Critical Angle Determination in TIRF Microscopy
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Solution Overview
Problem
Existing methods for determining the critical angle of total internal reflection in TIRF microscopy are not easily implementable and require additional optical elements, lacking robustness and efficiency.
Innovation Solution
A method that illuminates a sample with an excitation light beam, captures images at various angles of incidence, and determines the critical angle through contrast analysis of these images, using existing image sensors without additional optical elements, allowing for automatic or semi-automatic operation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If additional optical elements are used to determine the critical angle, then measurement precision is improved, but device complexity increases
Solution Approach 1:
The image sensor serves dual purposes: capturing sample images for scientific analysis and simultaneously providing the data needed to determine the critical angle. The system uses its own existing components (image sensor, illumination beam, sample holder) to perform the critical angle measurement without requiring external specialized optical elements, thereby achieving self-service and eliminating the need for additional optical components.
2Measurement precision
If additional optical elements are used to determine the critical angle, then measurement precision is improved, but ease of operation deteriorates
Solution Approach 1:
The system automatically determines the critical angle by analyzing images captured by the existing image sensor. The processor automatically calculates the critical angle from the captured images using the relationship between illumination angle and image brightness/contrast, eliminating the need for manual operation of additional optical elements and simplifying the user workflow.
Solution Approach 2:
The system captures images at varying illumination angles and uses the image brightness and contrast information as feedback to determine the critical angle. The processor analyzes the relationship between the illumination angle and the captured image characteristics to automatically identify the critical angle, creating a closed-loop measurement process that improves ease of operation.
3Reliability
If pupil plane evaluation is used to increase robustness, then reliability is improved, but device complexity increases
Solution Approach 1:
The existing image sensor, which is already part of the microscope system for sample imaging, is used to capture the images needed for critical angle determination. The system leverages its own imaging capability to perform the robustness verification by comparing images at different illumination angles, eliminating the need for separate pupil plane imaging elements while maintaining reliability.
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 robust and efficient determination of the critical angle without additional optical elements, utilizing existing image sensors to analyze image contrast and intensity ratios, thereby improving the precision and simplicity of the process.
Implementation Method 1
an excitation light beam is typically totally reflected at a boundary from a coverslip to a sample to be examined. This creates a so-called evanescent (decaying) field in the sample
Implementation Method 2
The excitation light beam is refracted at the interface between the surrounding medium (e.g. air) and the immersion oil and then strikes an interface between the specimen slide and the sample
Data Source
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AI summary
Methods and devices for determining a critical angle of total internal reflection, particularly in TIRF microscopy, are provided. For this purpose, a sample (15) is illuminated at an angle of incidence (117) and an image of the sample is recorded, for example, using an image sensor (110). Based on images taken at different illumination angles, the critical angle is then determined, for example, by contrast analysis.