Super-resolution three-dimensional (3D) optical microscopy has incomparable advantages over other high-resolution microscopic technologies, such as electron microscopy and atomic force microscopy, in the study of biological molecules, pathways and events in live cells and tissues. We present a novel approach of structured illumination microscopy (SIM) by using a digital micromirror device (DMD) for fringe projection and a low-coherence LED light for illumination. The lateral resolution of 90 nm and the optical sectioning depth of 120 μm were achieved. The maximum acquisition speed for 3D imaging in the optical sectioning mode was 1.6×107 pixels/second, which was mainly limited by the sensitivity and speed of the CCD camera. In contrast to other SIM techniques, the DMD-based LED-illumination SIM is cost-effective, ease of multi-wavelength switchable and speckle-noise-free. The 2D super-resolution and 3D optical sectioning modalities can be easily switched and applied to either fluorescent or non-fluorescent specimens.

Optical-sectioned images with super-resolution enhancement is achieved by combining the information in data obtained from a tunable structured illumination microscope. The system can easily switch between two fringe patterns required for optical sectioning and super-resolution. OCIS codes: (110.1758) Computational Imaging; (180.6900) Three-dimensional Microscopy; (100.6640) Super-resolution. 1. Introduction Conventional structured illumination microscopy (SIM) systems [1]-[3], can only produce two-dimensional (2D) patterns, which are not able to provide simultaneously the highest improvement in both the lateral and axial resolution. In fact, the performance of 2D-SIM depends on the modulation frequency of the illumination pattern; this value determines the resolution enhancement either laterally or axially [4]. To provide resolution enhancement in three dimensions, a three-dimensional (3D) structured pattern that includes both lateral and axial variations in the excitation illumination is required [5]. Gustafsson et al. [5] used three-wave (3W) interference to produce an interference pattern that modulates the object at two different lateral frequencies; the highest modulation frequency provides super-resolution performance while the lower modulation frequency, whose value is exactly equal to half of the highest modulation frequency, produces optical sectioning capability. However, these two modulation frequencies are fixed by the illumination system and they are completely dependent on each other; as a result there is no flexibility in choosing two independent different modulation frequencies. Here, we address the resolution enhancement's issue in 3D by using a 2D-SIM system whose modulation frequency is easily adjustable. In this tunable-frequency SIM system [6,7], a Fresnel biprism is illuminated by a wavefront emerging from one incoherent linear source (e.g. a slit) and it produces an axially-extended pattern in which the modulation frequency changes with the location of the Fresnel biprism along the optical axis. In this contribution, we present a computational method that takes advantage of the tunable-frequency SIM by recording phase-shifted images for two independent modulation frequencies (three images for each frequency of the same field of view). The lower spatial modulation frequency provides the optical sectioning (OS) capability by filling the missing cone and the other one produces super-resolution (SR) performance by almost doubling the cutoff frequency (í µí±“ ") of the conventional wide-field system. Combining information from two different datasets has been used in other applications such as in the HiLo imaging system [9] and in by the FABEMD algorithm [10]. The former uses uniform and speckle illumination to produce OS with a resolution comparable to deconvolution microscopy (i.e. without SR), and the latter is a double-shot SIM to generate an OS image without SR. Our method combines the information in the six intermediate SIM images and provides simultaneous OS and SR in the reconstructed 3D...

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The E. coli single‐stranded DNA‐binding protein (SSB) is essential to viability. It plays key roles in DNA metabolism where it binds to nascent single strands of DNA and to target proteins known as the SSB interactome. There are >2,000 tetramers of SSB per cell with 100–150 associated with the genome at any one time, either at DNA replication forks or at sites of repair. The remaining 1,900 tetramers could constantly diffuse throughout the cytosol or be associated with the inner membrane as observed for other DNA metabolic enzymes. To visualize SSB localization and to ascertain potential spatiotemporal changes in response to DNA damage, SSB‐GFP chimeras were visualized using a novel, super‐resolution microscope optimized for the study of prokaryotic cells. In the absence of DNA damage, SSB localizes to a small number of foci and the excess protein is associated with the inner membrane where it binds to the major phospholipids. Within five minutes following DNA damage, the vast majority of SSB disengages from the membrane and is found almost exclusively in the cell interior. Here, it is observed in a large number of foci, in discreet structures or, in diffuse form spread over the genome, thereby enabling repair events.