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. A fast method for correlating scanning electron microscopy (SEM) and fluorescence images when handling subcellular objects has been acheived. Yu Sun of the University of Toronto, Canada, and co-workers in China and Canada developed the image correlation technique to improve the implementation of a method for extracting DNA from cells. Such correlation is essential, because SEM images lack sufficient contrast to enable structures of interest to be identified and because of the need to use low-energy (and hence low-resolution) SEM imaging to avoid damaging DNA. Their correlation technique employs image processing algorithms to automatically correlate the two microscopy techniques and is an order of magnitude faster than manual image correlation by skilled operators. The researchers have demonstrated the method's usefulness by using it to extract DNA from cells under SEM imaging.