Fluorescence and SEM correlative microscopy for nanomanipulation of subcellular structures

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 hepatic sinusoidal endothelial lining and colorectal liver metastases

Colorectal cancer (CRC) is a common malignant disease and the severe nature of cases in men and women who develop colorectal cancer makes this an important socio-economic health issue. Major challenges such as understanding and modeling colorectal cancer pathways rely on our understanding of simple models such as outlined in this paper. We discuss that the development of novel standardized approaches of multidimensional (correlative) biomolecular microscopy methods facilitates the collection of (sub) cellular tissue information in the early onset of colorectal liver metastasis and that this approach will be crucial in designing new effective strategies for CRC treatment. The application of X-ray micro-computed tomography and its potential in correlative imaging of the liver vasculature will be discussed.

Microfluidic bacterial traps for simultaneous fluorescence and atomic force microscopy

The atomic force microscope has become an established research tool for imaging microorganisms with unprecedented resolution.However,its use in microbiology has been limited by the difficulty of proper bacterial immobilization.Here,we have developed a microfluidic device that solves the issue of bacterial immobilization for atomic force microscopy under physiological conditions.Our device is able to rapidly immobilize bacteria in well-defined positions and subsequently release the cells for quick sample exchange.The developed device also allows simultaneous fluorescence analysis to assess the bacterial viability during atomic force microscope imaging.We demonstrated the potential of our approach for the immobilization of rod-shaped Escherichia coli and Bacillus subtilis.Using our device,we observed buffer-dependent morphological changes of the bacterial envelope mediated by the antimicrobial peptide CM15.Our approach to bacterial immobilization makes sample preparation much simpler and more reliable,thereby accelerating atomic force microscopy studies at the single-cell level.