Adaptive SMLM Illumination for Uniform Molecule Localization
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Solution Overview
Problem
Existing single molecule localization microscopy (SMLM) techniques face challenges in achieving uniform spatial resolution due to heterogeneous fluorescent molecule densities, leading to image quality issues such as overlapping point spread functions (PSFs) and non-uniform illumination, which affects the precision of molecule localization.
Innovation Solution
Adapting the irradiance of the illuminating light beam based on the local density of fluorescent molecules within the sample, using a scanning unit that adjusts the beam diameter to optimize scanning efficiency and minimize overlapping PSFs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Area of stationary object
If uniform illumination is used to illuminate the sample, then the field of view is widened, but the spatial resolution becomes non-uniform across regions with different molecule densities
Solution Approach 1:
The patent applies local quality by adapting the irradiance of the illumination beam to match the local density of fluorescent molecules in different regions of the sample. High-density regions receive lower irradiance to prevent overlapping PSFs, while low-density regions receive higher irradiance to ensure sufficient signal, thereby achieving uniform spatial resolution across the entire field of view.
Solution Approach 2:
The patent implements dynamics by making the illumination irradiance adjustable and adaptable based on local molecule density. The system dynamically modifies the irradiance distribution across the field of view to optimize the single-molecule emission condition in each region, transitioning from static uniform illumination to dynamic localized illumination.
2Quantity of substance
If high irradiance is used to illuminate regions with high molecule density, then more molecules can be detected, but overlapping PSFs occur reducing localization precision
Solution Approach 1:
The patent applies local quality by tailoring the irradiance level to the specific local density of fluorescent molecules. In high-density regions, lower irradiance is applied to maintain the single-molecule emission condition and prevent overlapping PSFs, thereby preserving localization precision while still detecting a sufficient number of molecules.
Solution Approach 2:
The patent implements parameter changes by adjusting the irradiance parameter based on local molecule density. The system modifies the illumination intensity to optimize the balance between detecting enough molecules and maintaining precise localization, adapting the irradiance level to each region's specific characteristics.
3Measurement precision
If low irradiance is used to avoid overlapping PSFs in high density regions, then localization precision is maintained, but scanning efficiency decreases
Solution Approach 1:
The patent applies local quality by optimizing the irradiance level for each specific region based on its molecule density. Low-density regions receive higher irradiance to maximize signal intensity and scanning efficiency, while high-density regions receive lower irradiance to maintain localization precision, thereby achieving optimal performance across the entire sample.
Solution Approach 2:
The patent implements dynamics by making the irradiance level adaptive rather than static. The system dynamically adjusts the illumination intensity according to the local density distribution, allowing high scanning efficiency in low-density regions while maintaining precision in high-density regions, thus optimizing overall productivity.
4Device complexity
If Gaussian-shaped illumination is used, then the system is simple to implement, but the excitation is non-uniform limiting the exploitable field of view
Solution Approach 1:
The patent applies local quality by creating a non-uniform irradiance distribution that is optimized for the specific sample characteristics. Instead of using simple Gaussian illumination, the system implements a tailored illumination profile that provides uniform excitation across the field of view by compensating for the heterogeneous density distribution of fluorescent molecules.
Solution Approach 2:
The patent implements dynamics by making the illumination profile adaptable to the sample's density distribution. The system dynamically adjusts the spatial distribution of irradiance to optimize both the field of view and the uniformity of excitation, transitioning from a fixed Gaussian profile to a dynamically optimized illumination pattern.
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 approach enables precise localization of single fluorescent molecules across varying densities, improving image quality and scanning efficiency by ensuring only one molecule emits per focal volume, reducing artifacts and enhancing super-resolution imaging.
Implementation Method 1
scanning the sample using a light beam at a first wavelength having a predetermined irradiance... acquiring a first image and a second image at a second wavelength
Data Source
AI summary
An apparatus for localizing single fluorescent molecules comprised in a sample using single molecule localization microscopy. The apparatus comprises a scanning unit configured to scan the sample using a light beam at a first wavelength having a predetermined irradiance, an image acquisition unit configured to acquire a first image and a second image at a second wavelength, a localization unit configured to localize the single fluorescent molecules in the second image. Additionally, the apparatus comprises a determination unit configured to determine, based on the first image, one or more local densities of the single fluorescent molecules; and a calculation unit configured to calculate one or more local irradiances of the light beam as a predetermined function of each one of the one or more local densities of the single fluorescent molecules. The scanning unit is further configured to scan the sample using the one or more local irradiances of the light beam.