Abstract
A challenge in biological imaging is to capture high-resolution images at fast frame rates in live cells. The “instant structured illumination microscope” (iSIM) is a system designed for this purpose. Similarly to standard structured illumination microscopy (SIM), an iSIM provides a twofold improvement over widefield microscopy, in x, y and z, but also allows much faster image acquisition, with real-time display of super-resolution images.The assembly of an iSIM is reasonably complex, involving the combination and alignment of many optical components, including three micro-optics arrays (two lenslet arrays and an array of pinholes, all with a pitch of 222μm) and a double-sided scanning mirror. In addition, a number of electronic components must be correctly controlled. Construction of the system is therefore not trivial, but is highly desirable, particularly for live-cell imaging.We report, and provide instructions for, the construction of an iSIM, including minor modifications to a previous design in both hardware and software. The final instrument allows us to rapidly acquire fluorescence images at rates faster than 100fps, with approximately twofold improvement in resolution in both x–y and z; sub-diffractive biological features have an apparent size (full width at half maximum) of 145nm (lateral) and 320nm (axial), using a 1.49 NA objective and 488nm excitation.
Highlights
Among the methods of acquiring super-resolution fluorescence images [1,2], structured illumination microscopy (SIM) offers a relatively modest, twofold resolution improvement over widefield microscopy [3]
As SIM uses only a relatively small number of widefield images to capture the information required to improve resolution, it is in principle more suitable for live sample imaging; SIM offers the advantages of fast acquisition over a large area and weaker irradiation of the sample compared to alternative techniques such as stimulated emission depletion [4] and single-molecule localisation [5,6,7], and it is compatible with all fluorophores used in widefield and confocal imaging
The process of scanning and capturing an image at every position was very slow, and not suitable for live cell imaging. The speed of this type of imaging was improved to several frames per second in the MSIM by using a digital mirror device (DMD) to generate multiple excitation foci, which were scanned across the sample [13]
Summary
Among the methods of acquiring super-resolution fluorescence images [1,2], structured illumination microscopy (SIM) offers a relatively modest, twofold resolution improvement over widefield microscopy [3]. By scanning a displaced pinhole, and appropriately shifting and summing the resulting images, much more of the emission signal can be used, and the narrower PSF retained; the appropriate shift is 1⁄2 the pinhole displacement for each image This principle was first discussed in 1988 [11], and further developed into ‘‘image scanning microscopy’’ [12], in which a focussed excitation laser was scanned over the sample, and an image taken at each scanning focus position. The process of scanning and capturing an image at every position was very slow, and not suitable for live cell imaging The speed of this type of imaging was improved to several frames per second in the MSIM (multifocal SIM) by using a digital mirror device (DMD) to generate multiple excitation foci, which were scanned across the sample [13]. We chose to build an iSIM at the University of Leeds (UoL), in order to take advantage of its speed and resolution, and its compatibility with standard sample and fluorophore properties, and this chapter is aimed at providing detailed methods for those who wish to build a similar instrument
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