Abstract
We have developed a 3D super-resolution microscopy method that enables deep imaging in cells. This technique relies on the effective combination of multifocus microscopy and astigmatic 3D single-molecule localization microscopy. We describe the optical system and the fabrication process of its key element, the multifocus grating. Then, two strategies for localizing emitters with our imaging method are presented and compared with a previously described deep 3D localization algorithm. Finally, we demonstrate the performance of the method by imaging the nuclear envelope of eukaryotic cells reaching a depth of field of ~4µm.
Highlights
Fluorescence microscopy has proven to be a powerful tool in biology and physical sciences
We have developed a 3D super-resolution microscopy method that enables deep imaging in cells
This technique relies on the effective combination of multifocus microscopy and astigmatic 3D single-molecule localization microscopy
Summary
Fluorescence microscopy has proven to be a powerful tool in biology and physical sciences. The multifocus microscope based on diffractive optics was first described in 2013 by Abrahamsson et al [9] This technology enables the simultaneous imaging of multiple (typically 9 and up to 25) -spaced focal planes on a single camera. We demonstrate the effective combination of multiple plane imaging using MFM and axial localization of single emitters by PSF engineering (3D-MF-SMLM) This implementation enables deep 3D super-resolved imaging with high localization precision in the three directions with fast localization algorithm processing. In our implementation of the multiple focus microscope, a long focal length (f = 1000 mm) cylindrical lens is positioned in front of the em-CCD camera This optical element introduces astigmatism in the emitted light wavefront and enables to break the axial symmetry of the PSF [4] (Fig. 1(c) 1(d)). The fluorescence signal emitted from the sample was collected by the objective lens, separated from the excitation wavelengths through a four-band dichroic mirror (zt405/488/561/638rpc - Chroma, USA) and filtered using a bandpass filter (ET600/50m, Chroma, USA)
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