TIRFM Microscope Spatial Filter for Light Utilization
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing TIRFM microscopes face inefficiencies with incoherent light sources, where only a small portion of emitted light is utilized due to poor etendue matching, and laser sources are costly with potential image disruptions from coherence effects.
Innovation Solution
A TIRFM-capable microscope with a spatial filter device configured to project two-dimensional patterns onto the excitation light, allowing for adjustable annular or circular patterns to control evanescent field penetration and switch between TIRF and epi-illumination modes, using incoherent light sources with programmable spatial light modulators to optimize light usage and reduce coherence issues.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If an incoherent light source is used with an annular aperture to achieve TIRF illumination, then the critical angle for total internal reflection is obtained, but a large portion of the emitted light is not utilized due to poor etendue matching
Solution Approach 1:
The patent employs a spatial light modulator that can dynamically change the transmission pattern from an annular pattern (for TIRF illumination) to a circular pattern (for epifluorescence illumination). This dynamic adaptability allows the system to optimize light utilization for different imaging modes, resolving the contradiction between achieving critical angle illumination and maximizing light usage efficiency.
2Loss of energy
If a laser light source is used to improve light utilization efficiency, then almost all emitted light reaches the sample interface, but the cost increases and diffraction and interference effects disrupt the image
Solution Approach 1:
The patent changes the coherence parameter of the light source by using an incoherent LED light source instead of a coherent laser source. Combined with the spatial light modulator that shapes the illumination pattern, this achieves sufficient light directionality without the harmful diffraction and interference effects of coherent light, while maintaining cost-effectiveness.
3Measurement precision
If the spatial filter device is configured for TIRF illumination with an annular pattern, then high-resolution imaging is achieved, but the microscope cannot operate in epifluorescence mode
Solution Approach 1:
The spatial light modulator is configured to switch between different transmission patterns: an annular pattern for TIRF illumination (providing high spatial resolution) and a circular pattern for epifluorescence illumination (providing mode versatility). This dynamic reconfiguration capability resolves the contradiction between achieving high resolution and maintaining operational flexibility.
4Adaptability or versatility
If the spatial filter device is configured for epifluorescence illumination with a circular pattern, then operational versatility is improved, but TIRF illumination capability is lost
Solution Approach 1:
The spatial light modulator enables dynamic switching between operational modes by changing the transmission pattern. When epifluorescence illumination is needed, it switches to a circular pattern for versatility; when TIRF illumination is needed, it switches to an annular pattern for high spatial resolution. This resolves the contradiction between versatility and resolution by making both achievable at different times.
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 configuration enhances light yield and flexibility, allowing for high-resolution TIRF imaging while maintaining efficient light usage and minimizing disruptions, enabling operation as both TIRF and epifluorescence microscopes with adjustable penetration depth and spatial resolution.
Implementation Method 1
Total internal reflection fluorescence microscopy (TIRFM), i.e., microscopy with TIRF illumination, involves the excitation of a fluorescence of a specimen or a sample using an evanescent field. To generate the evanescent field, light is totally reflected on the inside of a reflective element, for example a cover glass, at the interface to the sample.
Implementation Method 2
This takes advantage of the fact that light in a medium with a higher refractive coefficient n1 incident at a shallow angle on an interface with a medium with a lower refractive coefficient n2 is totally reflected when this angle of incidence θ1, calculated from the normal to the interface, exceeds a critical angle
Implementation Method 3
a light field forms in the sample on the glass beyond the interface, which light field exponentially evanesces perpendicularly to the interface, with a typical penetration depth for visible light of 100-200 nm
Implementation Method 4
If fluorescing molecules that can absorb light of the radiated wavelength are located in this region, they are excited to emit fluorescent light. Such fluorescent light is known as total internal reflection fluorescence (TIRF).
Data Source
AI summary
A TIRFM-capable microscope including: a light source that generates/emits incoherent excitation light onto an optical path that includes a first projection lens system, a spatial filter, a second projection lens system and an objective. The TIRFM-capable microscope also includes a controller; wherein the first projection lens system projects excitation light onto the spatial filter that filters the excitation light with two-dimensional patterns, the spatial filter lies in a plane conjugate to a back focal plane of the objective which includes an objective lens that directs excitation light onto and receives fluorescent light from the sample, wherein, for a numerical aperture NAObj of the objective and a refractive index nspec of the sample NAObj>nspec, and the controller activates the spatial filter to select/generate various two-dimensional patterns and selects/adjusts the position/shape/size of the pattern such that TIRF illumination of the sample is generated.