Comprehensive Notes on Micro-Computed Tomography (µCT)
Introduction to µCT
Overview
Micro-computed tomography (µCT) is a technique used to estimate the local absorbance of a structure. It does not directly form an image but rather uses mathematical predictions to reconstruct a 3D object.
X-Ray Properties
X-rays have a short wavelength and high energy, allowing them to penetrate even thick materials. Unlike visible light, it is not possible to focus X-rays using lenses.
Interaction with Materials
When X-rays pass through a material, denser materials absorb more radiation compared to less dense materials (e.g., bones absorb more than soft tissues). This differential absorption reveals the inner structure of biological samples.
The X-ray source involves the interaction between a metallic target and high-energy electrons from an electron gun, which includes focusing lenses.
X-Ray Generation
The voltage applied affects the acceleration of the electron beam. The size of the X-ray spot can be modified by adjusting the distance of the magnetic lens. Transmission targets offer higher magnification, facilitating the examination of smaller samples compared to reflection targets.
Mechanisms for Generating X-rays:
Bremsstrahlung Radiation:
High-speed electrons are deflected by atoms in a metal target, losing kinetic energy.
The lost energy is released as a photon (X-ray).
Electrons can be deflected by different amounts, resulting in a continuous spectrum of X-rays.
Characteristic Emission:
A high-speed electron collides with an electron in the target atom.
This collision knocks out an electron from its orbital, creating a vacancy.
An electron from a higher-energy shell fills the lower-energy shell.
This transition releases energy as an X-ray photon with a specific energy level characteristic of the target material.
Energy Spectrum and Decay
There's a cut-off energy below which X-rays cannot escape the source. Different metals exhibit different exponential decay rates, leading to varying characteristic slopes in the energy spectrum. Although the decay is exponential, it never reaches zero.
Energy Considerations for Biological Samples
Reducing beam energy can be advantageous for biological samples because lower energy X-rays are absorbed more effectively by soft tissues.
X-Ray Detection
Most modern X-ray systems use a Digital Flat Panel Detector, which typically has two layers:
A scintillator crystal that absorbs X-rays and re-emits them as visible light.
Photodiodes (in pixels) that detect the visible light.
A digital flat panel acts as a large digital camera sensor (e.g., mm) with a scintillator in front.
Signal-to-Noise Ratio (SNR)
Image quality is limited by signal strength (number of photons reaching the detector). Longer integration times can reduce noise.
To improve the SNR, a brighter X-ray source or longer exposure time can be used to increase the X-ray flux (number of X-ray photons reaching the detector).
The SNR improves with the square root of flux. For instance, an image that takes 16 times longer to acquire will have 4 times less noise. This relationship can be expressed as: .
Time-Limiting Factors
Acquisition time is a significant limiting factor in µCT imaging. Synchrotron beamlines can provide much shorter acquisition times due to their super-bright X-ray sources.
Resolution and Magnification
For very small samples, placing them close to the beam enhances magnification. Alternatively, using a larger detector allows the sample to be placed further away, though this is a more expensive solution.
Resolution is limited by the size of the image and the pixels that make up that image. A large focal spot can blur the image.
Image Reconstruction
The sum of all integrated information from multiple projections, considering both thickness and density of the materials, is used to generate a mathematical interpretation of the object's internal structure.
Each row represents a forward projection. Less energy is absorbed at the edges, resulting in brighter areas, whereas denser regions absorb more energy. A high-pass filter is applied to the original sample data to reduce noise and improve image resolution.
Scanning and Stability
The sample is rotated in small steps to cover every angle. Sample stability is crucial for obtaining good projection images, especially for rigid samples that must remain still during the scan.
Tomography
The resulting computed image set is called a tomogram where:
Typical resolution ranges from 1.5 - 125µm (isometric voxels).
Sample sizes can range from mm to ~300 mm.
A typical system might include:
Electron beam x-ray source: 225kV, 225W.
mm detector with scintillator.
Each pixel is 150µm.
Can capture up to 15 frames per second.
*'Voxel' is short for volume pixel, and a tomogram is based on isometric voxels.
Variants and Applications
Many machine variants exist, ranging from smaller to larger, with a wide range of applications. Synchrotron-based microtomography allows for in vivo time-resolved imaging using very bright X-ray sources.
For example, the thorax of tethered blowflies flying in the TOMCAT beamline of the Swiss Light Source (a 3rd generation synchrotron) was imaged. X-rays were converted to visible light using a Ce-doped LuAG scintillator and imaged using a 12-bit CMOS detector @ 2,500 Hz while the insects were spun at 347° s-1.