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 68634 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.

The 4D nucleome: Evidence for a dynamic nuclear landscape based on co‐aligned active and inactive nuclear compartments

“…The further development of super‐resolved localization microscopy for multicolor, live cell technology for the 4D mapping of DNA together with its architectural and functional proteins constitutes a huge challenge. Exploitation of the entire set of available methods, including novel correlative fluorescence – electron‐microscopic (CFEM) approaches[39,156–159], apparently provides the best safeguard against misleading interpretations resulting from artifacts of each individual method.…”

Stochastic Micro-Pattern for Automated Correlative Fluorescence - Scanning Electron Microscopy

“…Alignment accuracy is defined by the objective magnification that is used and the pixel size of the camera. Assuming a pixel resolution of 200 nm, which is at the diffraction limit, our approach can achieve an alignment accuracy of ~80 nm, and is thus as good as previously described conventional correlative light-electron microscopy approaches 23 28 29 . Intriguingly, reports using resin sections show that fiducial landmarks are suitable to align florescent images and transmission electron micrographs with a precision of ~100 nm 30 , and this alignment precision could be further increased to 20–30 nm when combined with super-resolution fluorescence microscopy 17 31 .…”

In Situ Detection of Complex DNA Damage Using Microscopy: A Rough Road Ahead

“…Similar to the other scanning probe microscopy techniques, SEM is limited to the study of samples surfaces. Thus, SEM is not suitable for in situ DNA detection, but it can be used in combination with fluorescence microscopy [117]. The reader may refer to the online SCIENCE photo LIBRARY for examples of SEM-based chromosome images [118].…”