# Axial X-Ray Microscopy in Nanotomography

## TL;DR

A new X-ray microscopy method allows detailed 3D imaging of living cells without staining or slicing, enabling clearer views of tiny cell structures.

## Contribution

A novel axial X-ray tomography method for living cells that avoids staining and slicing, validated with nanoscale resolution.

## Key findings

- The method resolves subcellular features down to 140 nm with the ×46 microscope and 50 nm with the ×345 microscope.
- Low-contrast intracellular structures and 50–100 nm organelles were successfully detected.
- The technique was validated using numerical simulations and applied to Convallaria and mouse cerebellar granule cells.

## Abstract

We developed a new tomography method for living cells using a special X-ray microscope. Our method is free from limitations related to the complicated preparation steps needed in other techniques—no staining of selected cell organelles (as in optical microscopy) and no slicing cells into ultra-thin sections (as in electron microscopy). Here is how it works: We scan the cell point by point with a focused X-ray beam. The signal from the focused spot is much stronger than from surrounding areas. This allows us to use mathematical techniques to reconstruct detailed images of the cell’s tiniest structures with the clarity we need.

Background/Objectives: This article develops theory and methods for 3D tomographic imaging of absorption coefficient distributions using axial scanning with EUV microscopes at 46× and 345× magnification. Unlike conventional CT that requires sample rotation, axial scanning moves cells through the microscope focus. The aim is tomographic reconstruction of living cell fine structure without the organelle staining used in optical fluorescence microscopy or ultra-thin cell slicing as in electron microscopy. Methods: By generalizing the geometric-optical approximation for small absorption coefficient inhomogeneities in absorbing media, we derived a new explicit tomography equation and solution algorithm validated through numerical simulation. The approach was applied to Convallaria cell analysis using the ×46 microscope. For the ×345 microscope, we developed an alternative method where the kernel of the tomography integral equation was determined experimentally using gold nanospheres with known absorption coefficient, shape, and position. This method was tested through modeling and applied to diagnostics of Convallaria and mouse cerebellar granule cells. Results: The developed methods resolve subcellular features down to 140 nm using the ×46 microscope and 50 nm using the ×345 microscope. Thin low-contrast intracellular structures and individual 50–100 nm organelles were detected. Conclusions: Methods for retrieving absorption coefficient distributions in cone-beam geometry based on geometric-optical theory generalization and on calibration by gold nanoparticles have been developed and validated through numerical simulation and cell analysis. These methods demonstrate for the first time the effectiveness of axial nanotomography using multilayer mirror microscopes for cell diagnostics.