Abstract
Structured illumination microscopy (SIM) is a powerful super-resolution (SR) microscopy technique which is applicable to a wide variety of biological systems because it does not impose photophysics requirements on the sample. Nevertheless, current ferroelectric or liquid crystal display spatial light modulator (SLM) based SIM instruments are expensive and slow compared with digital micromirror device (DMD) implementations, while DMD implementations either rely on incoherent projection which results in an order of magnitude lower signal-to-noise, or utilize coherent light at only a single wavelength. The primary obstacle to realizing a multicolor coherent DMD SIM microscope is the blazed grating effect due to the tilted micromirrors, and the lack of efficient quantitative approaches for dealing with such systems. To address this challenge, we develop a variety of quantitative tools which are applicable to any experiment relying on a DMD as an active diffractive element, including a closed form solution of the blaze and diffraction conditions, a forward model of DMD diffraction, and a forward model of coherent pattern projection. We demonstrate their power by using periodic patterns to directly map the optical transfer function of our microscope, an approach that was previously computationally infeasible. Finally, we apply these techniques to SIM by identifying experimentally feasible configuration for combinations of three and four common fluorophore wavelengths. Based on these advances, we constructed a custom DMD SIM microscope using three wavelengths and coherent light and validated this instrument by demonstrating resolution enhancement for known calibration samples, fixed cells, and live cells. This low-cost setup opens the door to applying SIM more broadly in live cell and other time-resolved experiments.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
New closed-form joint solution of diffraction and blaze conditions, experimental verification of DMD and SIM pattern forward models for three wavelengths at pupil plane, reworked optical transfer function model to account for excitation polarization, updated numerical simulations to compare to incoherent SIM, and added three color live-cell SIM experiments.
