Biplane Microscopy System for 3D Nanometer Tracking
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Solution Overview
Problem
Current microscopy techniques face limitations in achieving 3D imaging with sub-100 nanometer resolution and temporal resolution below 1 millisecond, constraining the visualization of dynamic sub-cellular processes and spatial localization accuracy.
Innovation Solution
A biplane microscopy system that simultaneously detects luminescence light from two focal planes, allowing for 3D imaging and particle tracking with nanometer accuracy and sub-millisecond temporal resolution, using a sample stage, activation and readout light sources, beam splitting, and a camera to create 3D data sets and track particles in real-time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional microscopy techniques are used, then imaging is simpler and faster, but spatial resolution is limited to 200-250 nanometers in the focal plane
Solution Approach 1:
The patent divides the imaging system into multiple independent modules: illumination system with multiple wavelengths, detection system with multiple cameras, and control system. Each module can be optimized independently while working together to achieve super-resolution 3D imaging, resolving the contradiction between enhanced measurement precision and device complexity
Solution Approach 2:
The patent transitions from 2D conventional microscopy to 3D super-resolution imaging by introducing axial detection capability through multiple detection planes. This dimensional expansion enables sub-100 nanometer resolution in three dimensions while maintaining systematic control through the multi-module architecture
2Measurement precision
If axial scanning is performed to achieve 3D imaging, then spatial resolution improves, but temporal resolution deteriorates to above 1 millisecond
Solution Approach 1:
The patent pre-configures multiple detection planes at different axial positions before imaging begins. This eliminates the need for sequential axial scanning during image acquisition, allowing simultaneous capture of 3D information and achieving temporal resolution below 1 millisecond while maintaining sub-100 nanometer axial resolution
Solution Approach 2:
The patent implements continuous 3D imaging by maintaining all detection planes active simultaneously during the entire acquisition process. This continuous multi-plane detection eliminates temporal gaps between axial scans, achieving both high axial resolution and sub-millisecond temporal resolution for dynamic biological processes
3Measurement precision
If photo-sensitive probes are used for localization, then spatial resolution improves to sub-100 nanometers, but the complexity of probe preparation and activation increases
Solution Approach 1:
The patent employs photo-sensitive probes with multiple functional states: activation at specific wavelengths, emission at different colors, and switchable on/off states. This multi-functionality allows a single probe type to enable various imaging modes (multi-color, super-resolution, 3D) without requiring different probe preparations, reducing overall complexity while achieving sub-100 nanometer localization accuracy
Solution Approach 2:
The patent exploits changes in probe parameters (fluorescence wavelength, activation state, emission intensity) through controlled photo-physical transitions. By manipulating these parameters via specific illumination wavelengths, the system achieves high localization accuracy without complex probe chemistry, resolving the contradiction between measurement precision and preparation complexity
4Adaptability or versatility
If multiple detection planes are implemented simultaneously, then 3D imaging capability improves, but the complexity of data processing and analysis increases
Solution Approach 1:
The patent incorporates feedback mechanisms in the control system that use detected signals from multiple planes to automatically adjust illumination, synchronize camera acquisition, and guide data reconstruction. This closed-loop control simplifies the processing of multi-plane data by coordinating acquisition parameters and enabling automated 3D reconstruction algorithms
Solution Approach 2:
The patent introduces intermediate processing layers including reference plane detection for alignment, calibration samples for parameter determination, and software intermediaries for coordinate transformation. These intermediaries bridge the complexity of multi-plane data by providing systematic methods for registration, reconstruction, and analysis
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
Enables 3D imaging with a resolvable volume 100-fold smaller than conventional microscopy and achieves unprecedented temporal and spatial resolution, allowing for the visualization of dynamic sub-cellular processes with improved accuracy and speed.
Implementation Method 1
illuminating a sample with an activation light activates probes of at least one probe subset of photo-sensitive luminescent probes
Implementation Method 2
illuminating the sample with a readout light causes luminescence light from the activated probes
Data Source
AI summary
A microscopy system is configured for creating 3D images from individually localized probe molecules. The microscopy system includes a sample stage, an activation light source, a readout light source, a beam splitting device, at least one camera, and a controller. The activation light source activates probes of at least one probe subset of photo-sensitive luminescent probes, and the readout light source causes luminescence light from the activated probes. The beam splitting device splits the luminescence light into at least two paths to create at least two detection planes that correspond to the same or different number of object planes of the sample. The camera detects simultaneously the at least two detection planes, the number of object planes being represented in the camera by the same number of recorded regions of interest. The controller is programmable to combine a signal from the regions of interest into a 3D data.