Structured Illumination Microscopy (SIM)

Introduction

Conventional optical microscopes have a resolving power limited by the diffraction limit, first described by Ernst Abbe in the 19th century, which is approximately half the wavelength of the light used. Although this restriction makes it impossible to visualize objects less than about 200 nm, organelle membranes, cytoskeletal filaments, and protein complexes are all found below this size. It was only in the early 2000s that Gustafsson and others introduced Structured Illumination Microscopy (SIM), which overcomes this limitation by encoding high-fidelity spatial data into the image being studied by using patterned illumination.

Principle of Operation

The phenomenon behind the SIM working principle lies in the fact that the patterns of known illuminations interfere with the fine structural detail of the specimen and lead to the formation of Moiré fringes that hold the sub-diffraction spatial information. A sinusoidal grid pattern is normally projected on the sample due to varying angles and phases. SIM can be used to effectively increase both lateral and axial resolution by combining data from more than one orientation. The advantage of the technique is that it can utilise the response of linear optical sources, which enables it to be utilised with common dyes and fluorescent proteins, unlike nonlinear techniques, such as STED or PALM/STORM.

Equipment and Methodology

The device records a sequence of images (usually nine to fifteen in each plane) using patterns of isochronic light illumination. Fourier-based reconstruction algorithms are advanced reconstruction algorithms that retrieve high-resolution data and recreate an ultimate super-resolved image. In the case of live-cell imaging, small-intensity light and brief gain acquisition mechanisms are employed to reduce the effects of photobleaching and phototoxicity. Accuracy and reconstruction of the system and reconstruction can be guaranteed using fluorescent bead standards.

Applications and Industry Use

In biomedical diagnostics and pathology, SIM can be used to create improved contrast of tissue morphology and markers of diseases. Since SIM can image in multi-color and reconstruct in 3D, it is a transition between the traditional methods of using fluorescence microscopy and the more advanced and difficult methods such as STORM and STED. 

Common Challenges and Troubleshooting

Although SIM has a high resolution-accessibility ratio, optical alignment, pattern contrast, and sample stability need very tight control. The artefacts can be due to poor projection of patterns, movement of samples, or photobleaching in the process of capturing the image. Signal-to-noise ratios may also be low, resulting in reconstruction errors, or the calibration parameters may be incorrect. To reduce these problems, scientists are forced to apply stable fluorophores, optics of high quality, and sophisticated computational correction algorithms. Having a system that is regularly calibrated and using vibration isolation stages is necessary to achieve an accurate data acquisition; this is particularly necessary in 3D SIM use.

Importance in Modern Microscopy

SIM has become an essential part of optical imaging in the modern world because it enables super-resolution without the destruction of sample viability and without the use of specialist fluorophores. Its ability to be used with live-cell imaging, as well as its ability to perform multicolour, volumetric imaging, makes it the best choice in dynamic biological studies.