PMID- 28223507 OWN - NLM STAT- MEDLINE VI - 114 IP - 10 TI - Imaging mRNA and protein interactions within neurons. PG - E1875-E1884 LA - eng PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PL - United States TA - Proc Natl Acad Sci U S A JT - Proceedings of the National Academy of Sciences of the United States of America JID - 7505876 IS - 1091-6490 (Electronic) LID - 10.1073/pnas.1621440114 [doi] FAU - Eliscovich, Carolina AU - Eliscovich C AD - Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461. FAU - Shenoy, Shailesh M AU - Shenoy SM AD - Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461. AD - Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461. FAU - Singer, Robert H AU - Singer RH AUID- ORCID: 0000-0002-6725-0093 AD - Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461; robert.singer@einstein.yu.edu. AD - Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461. AD - Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147. IS - 0027-8424 (Linking) RN - 0 (Fluorescent Dyes) RN - 0 (Proteins) RN - 0 (RNA, Messenger) RN - 0 (RNA-Binding Proteins) SB - IM MH - Fluorescent Dyes/chemistry MH - Gene Expression Regulation/genetics MH - In Situ Hybridization, Fluorescence/methods MH - Neurons/*chemistry/ultrastructure MH - Proteins/genetics/*isolation & purification MH - RNA, Messenger/genetics/*isolation & purification MH - RNA-Binding Proteins/genetics/*isolation & purification MH - Single Molecule Imaging/methods OTO - NOTNLM OT - *chromatic aberration correction OT - *smFISH-IF OT - *super registration PMC - PMC5347572 DCOM- 20180410 LR - 20200716 DP - 20170307 DEP - 20170221 AB - RNA-protein interactions are essential for proper gene expression regulation, particularly in neurons with unique spatial constraints. Currently, these interactions are defined biochemically, but a method is needed to evaluate them quantitatively within morphological context. Colocalization of two-color labels using wide-field microscopy is a method to infer these interactions. However, because of chromatic aberrations in the objective lens, this approach lacks the resolution to determine whether two molecules are physically in contact or simply nearby by chance. Here, we developed a robust super registration methodology that corrected the chromatic aberration across the entire image field to within 10 nm, which is capable of determining whether two molecules are physically interacting or simply in proximity by random chance. We applied this approach to image single-molecule FISH in combination with immunofluorescence (smFISH-IF) and determined whether the association between an mRNA and binding protein(s) within a neuron was significant or accidental. We evaluated several mRNA-binding proteins identified from RNA pulldown assays to determine which of these exhibit bona fide interactions. Surprisingly, many known mRNA-binding proteins did not bind the mRNA in situ, indicating that adventitious interactions are significant using existing technology. This method provides an ability to evaluate two-color registration compatible with the scale of molecular interactions.