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Advanced 3D Analysis and Optimization of Single‐Molecule FISH in Drosophila Muscle
Author(s) -
Noma Akiko,
Smith Carlas S.,
Huisman Maximiliaan,
Martin Robert M.,
Moore Melissa J.,
Grunwald David
Publication year - 2018
Publication title -
small methods
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.66
H-Index - 46
ISSN - 2366-9608
DOI - 10.1002/smtd.201700324
Subject(s) - organelle , in situ hybridization , messenger rna , fluorescence in situ hybridization , microbiology and biotechnology , rna , biology , biophysics , nucleic acid , computational biology , biochemistry , gene , chromosome
Single‐molecule fluorescence in situ hybridization (smFISH) provides direct access to the spatial relationship between nucleic acids and specific subcellular locations. The ability to precisely localize a messenger RNA can reveal key information about its regulation. Although smFISH is well established in cell culture or thin sections, the utility of smFISH is hindered in thick tissue sections due to the poor probe penetration of fixed tissue, the inaccessibility of target mRNAs for probe hybridization, high background fluorescence, spherical aberration along the optical axis, and the lack of methods for image segmentation of organelles. Studying mRNA localization in 50 µm thick Drosophila larval muscle sections, these obstacles are overcome using sample‐specific optimization of smFISH, particle identification based on maximum likelihood testing, and 3D multiple‐organelle segmentation. The latter allows independent thresholds to be assigned to different regions of interest within an image stack. This approach therefore facilitates accurate measurement of mRNA location in thick tissues.