Quantitative three-dimensional Laboratory X-Ray Microscopy

The goal of the present proposal is to provide quantitative three-dimensional water-window x-ray microscopy of intact cells in their near-native state with true isotropic high resolution.

We developed the first laboratory water-window x-ray microscope and this instrument recently reached a milestone:  Hundreds of high-resolution 2D images can now be recorded in a repeatable and reliable way, allowing for quantitative evaluation of biological processes.

In order to do full 3D imaging with 30-40 nm isotropic resolution in a similar fashion for adherent cells, we need to remove the missing-wedge and depth-of-focus problems in the tomography.

We propose doing this by a new sample preparation process, including surface modification of TEM grids and acoustic positioning of cells, in combination with focus-stack tomography. This new method will be applicable for all water-window x-ray microscopes. The method will be tested by imaging nanoparticle uptake, interaction, accumulation and excretion in macrophages.