Abstract
Fluorescence microscopy has been widely accessible and indispensable in cell biology research. This technique enables researchers to label targets, ranging from individual entities to multiple groups, with fluorescent markers. It offers precise determinations of localization, size, and shape, along with accurate quantifications of fluorescence signal intensities. Furthermore, an ideal fluorescence microscope can achieve approximately 250 nm in lateral and 600 nm in axial resolution. Despite its integral role in these measurements, the calibration of fluorescence microscopes is often overlooked. This protocol introduces the use of 3D-Speckler (3D fluorescence speckle analyzer), a semi-automated software tool we have recently developed, for calibrating fluorescence microscopy. Calibration of fluorescence microscopy includes determining resolution limits, validating accuracy in size measurements, evaluating illumination flatness, and determining chromatic aberrations. 3D-Speckler is user-friendly and enables precise quantification of fluorescence puncta, including nanoscale 2D/3D particle size, precise locations, and intensity information. By utilizing multispectral fluorescence beads of known sizes alongside 3D-Speckler, the software can effectively calibrate imaging systems. We emphasize the importance of routine calibration for imaging systems to maintain their integrity and reproducibility, ensuring accurate quantification. This protocol provides a detailed step-by-step guide on using 3D-Speckler to calibrate imaging systems. Key features • Semi-automated particle detection. • Accurate three-dimensional measurement of fluorescent particle sizes. • High-precision three-dimensional localization of fluorescent particles. • Precision analysis of point spread function and chromatic aberration in fluorescence microscopy.