Fluorescence and SEM correlative microscopy for nanomanipulation of subcellular structures

Abstract

Nanomanipulation under scanning electron microscopy (SEM) enables direct interactions of a tool with a sample. We recently developed a nanomanipulation technique for the extraction and identification of DNA contained within sub-nuclear locations of a single cell nucleus. In nanomanipulation of sub-cellular structures, a key step is to identify targets of interest through correlating fluorescence and SEM images. The DNA extraction task must be conducted with low accelerating voltages resulting in low imaging resolutions. This is imposed by the necessity of preserving the biochemical integrity of the sample. Such poor imaging conditions make the identification of nanometer-sized fiducial marks difficult. This paper presents an affine scale-invariant feature transform (ASIFT) based method for correlating SEM images and fluorescence microscopy images. The performance of the image correlation approach under different noise levels and imaging magnifications was quantitatively evaluated. The optimal mean absolute error (MAE) of correlation results is 68±34 nm under standard conditions. Compared with manual correlation by skilled operators, the automated correlation approach demonstrates a speed that is higher by an order of magnitude. With the SEM-fluorescence image correlation approach, targeted DNA was successfully extracted via nanomanipulation under SEM conditions.