Structured illumination light sheet microscopy (SI-LSM) is an optical imaging technique used for achieving volumetric imaging with high temporal and spatial resolution in all three dimensions. It combines the ability of light sheet microscopy to maintain spatial resolution throughout relatively thick samples with the higher axial and spatial resolution characteristic of structured illumination microscopy. SI-LSM can achieve lateral resolution below 100 nm in biological samples hundreds of micrometers thick. SI-LSM is most often used for fluorescent imaging of living biological samples, such as cell cultures. It is particularly useful for longitudinal studies, where high-rate imaging must be performed over long periods of time without damaging the sample. The two methods most used for fluorescent imaging of 3D samples – confocal microscopy and widefield microscopy – both have significant drawbacks for this type of application. In widefield microscopy, both in-focus light from the plane of interest as well as out-of-focus light from the rest of the sample is acquired together, creating the "missing cone problem" which makes high resolution imaging difficult. Although confocal microscopy largely solves this problem by using a pinhole to block unfocused light, this technique also inevitably blocks useful signal, which is particularly detrimental in fluorescent imaging when the signal is already very weak. In addition, both widefield and confocal microscopy illuminate the entirety of the sample throughout imaging, which leads to problems with photobleaching and phototoxicity in some samples. While light-field microscopy alone can address most of these issues, its achieved resolution is still fundamentally limited by the diffraction of light and it is unable to achieve super-resolution. SI-LSM works by using a patterned rather than uniform light sheet to illuminate a single plane of a volume being imaged. In this way, it maintains the many benefits of light-sheet microscopy while achieving the high resolution of structured illumination microscopy.
Background and Theory The theory behind SI-LSM is best understood by considering the separate development of structured illumination and light sheet microscopy.
Structured Illumination Microscopy
Structured illumination microscopy (SIM) is a method of super-resolution microscopy which is performed by acquiring multiple images of the same sample under different patterns of illumination, then computationally combining these images to achieve a single reconstruction with up to 2x improvement over the diffraction limited lateral resolution. The theory was first proposed and implemented in a 1995 paper by John M. Guerra in which a silicon grating with 50 nm lines and spaces was resolved with 650 nm wavelength (in air) illumination structured by a transparent replica proximal to said grating. The name "structured illumination microscopy" was coined in 2000 by M.G.L. Gustafsson. SIM takes advantage of the "Moiré Effect", which occurs when two patterns are multiplicatively superimposed. The superimposition causes "Moiré Fringes" to appear, which are coarser than either original pattern but still contain information about the high frequency patterns which would otherwise not be visible. The theory behind SIM is best understood in the Fourier or frequency domain. In general, imaging systems can only resolve frequencies below the diffraction limit. Thus, in the Fourier domain, all recorded frequencies from the imaged sample would reside within a circle of a fixed radius. Any frequencies outside this limit cannot be resolved. However, the frequency spectrum can be shifted by imaging the sample with patterned illumination. Most often, the pattern is a 1D sinusoidal gradient, such as the pattern used to create the Moiré fringes in the above image. Because the Fourier transform of a sinusoid is a shifted delta function, the transform of this pattern will consist of three delta functions: one at the zero frequency and two corresponding to the positive and negative frequency components of the sinusoid (see below image). When the target is illuminated using this pattern, the target and illumination pattern are multiplicatively superimposed, which means the Fourier transform of the resulting image is the convolution of the individual transforms of the target and the illumination pattern. Convolving any function with a delta function has the effect of shifting the center of the original function to the location of the delta function. Thus, in this situation, the frequency spectrum of the target is shifted and frequencies that were previously too high to resolve now lie within the circle of resolvable frequencies. The result is that for a single image acquisition with SIM, the frequency components from three separate regions in the Fourier domain (corresponding to the center and the positive and negative shifts) are all captured together. Finally, because rotation in the spatial domain results in the same rotation in the Fourier domain, high frequencies over the full 360° can be captured by rotating the illumination pattern. Figure b) in the image below shows which frequency components would be captured by acquiring 4 separate images and rotating the illumination pattern by 45° in between each acquisition. Once all images have been captured, a single final image can be computationally reconstructed. Using this technique, resolution can be improved up to 2x over the diffraction limit. This 2x limit is imposed because the illumination pattern itself is still diffraction limited.
The concepts behind 2D SIM can be expanded to 3D volumetric imaging. By using three mutually coherent beams of excitation light, interference patterns with multiple frequency components can be created in the imaged sample. This ultimately makes it possible to perform 3D reconstructions with up to 2x improved resolution along all three axes. However, due to the strong scattering coefficient of biological tissues, this theoretical resolution can only be achieved in samples thinner than about 10 um. Beyond that, the scattering leads to an excess of background signal which makes accurate reconstruction impossible.
Light Sheet Microscopy
… excerpt ends here. Continue reading the full article.
