From Hardware to Software: How ScanImage Transformed Laser Scanning Microscopy Control

Pologruto TA, Sabatini BL, Svoboda K. ScanImage: flexible software for operating laser scanning microscopes. Biomed Eng Online 2003;2:13. doi: 10.1186/1475-925X-2-13.

Background: Laser scanning microscopy (LSM), including confocal and two-photon excitation modalities, is central to modern biological imaging. Despite the widespread availability of commercial systems, many research applications require custom-designed microscopes that integrate specialized optics and scanning controls. However, the complexity of developing custom data acquisition hardware and control software has historically limited such innovations.

Hypothesis: This study hypothesized that a fully software-based approach could efficiently control a laser scanning microscope, eliminating the need for specialized data acquisition hardware while maintaining high image quality and real-time performance.

Methods: The authors developed and implemented ScanImage, a software package written in MATLAB 6.1, to operate a laser scanning microscope using standard multifunction data acquisition boards (PCI-6110, National Instruments) for mirror control and signal collection. Scanning and focusing were managed through a three-axis motor controller (Sutter MP285) via a serial communication interface. Performance benchmarking was conducted on a Windows 2000 PC equipped with an 800 MHz Pentium III processor and 512 MB RAM.

Results: The software achieved near-ideal signal-to-noise characteristics at a sampling rate of 1.25 MHz and pixel times as short as 3 μs. Simulations and experimental data demonstrated that digital integration produced high apparent quantum efficiency and low photon bleedthrough (~6–10%). ScanImage maintained real-time image refresh across multiple fluorescence channels without data loss.

Conclusions: This study demonstrated that software-driven data acquisition and control can replace complex hardware in laser scanning microscopy. ScanImage provides a flexible, efficient and open-source solution enabling rapid customization and integration into advanced imaging experiments.