ScanImage is a flexible, open-source software package designed to control laser scanning microscopes, enabling users to operate these instruments without the need for custom data acquisition hardware. The software, written in MATLAB, uses standard multifunction boards for data acquisition and scanning control, placing the burden of signal integration and image processing on the computer's CPU. This approach simplifies the design and construction of custom microscopes, making it easier to implement specialized designs for challenging applications. ScanImage supports multi-channel acquisition, 3D image acquisition, and automated stage controls, including ROI selection and focus adjustment. The software also includes a focus mode for real-time image observation and can handle various pixel times, achieving near-ideal signal-to-noise ratios. ScanImage is freely available under an OSI Certified open-source license and can be downloaded from the provided website.ScanImage is a flexible, open-source software package designed to control laser scanning microscopes, enabling users to operate these instruments without the need for custom data acquisition hardware. The software, written in MATLAB, uses standard multifunction boards for data acquisition and scanning control, placing the burden of signal integration and image processing on the computer's CPU. This approach simplifies the design and construction of custom microscopes, making it easier to implement specialized designs for challenging applications. ScanImage supports multi-channel acquisition, 3D image acquisition, and automated stage controls, including ROI selection and focus adjustment. The software also includes a focus mode for real-time image observation and can handle various pixel times, achieving near-ideal signal-to-noise ratios. ScanImage is freely available under an OSI Certified open-source license and can be downloaded from the provided website.