ScanImage is a flexible, open-source software package for operating laser scanning microscopes. It allows for custom design without the need for specialized hardware. The software uses standard data acquisition boards and a computer's CPU to handle signal integration and image processing. It is written in MATLAB, making it compatible with high-level image analysis environments. The software controls a laser-scanning microscope, generating analog voltage waveforms to drive scan mirrors, acquiring data from photomultiplier tubes, and processing signals to produce images. It supports multiple input channels and is designed to be easily expandable. The performance of ScanImage was benchmarked under various conditions, showing minimal delay in image processing and display. ScanImage uses a sampling frequency of 1.25 MHz, which allows nearly ideal imaging for pixel times as short as 3 µs. The software also handles bleedthrough between pixels and provides high-quality images with near-ideal noise properties and real-time refresh rates. It supports multi-channel acquisition, 3D image acquisition, and automated stage controls. ScanImage stores data in TIF files, which are compatible with various image-processing programs. The software is modular, allowing users to develop and test data analysis functions easily. It also supports controlling additional devices and has a user-friendly interface for configuring different microscopes and experimental setups. ScanImage is freely available as open-source software and can be accessed from the provided URL. It is designed to be flexible and customizable, making it suitable for a wide range of applications in biological imaging. The software is supported by a community of researchers and is continually updated to improve performance and functionality.ScanImage is a flexible, open-source software package for operating laser scanning microscopes. It allows for custom design without the need for specialized hardware. The software uses standard data acquisition boards and a computer's CPU to handle signal integration and image processing. It is written in MATLAB, making it compatible with high-level image analysis environments. The software controls a laser-scanning microscope, generating analog voltage waveforms to drive scan mirrors, acquiring data from photomultiplier tubes, and processing signals to produce images. It supports multiple input channels and is designed to be easily expandable. The performance of ScanImage was benchmarked under various conditions, showing minimal delay in image processing and display. ScanImage uses a sampling frequency of 1.25 MHz, which allows nearly ideal imaging for pixel times as short as 3 µs. The software also handles bleedthrough between pixels and provides high-quality images with near-ideal noise properties and real-time refresh rates. It supports multi-channel acquisition, 3D image acquisition, and automated stage controls. ScanImage stores data in TIF files, which are compatible with various image-processing programs. The software is modular, allowing users to develop and test data analysis functions easily. It also supports controlling additional devices and has a user-friendly interface for configuring different microscopes and experimental setups. ScanImage is freely available as open-source software and can be accessed from the provided URL. It is designed to be flexible and customizable, making it suitable for a wide range of applications in biological imaging. The software is supported by a community of researchers and is continually updated to improve performance and functionality.