Outline of the Correlative Workflow.

Abstract

<p>A. The flowchart lists the general steps involved in the correlative workflow. B. Left panel: installation of the anesthetized mouse on the LM stage. The ear with the transplanted fluorescent tumor cells (right panel, cartoon) is mounted in a custom-built holder. C. Z-projection of a typical 2PEM dataset obtained from the mouse ear that was injected with GFP-expressing tumor cells (green). SHG signal of the collagen fibers is shown in blue. Evans Blue stains blood vessels and is depicted in red. Scale bar: 100 µm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s003" target="_blank">Movie S1</a>. D. Cartoon of the NIRB process representing the imaged volume containing a vessel (red), collagen fibers (blue), and tumor cells (green). Our NIRB-procedure entails drawing a frame at the surface of the skin with a high-powered laser, above and away from the ROI. The imaged volumes presented in this work were in between 60 and 200 µm in depth and ranging from 270 to 440 µm in xy. E. Following NIRB, the same volume is imaged again with 2PEM. The NIRB marks visible in this z-projection are traced in white. Scale bar: 100 µm. Asterisks point to hair follicles. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s003" target="_blank">Movie S1</a>. F. After chemical fixation of the mouse ear sample, NIRB markings remain temporarily autofluorescent and their location can be mapped. Asterisks pointing to hair follicles (as in E). G. Following EM processing, the embedded sample can be trimmed and sectioned by ultramicrotomy. Serial thick sections (500 nm) are placed on glass slides to be imaged by light microscopy (‘sections on LM slide’). Serial thin sections (60 to 240 nm) are mounted on slot grids (‘sections on TEM grid’) allowing for TEM observation and/or electron tomography.</p