Foundations and Structural Applications of Fluorescence Optical Imaging in Biological Systems

Fluorescence optical imaging has become a fundamental methodology for investigating the structural organization of biological systems across multiple spatial scales. Owing to its high sensitivity, molecular specificity, and compatibility with advanced optical platforms, fluorescence imaging enables precise visualization of cellular and subcellular architecture within complex biological environments. This review focuses on the foundational principles and structural applications of fluorescence optical imaging in biological research. Core photophysical concepts, excitation strategies, optical components, and factors influencing image quality are discussed to establish a conceptual framework for fluorescence-based microscopy. The review systematically examines major fluorescence imaging modalities, including widefield, confocal, total internal reflection fluorescence, multiphoton, and light-sheet microscopy, with emphasis on their structural imaging capabilities. Advances in super-resolution techniques such as stimulated emission depletion, photoactivated localization microscopy, stochastic optical reconstruction microscopy, and structured illumination microscopy are highlighted for their role in overcoming diffraction-limited resolution. The development and application of fluorescent probes, genetically encoded reporters, and nanomaterial-based labels are critically reviewed in the context of structural visualization. Collectively, this article provides a comprehensive overview of fluorescence optical imaging as a structural biology tool, offering insights into its methodological strengths, limitations, and contributions to highresolution structural analysis in modern biological sciences.