Abstract
Structured illumination microscopy (SIM) is a widely used imaging technique that doubles the effective resolution of widefield microscopes. Most current implementations rely on diffractive elements, either gratings or programmable devices, to generate structured light patterns in the sample. These can be limited by spectral efficiency, speed, or both. Here we introduce the concept of fiber SIM that allows for camera frame rate limited pattern generation and manipulation over a broad wavelength range. Illumination patterns are generated by coupling laser beams into radially opposite pairs of fibers in a hexagonal single mode fiber array where the exit beams are relayed to the microscope objective's back focal plane. The phase stepping and rotation of the illumination patterns are controlled by fast electro-optic devices. We achieved a rate of 111 SIM frames per second and imaged with excitation patterns generated by both 488 nm and 532 nm lasers.
Highlights
Understanding biology at the microscopic scale with chemical specificity is one of the de-facto driving forces behind fluorescence microscopy [1,2]
The excitation beam is relayed using two 180 mm achromatic lenses (AC508-180-A-ML, ThorLabs), RL 2 and RL 3, to the first dichroic mirror, DiM1, (Di03-R405/488/532/635-t3-25x36, Semrock), which is used to compensate for phase lag between the S and P components of the excitation light generated by the main imaging dichroic, DiM 2, (Di03-R405/488/532/635-t1-25x36, Semrock) [17]
The same bead set was evaluated with Structured illumination microscopy (SIM), and the average full width half max (FWHM) was found to be 97 nm with a standard deviation of 1.8 nm
Summary
Understanding biology at the microscopic scale with chemical specificity is one of the de-facto driving forces behind fluorescence microscopy [1,2]. Many interesting biological structures, such as the arrangement of the nuclear pore complex, lie beyond this limit Imaging modalities such as electron microscopy can be used to study structures at these length scales, but they lack chemical specificity, and with it, the ability to infer information about their function. A handful of techniques were introduced that fundamentally changed how the sub-diffractive arrangement and function of biological structures were studied [3,4,5,6,7,8]. They are generally referred to as "super resolution microscopy" methods. In this regard, structured illumination microscopy (SIM) remains the dominant imaging method for dynamic cellular imaging at a resolution of ∼100 nm
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