. This resource is essential for researchers who perform quantitative fluorescence imaging, including those using advanced techniques like Förster Resonance Energy Transfer (FRET) or Fluorescence Recovery After Photobleaching (FRAP). It guides them in optimizing their imaging systems and experimental protocols to achieve reliable and reproducible quantitative data. Understanding these principles is crucial for drawing valid conclusions from microscopy experiments. As a peer-reviewed publication in a leading journal, the article offers authoritative insights into best practices for quantitative fluorescence microscopy. It helps researchers to critically evaluate their imaging data, identify potential sources of error, and implement strategies to improve the quality and robustness of their quantitative analyses.