Fast structured illumination microscopy using rolling shutter cameras

Abstract

Spatial light modulators (SLM) update in a synchronous manner, whereas the data readout process in fast structured illumination systems is usually done using a rolling shutter camera with asynchronous readout. In structured illumination microscopy (SIM), this leads to synchronization problems causing a speed limit for fast acquisition. In this paper we present a configuration to overcome this limit by exploiting the extremely fast SLM display and dividing it into several segments along the direction of the rolling shutter of the sCMOS camera and displaying multiple SLM frames per camera acquisition. The sCMOS runs in continuous rolling shutter mode and the SLM keeps the readout-line always inside a dark region presenting different SIM patterns before and after the readout/start-exposure line. Using this approach, we reached a raw frame rate of 714 frames per second (fps) resulting in a two-beam SIM acquisition rate of 79 fps with a region of interest (ROI) of 16.5 × 16.5 μm2.

High‐Speed Large‐Field Multifocal Illumination Fluorescence Microscopy

“…Structured illumination microscopy (SIM) arguably offers the highest degree of parallelization as only a relatively small number of wide‐field images are required for rendering a super‐resolution image. State‐of‐the‐art SIM can achieve imaging rates of 79 frames per second for a region of interest (ROI) of 16.5 × 16.5 µm 2 . Recent progress on multifocal SIM enabled 3D interrogation of live organisms at frame rates of ∼1 Hz, covering a FOV in the 50–100 µm range .…”

“…State-of-the-art SIM can achieve imaging rates of 79 frames per second for a region of interest (ROI) of 16.5 × 16.5 µm 2 . [34] Recent progress on multifocal SIM enabled 3D interrogation of live organisms at frame rates of ∼1 Hz, covering a FOV in the 50-100 µm range. [35][36][37] However, the existing scanning-based parallel acquisition methods are only applicable for imaging very small areas with high resolution, so that wide-field approaches remain the method of choice for large-scale imaging.…”

High speed multi-plane super-resolution structured illumination microscopy of living cells using an image-splitting prism

“…dyes that are photo-switchable or inherently blinking) are required for the linear 2x resolution enhancement. The combination of these features makes SR-SIM a very fast super-resolution imaging technique, with current instruments [8,9] providing 2D imaging with approximately 100 nm spatial resolution in the lateral direction at video-rate speed and even faster.…”

Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength

“…Therefore, SIM has been successfully used in various biological studies based on imaging. By using a programmable spatial light modulator (SLM) rapid control of the structured illumination pattern is achieved [3–7]. …”