Total Internal Reflection Microscopy for Large-Sample 3D Layer Imaging
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Solution Overview
Problem
Conventional microscopy techniques struggle to achieve high axial and lateral resolution for large biological samples, often requiring sectioning and fixation which alters sample morphology and limits the visualization of complex internal structures.
Innovation Solution
A method and system using total internal reflection microscopy with a light coupling medium having a refractive index higher than the sample, allowing imaging of consecutive top layers via an evanescent field, followed by layer removal and image combination to generate a three-dimensional image.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If physical slicing methods are used to image large samples, then axial resolution is improved, but sample morphology is altered and processing complexity increases
Solution Approach 1:
The patent applies segmentation by dividing the large sample into multiple sequential layers that are imaged individually through the light coupling medium. Each layer is captured separately and then computationally reconstructed into a complete 3D image, avoiding the need for physical slicing while maintaining axial resolution.
Solution Approach 2:
The patent introduces a light coupling medium as an intermediary between the objective lens and the sample surface. This medium enables optical access to deep layers of large samples without requiring physical sectioning, thereby preserving sample morphology while achieving high axial resolution through optical sectioning.
2Ease of operation
If physical slicing and fixation are applied to prepare samples, then imaging is enabled, but native structures are altered
Solution Approach 1:
The patent replaces mechanical slicing and fixation methods with an optical approach using total internal reflection microscopy. The light coupling medium enables optical sectioning and imaging of intact sample layers without mechanical intervention, preserving the native structure and composition of the sample.
3Length of stationary object
If longer wavelengths are used to image thicker samples, then penetration depth is improved, but lateral resolution deteriorates
Solution Approach 1:
The patent changes the illumination parameter by using evanescent wave excitation at the interface between the light coupling medium and sample. This creates an exponentially decaying light field that provides both deep penetration into the sample and high lateral resolution, overcoming the trade-off between penetration depth and resolution associated with longer wavelengths.
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 high axial and lateral resolution imaging of large samples up to millimeters or centimeters thick, preserving native structures and reducing bleaching, with improved resolution through evanescent field excitation and super resolution techniques.
Implementation Method 1
excitation energy for imaging the top layer is provided via an evanescent field formed in the top layer of the sample at the interface with the light coupling medium
Implementation Method 2
wherein excitation energy for imaging the top layer is provided via an evanescent field formed in the top layer of the sample at the interface with the light coupling medium
Data Source
Figure 1
Figure 2a~2d
Figure 3
AI summary
A method for total internal reflection microscopy imaging of a sample and a system for total internal reflection microscopy imaging to carry out the method steps, are described, the method comprising the steps of (a) providing the sample; (b) placing a light coupling medium over a surface of the sample, the light coupling medium having a light incoupling portion and a light outcoupling portion, wherein the light outcoupling portion of the light coupling medium is in optical contact with the surface of a top layer of the sample; (c) imaging the top layer of the sample through the light coupling medium, to obtain an image of the top layer; (d) removing at least partially the top layer, thereby revealing a new top layer of the sample; and repeating steps (b) to (d) for one or more new top layers; wherein the light coupling medium has a refractive index of at least 1.4, and wherein the refractive index of the light coupling medium is higher than a refractive index of the sample; wherein excitation energy for imaging the top layer is provided via an evanescent field formed in the top layer of the sample at the interface with the light coupling medium; and wherein combining the obtained images generates a three-dimensional image of the sample.