Abstract
Background: Super-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. Results: Here 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. Conclusions: Our 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.
Item Type: | Journal article |
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Faculties: | Veterinary Medicine > Department of Veterinary Medicine |
Subjects: | 600 Technology > 610 Medicine and health |
URN: | urn:nbn:de:bvb:19-epub-33967-6 |
ISSN: | 1471-213X |
Language: | English |
Item ID: | 33967 |
Date Deposited: | 15. Feb 2017, 16:02 |
Last Modified: | 04. Nov 2020, 13:11 |