Super-resolved structured illumination microscopy (SR-SIM) is among the fastest fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-build instruments are able to deliver two-fold resolution enhancement with high acquisition speed. SR-SIM is usually a two-step process, with raw-data acquisition and subsequent, time-consuming post-processing for image reconstruction. In contrast, wide-field and (multi-spot) confocal techniques produce high-resolution images instantly. Such immediacy is also possible with SR-SIM, by tight integration of a video-rate capable SIM with fast reconstruction software. Here we present instant SR-SIM by VIGOR (Video-rate Immediate GPU-accelerated Open-Source Reconstruction). We demonstrate multi-color SR-SIM at video frame-rates, with less than 250 ms delay between measurement and reconstructed image display. This is achieved by modifying and extending high-speed SR-SIM image acquisition with a new, GPU-enhanced, network-enabled image-reconstruction software. We demonstrate high-speed surveying of biological samples in multiple colors and live imaging of moving mitochondria as an example of intracellular dynamics.

† These authors contributed equally.visualize living cells with super-resolution at multiple reconstructed frames per second in real time. We demonstrate the versatility of this new platform by imaging cellular organelle dynamics based on live-cell fluorescent stains as well as with fluorescent protein stained samples.

Digital micromirror devices (DMDs) are spatial light modulators that employ the electro-mechanical movement of miniaturized mirrors to steer and thus modulate the light reflected off a mirror array. Their wide availability, low cost and high speed make them a popular choice both in consumer electronics such as video projectors, and scientific applications such as microscopy. High-end fluorescence microscopy systems typically employ laser light sources, which by their nature provide coherent excitation light. In super-resolution microscopy applications that use light modulation, most notably structured illumination microscopy (SIM), the coherent nature of the excitation light becomes a requirement to achieve optimal interference pattern contrast. The universal combination of DMDs and coherent light sources, especially when working with multiple different wavelengths, is unfortunately not straight forward. The substructure of the tilted micromirror array gives rise to a blazed grating, which has to be understood and which must be taken into account when designing a DMD-based illumination system. Here, we present a set of simulation frameworks that explore the use of DMDs in conjunction with coherent light sources, motivated by their application in SIM, but which are generalizable to other light patterning applications. This framework provides all the tools to explore and compute DMD-based diffraction effects and to simulate possible system alignment configurations computationally, which simplifies the system design process and provides guidance for setting up DMD-based microscopes. This article is part of the Theo Murphy meeting ‘Super-resolution structured illumination microscopy (part 1)’.

Optical nanoscopy is rapidly gaining momentum in the life sciences. Current instruments are, however, often large and expensive, and there is a substantial delay between raw data collection and super-resolved image display. Here, we describe the implementation of a compact, cost-effective, high-speed, structured illumination microscope (SIM), which allows for video-rate superresolved image reconstructions at imaging rates up to 60 Hz. The instrument is based on a digital micromirror device (DMD) and a global-shutter camera, which enables faster pattern cycles and higher duty cycles than commonly used liquid crystal-based spatial light modulators. In order to utilize a DMD for creating illumination patterns by the coherent superposition of laser beams, we carefully studied its blazed grating effect Through both simulation and experimental determination of system parameters, we identified and optimized its alignment for optimal SIM pattern contrast. Raw image data are collected using inexpensive industry-grade CMOS cameras, while a parallel-computing platform allowed us to reconstruct and visualize living cells in real time. We demonstrate the performance of this system by imaging submicron-sized fluorescent beads diffusing in an aqueous solution, resolving bead−bead interactions in real time. We show that the system is sensitive enough to image intracellular vesicles labeled with fluorescent proteins in fixed cells. We also image dynamic fluctuations of the endoplasmic reticulum (ER), as well as the movement of mitochondria in living osteosarcoma cells, where the cellular organelles are labeled with live cell fluorescent stains.