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. 2016 Nov 3;4:120. doi: 10.3389/fcell.2016.00120

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Copyright © 2016 Clark and Petty.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Example of Ratio Imaging Microscopy. A nylon mesh was non-specifically coated with Alexa Fluor® 488-labeled antibodies and Alexa Fluor® 568-labeled antibodies then air-dried. (A) shows the meshwork using bright-field microscopy; the overlapping regions should be noted. (B,C) show the green and red fluorescence emission channels, respectively. The ratio image of Alexa Fluor® 488 emission divided by Alexa Fluor® 568 emission was calculated, which is shown in (D). It should be noted that the pathlength differences in areas of mesh overlap disappear and that the non-specific staining in (B,C) disappear by ratioing to one. (Ratio scale bars are given along the right hand side of each panel.) (5x objective).