title: Construction of an instant structured illumination microscope.
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 222 μm) 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 100 fps, 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 145 nm (lateral) and 320 nm (axial), using a 1.49 NA objective and 488 nm excitation.
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK. Electronic address: a.curd@leeds.ac.uk.
authors.forename: Alistair
authors.initials: A
authors.lastname: Curd
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
authors.forename: Alexa
authors.initials: A
authors.lastname: Cleasby
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Makowska
authors.affiliation: Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: York
authors.affiliation: Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Hari
authors.initials: H
authors.lastname: Shroff
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
authors.forename: Michelle
authors.initials: M
authors.lastname: Peckham
mesh_terms: D007089:Image Enhancement; D021621:Imaging, Three-Dimensional / Q000295:instrumentation*; D057230:Limit of Detection; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D012984:Software
keywords: Construction; Fluorescence microscopy; Instant structured illumination microscope; Super-resolution