Abstract
BackgroundSuper-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) is well established on flat, adherent cells. However, blastomeres of mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to the movement during scanning and the large distance to the cover glass.ResultsHere we present a highly detailed protocol that allows performing 3D-SIM on blastomeres of mammalian embryos with an image quality comparable to scans in adherent cells. This protocol was successfully tested on mouse, rabbit and cattle embryos and on rabbit spermatozoa.ConclusionsOur protocol provides detailed instructions on embryo staining, blastomere isolation, blastomere attachment, embedding, correct oil predictions, scanning conditions, and oil correction choices after the first scan. Finally, the most common problems are documented and solutions are suggested. To our knowledge, this protocol presents for the first time a highly detailed and practical way to perform 3D-SIM on mammalian embryos and spermatozoa.
Highlights
Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-Structured illumination microscopy (SIM)) is well established on flat, adherent cells
We have developed a protocol for 3D structured illumination microscopy (3D-SIM) on mammalian embryos and generated high quality data for multiple studies [7, 8]
If antibodies for internuclear signals were located at the nuclear periphery (Fig. 6b) we used only 2 % bovine serum albumin (BSA) in phosphate buffered saline (PBS) as blocking buffer since other additives could reduce accessibility of chromatin for antibodies
Summary
Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) is well established on flat, adherent cells. With an axial resolution of about 100 nm and a lateral resolution of about 300 nm 3D structured illumination microscopy (3D-SIM) features an 8-fold volumetric resolution improvement over confocal microscopy [1, 2]. This improvement is achieved by redirecting the laser beams through a mobile grid and scanning each section 15 times with a slightly altered grid location and orientation (3 angles with 5 phases each). The signals produced at the borders of this mobile grid allow a software to compute super-resolution images [3,4,5,6] This scanning and calculating procedure has three requirements for specimens. This is especially important when scanning thick objects since the total exposure time throughout all sections increases with the total number of z-sections
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