3D Label Localization via Serial Sectioning in Electron Microscopy
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Solution Overview
Problem
Electron tomography is limited to analyzing extremely thin samples due to the need for electrons to pass through, causing sample damage and preventing the study of structures larger than the sample thickness, and requires high doses of electrons for imaging, leading to poor signal-to-noise ratios and damage minimization challenges.
Innovation Solution
The method involves taking images of the sample in a consistent orientation and removing surface layers between each image to track label positions, allowing for 3D reconstruction using either TEM or SEM, with the use of labels such as gold nanoparticles or luminescent labels to enhance contrast and reduce damage through lower energy particle beams.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If electron tomography is used to image samples, then high-resolution 3D reconstruction can be obtained, but the sample thickness must be extremely thin (less than 1 μm or even less than 100 nm)
Solution Approach 1:
The sample is conceptually divided into multiple thin layers along the beam direction. Each layer is imaged separately with a particle beam, and the images are then combined to reconstruct the 3D structure of the entire thicker sample. This segmentation allows the sample to exceed the thickness limit of conventional electron tomography while maintaining high resolution.
Solution Approach 2:
The invention transitions from conventional 2D projection imaging to 3D volumetric imaging by adding the depth dimension through sequential layer-by-layer imaging. Multiple images taken from different depth positions are integrated to create a comprehensive 3D reconstruction, enabling analysis of thicker samples in three dimensions.
2Measurement precision
If high doses of electrons are used for imaging in electron tomography, then image quality improves, but sample damage increases
Solution Approach 1:
The total electron dose is segmented and distributed across multiple imaging steps, with each thin layer receiving a lower electron dose. This distributes the cumulative damage across many small exposures rather than one high-dose exposure, reducing overall sample degradation while maintaining adequate signal quality.
Solution Approach 2:
Instead of applying a high electron dose to the entire thick sample at once, the method applies partial electron doses to successive thin layers. Each layer receives sufficient electrons for acceptable image quality, but the cumulative effect on any single region is reduced compared to imaging the entire sample simultaneously at high dose.
3Volume of moving object
If the sample thickness is increased to study larger structures, then the ability to study larger structures improves, but electron transmission decreases causing poor image quality
Solution Approach 1:
The thick sample containing large structures is segmented into multiple thin slices along the beam direction. Each slice is thin enough to allow good electron transmission and produce high-quality images, yet the collection of all slices collectively represents the complete large structure, enabling study of volumes exceeding conventional thickness limits.
Solution Approach 2:
The invention solves the volume limitation by transitioning from single-plane 2D imaging to multi-plane 3D imaging. By capturing images from multiple depth positions and integrating them, the method achieves volumetric imaging capability that preserves image quality while enabling study of larger overall structures through the additional depth dimension.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables the analysis of thicker samples with improved resolution and reduced damage by determining label positions in multiple layers and using lower energy particle beams, allowing for detailed 3D reconstruction of structures within biological samples.
Implementation Method 1
the labels are luminescent labels, the particle beam is a finely focused particle beam scanned over the surface of the sample and wherein the information comprises information proportional to the amount of light emitted from said labels in response to exposure to the particle beam
Implementation Method 2
As the interaction between the electrons passing through the sample and the atoms making up the sample is highly dependant on the number of protons Z of the atoms, especially local variations in atomic composition of the sample can be imaged in this way. As biological material comprises very little high-Z atoms, especially labelling with labels containing high-Z material is very effective.
Data Source
AI summary
The invention relates to a method in which labels are introduced in a sample, a flat surface is prepared on the sample and a series of images is made of the sample surface with e.g. a scanning electron microscope. The labels may be gold labels or e.g. fluorescent labels. By removing a surface layer between obtaining each image, labels at the surface in one image will be removed and will not be visible in a subsequent image. Thereby a 3D reconstruction of the position of labels in the sample can be made.