Abstract
In the context of various approaches to super-resolution microscopy, structured illumination microscopy (SIM) offers several advantages: it needs rather low light doses (with a low risk of phototoxicity or photobleaching), is comparably fast and flexible concerning the use of microscopes, objective lenses and cameras, and has potential for 3D imaging. This paper describes an experimental setup for SIM with first diffraction orders of a spectral light modulator (SLM) creating an interference pattern in two dimensions. We kept this system rather compact with a comparably large illuminated object field, validated it with nano-beads and applied it further to living cells for imaging the cytoskeleton, mitochondria or cell nuclei with a resolution slightly above 100 nm. Its advantages, challenges and limitations—concerning cameras, acquisition time, depth of imaging, light exposure, and combining it with further super-resolving methods—are discussed.
Highlights
Resolution in microscopy is well described by the Abbe or the Rayleigh criterion
Schock (Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Germany), and calibration slides were prepared as described in Reference [21]. 3T3 murine fibroblasts were cultivated as monolayers or multilayers on microscope cover slips in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum, pyruvate and antibiotics at 37 ◦ C and 5% CO2 . 72 h after seeding cells were incubated for 20 min with Tubulin TrackerTM Green at a concentration of 250 nM
structured illumination microscopy (SIM) is a promising approach in view of the low light exposure in optical microscopy
Summary
Resolution is determined by diffraction of a coherent light beam and results in ymin ≥ λ/2AN (with λ corresponding to the wavelength of light and AN to the numeric aperture of the microscope objective lens), whereas in the second case, ymin = 0.61 λ/AN results from the diffraction function of an incoherently luminescent spot (Airy disk). In both cases, values around 200 nm are attained for high numeric apertures (AN ≥ 1.30) and around 400–500 nm for moderate apertures (AN ≤ 0.60), e.g., upon imaging of larger objects by long distance objective lenses. In contrast to CLSM only the plane of detection has to be exposed to light, so that the total light exposure in 3D imaging is considerably
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