Enhanced Multicolor Structured Illumination Microscopy via Dispersion-Compensated Digital Micromirror Devices

Structured Illumination Microscopy (SIM) is unique in its combination of high spatial resolution, temporal resolution, minimal photodamage, and compatibility with a wide range of fluorescent dyes making it well-suited for live-cell imaging to provide valuable and detailed information into the dynamic processes within living cells. However, the adoption of SIM has been constrained by significant challenges associated with its illumination setup. SIM achieves super-resolution by using sinusoidal patterned illumination to shift high spatial-frequency information into a lower frequency range which makes it detectable within the microscope’s optical transfer function. Scientists first used  diffraction gratings to generate these illumination patterns, however, their limitations led to the adoption of liquid crystal on silicon for its higher speed and simpler implementation. Recently, Digital Micromirror Devices (DMDs) have emerged as a promising alternative due to their cost-effectiveness and high refresh rates and therefore better suited for high-speed SIM imaging. Despite these advantages, the application of DMDs in SIM faces a significant obstacle because the blazed grating structure of the DMDs causes strong angular dispersion for different wavelengths of light and that dispersion effect necessitates precise alignment of the input angle for each excitation wavelength which complicates the implementation of multicolor imaging. Previous approaches to address this issue have included using coherent light sources with integer-ratio wavelengths or setting up individual light paths for different excitation wavelengths. These methods, however, are challenging to scale and add significant complexity to the system. Alternatively, incoherent light sources can be used to avoid the diffraction effect, but this leads to reduced contrast in SIM patterns and inferior resolution.

To this end, a new study published in Optics Letters by a University of Chicago team led by Professor Norbert Scherer and his graduate student, Daozheng Gong, and collaborators Chufan Cai, Eli Strahilevitz, and Jing Chen developed a novel solution for multicolor DMD-SIM that can overcome the limitations posed by the DMD’s blazed grating effect and can enable super-resolution imaging with multiple color channels. The researchers proposed and implemented a multi-color DMD-SIM setup that employs a diffraction grating to counteract the DMD’s dispersion. This innovative approach allows for the use of various laser wavelengths commonly employed in biological imaging without the need for precise alignment of each wavelength. The team demonstrated the effectiveness of their setup by performing super-resolution SIM imaging of fluorescent beads and live cell samples with four color channels.

First, the research team performed experimental validation by using 100-nm-diameter multicolor fluorescent bead samples that served as control sample to test the resolution and accuracy of the new grating-DMD-SIM (gDMD-SIM) system. The team conducted both wide-field and SIM imaging of these beads across three color channels: red (642 nm), green (532 nm), and blue (488 nm). They found that their SIM reconstructed images displayed significant spatial resolution improvements compared to wide-field images. Fourier Ring Correlation analysis confirmed these improvements and showcased the system’s capability to achieve near-ideal resolution enhancements across multiple color channels. The authors afterward proceeded to test their gDMD-SIM system on live human BJ fibroblast cells. The cells were labeled with specific dyes to target different cellular structures: Hoechst for the nucleus, MitoTracker for mitochondria, CellMask Orange for actin, and Tubulin Tracker Deep Red for microtubules and their findings were striking with the SIM images provided significantly higher resolution and clearer details compared to wide-field images. In particular, the SIM images could distinctly resolve adjacent microtubules, actin filaments, and mitochondria, which usually appear as blurred structures in the wide-field images. Moreover, the gDMD-SIM system’s optical-sectioning filtered out-of-focus fluorescence and enhanced both image clarity and contrast which was evident in the images of the nucleus where the SIM images showed much clearer and more defined structures compared to the wide-field images. The researchers further evaluated the gDMD-SIM system’s performance via time-series imaging of live cells where they looked at the morphological changes of mitochondria and the fluctuation of microtubules using short exposure times of 30 ms per image. They found the time-series SIM images showed dynamic cellular processes with excellent clarity and detail. For example, their experiments showed the morphological changes in mitochondria and the interactions between microtubules and other cellular structures were well-resolved which demonstrated the system’s capability to capture high-resolution images of fast-moving biological processes.

In conclusion the study led by Professor Norbert Scherer and his team successfully addressed the longstanding challenge of angular dispersion caused by the blazed grating structure of DMDs. This is significant because the new compensatory diffraction grating to counteract the DMD’s angular dispersion effect allows for the simultaneous use of multiple laser wavelengths which for multicolor imaging can enable detailed investigation of complex biological systems where multiple molecular components and interactions must be visualized simultaneously. Moreover, the developed gDMD-SIM system can provide significant improvement in spatial resolution across multiple color channels which will allow scientists to observe finer details and interactions within cells and which results in better understanding of cellular structures and dynamics. Additionally, the system’s design allows for easy scalability to additional color channels and once the diffraction grating is properly aligned, new wavelengths can be incorporated with little adjustments. Furthermore, the high refresh rate of DMDs combined with the optimized diffraction compensation supports high-speed imaging and therefore capable of capturing dynamic processes and real-time cellular activities such as mitochondrial morphology changes and microtubule fluctuations without significant photobleaching or phototoxicity which is a major advantage. It is noteworthy to mention, the reported enhanced imaging capabilities can potentially be used to improve diagnostic methods in medical research and clinical applications. Indeed, the detailed visualization of cellular structures may provide better early detection and study of diseases at the cellular level which can expedite development of new and improved diagnostic techniques and new therapeutic discoveries.