Serial Block Face Imaging Artifact Compensation
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Solution Overview
Problem
Serial block face imaging techniques using particle-optical apparatuses with ultramicrotomes face challenges such as sample deformation, lateral shifts, surface roughness, and charging issues, which lead to artifacts and distortions in 3D reconstructions.
Innovation Solution
A method involving the use of a particle-optical apparatus with a movable sample stage and a microtome for precise cutting, where images are captured with different acceleration voltages to detect and compensate for lateral shifts, and additional steps to address surface roughness and charging, including local ionization to neutralize the sample surface.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a microtome blade is used to cut sample layers during serial block face imaging, then sequential imaging of sample surfaces can be performed to create 3D reconstructions, but lateral forces from the cutting blade cause sample blocks to shift or deform laterally, impairing the 3D reconstruction quality
Solution Approach 1:
The patent replaces the mechanical microtome cutting system with a focused ion beam (FIB) system that removes sample material through ion bombardment and sputtering. This substitution eliminates the lateral mechanical forces that cause sample shift and deformation, while still enabling sequential removal and imaging of sample layers for 3D reconstruction.
Solution Approach 2:
The patent introduces a detector system that monitors charging effects on the sample surface during FIB cutting and imaging. This intermediary detection mechanism allows for real-time identification of charging artifacts, enabling compensation measures to be taken that prevent degradation of imaging quality despite the non-conductive nature of resin-embedded samples.
2Loss of information
If the sample surface is imaged repeatedly during serial block face imaging, then 3D reconstruction data can be accumulated, but the sample surface becomes charged due to poor electrical conductivity of resin, leading to charging-governed signal amplification and image drift
Solution Approach 1:
The patent introduces a detector system that monitors charging effects on the sample surface during FIB cutting and imaging. This intermediary detection mechanism allows for real-time identification of charging artifacts, enabling compensation measures to be taken that prevent degradation of imaging quality despite the non-conductive nature of resin-embedded samples.
Solution Approach 2:
The patent employs low-vacuum or variable pressure conditions in the imaging chamber, allowing controlled introduction of gas molecules that can neutralize surface charges on the non-conductive resin sample. This parameter change in chamber pressure enables repeated imaging without accumulating charging effects that would cause image drift or signal amplification artifacts.
3Speed
If high acceleration voltage is used to image sample layers, then imaging speed and penetration depth are improved, but sample deformation and charging effects are exacerbated
Solution Approach 1:
The patent employs low-vacuum or variable pressure conditions in the imaging chamber, allowing controlled introduction of gas molecules that can neutralize surface charges on the non-conductive resin sample. This parameter change in chamber pressure enables repeated imaging without accumulating charging effects that would cause image drift or signal amplification artifacts.
Solution Approach 2:
The patent replaces the mechanical microtome cutting system with a focused ion beam (FIB) system that removes sample material through ion bombardment and sputtering. This substitution eliminates the lateral mechanical forces that cause sample shift and deformation, while still enabling sequential removal and imaging of sample layers for 3D reconstruction.
4Ease of operation
If the microtome blade is not oriented exactly at right angles to the optical axis, then cutting can still proceed, but the sample surface becomes inclined relative to the optical axis, causing sample regions to be out of focus and imaged unsharply
Solution Approach 1:
The patent replaces the mechanical microtome cutting system with a focused ion beam (FIB) system that removes sample material through ion bombardment and sputtering. This substitution eliminates the lateral mechanical forces that cause sample shift and deformation, while still enabling sequential removal and imaging of sample layers for 3D reconstruction.
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 method effectively identifies and compensates for artifacts, improving the accuracy and quality of 3D reconstructions by minimizing sample deformation, lateral shifts, and charging effects, thereby enhancing the reliability of the imaging process.
Implementation Method 1
The particles of a primary particle beam are directed onto a sample, wherein the particles of the primary particle beam interact with the sample material, so that interaction products are released from the sample
Implementation Method 2
the outermost sample layer is removed with the cutting device, in that a sample layer is thereby uncovered
Implementation Method 3
introducing gas locally onto the sample surface, in that the gas is thereby ionized and the charges on the sample surface are compensated for
Data Source
AI summary
A method includes capturing a first image of the sample via a detector, wherein the particles of the primary particle beam have a first average energy so that the interaction products detected by the detector predominantly contain sample information from a sample layer lying below the sample surface. The method also includes removing the outermost sample layer with the aid of the cutting device, and capturing a second image of the sample via the detector, wherein the particles of the primary particle beam have a second average energy so that the interaction products detected by the detector predominantly contain sample information from the surface layer of the sample. The method further includes calculating the lateral shift/lateral offset of the sample from a comparison of the first and second images, and compensating for the lateral offset.