mmSIM: An open toolbox for accessible structured illumination microscopy

Abstract

SummarySince the first practical super-resolution structured illumination fluorescence microscopes (SIM) were demonstrated more than two decades ago the method has become increasingly popular for a wide range of bioimaging applications. The high cost and relative inflexibility of commercial systems, coupled with the conceptual simplicity of the approach and the desire to exploit and customise existing hardware, have led to the development of a large number of home-built systems. Several detailed hardware designs are available in the scientific literature, complemented by open-source software tools for SIM image validation and reconstruction. However, there remains a lack of simple open-source software to control these systems and manage the synchronization between hardware components, which is critical for effective SIM imaging. This article describes a new suite of software tools based on the popular Micro-Manager package, which enable the keen microscopist to develop and run a SIM system. We use the software to control two custom-built, high-speed, spatial light modulator-based SIM systems, evaluating their performance by imaging a range of fluorescent samples. By simplifying the process of SIM hardware development, we aim to support wider adoption of the technique.

mmSIM: an open toolbox for accessible structured illumination microscopy

“…An additional secondary falling edge was then used to trigger the advance to the next frame. Once the primary signal was pulsed LOW, the bitplane sequence was reset to ensure a consistent ordering of the illumination patterns within each raw SIM image sequence of nine images (for an example SLM configuration 'repertoire' file, see [22]). The system is typically operated using the same set of nine SLM bitplanes for all excitation lasers which means that the shift of the first-order information passbands is the same for all wavelengths, whereas to achieve the theoretically maximal increase in spatial resolution requires a wavelength-dependent shift.…”

“…A configuration file for the system is provided as an example. In addition, a µManager startup script (MMStartup.bsh) [22] was used to set the region of interest of the camera to the area of the sample illuminated by the SLM as this cannot be set through the Device/Property browser and so could not be saved in the hardware configuration file.…”

“…The startup script [22] was used to send a TTL pulse from µManager via the DAQ to reset the SLM running order, ensuring a consistent ordering of raw images in each SIM dataset. It is also possible to assign the TTL reset trigger to a µManager custom shutter, as with the digital shutters for the laser lines.…”

“…To extend µManager with useful SIM functionality, we developed two additional plugins, mmLiveFFT and mmSIMCapture [22]. These plugins are easily installed by copying the corresponding .jar files directly into the Micro-Manager-2.0gamma mmplugins folder.…”

“…Data accessibility. All source code, documentation and files are available through GitHub (https://github.com/ ctr26/MMSIM) and archived using Zenodo (https://doi.org/10.5281/zenodo.4432274) [22].…”