Correlative Microscopy for Nanomanipulation of Sub-Cellular Structures

Nanomanipulation under scanning electron microscopy (SEM) has been demonstrated as an enabling technique for the manipulation and characterization of nanomate-rials. We recently developed nanomanipulation techniques for the extraction and identification of DNA contained within sub-nuclear locations of a single cell nucleus. In nanomanipulation of DNA, a key step is target identification through SEM-fluorescence correlative imaging. Existing image correlation techniques often require fiducial marks and/or manual feature selection or data training, which are unsuitable for DNA nanomanipulation. This paper presents an approach for correlating SEM-fluorescence microscopy images, proven effective in processing images taken under poor SEM imaging conditions imposed by the necessity of preserving DNA's biochemical integrity. The performance of the image correlation approach under different imaging conditions was quantitatively evaluated. Compared to manual correlation by skilled operators, the automated correlation approach demonstrated an order of magnitude higher speed. The SEM-fluorescence correlation approach enables targeted nanomanipulation of sub-cellular structures under SEM.