Dual-Axis Depletion Beams for 3D Super-Resolution Microscopy
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Solution Overview
Problem
Current super-resolution microscopy techniques face challenges in achieving high-resolution imaging in thick, optically inhomogeneous biological samples, particularly in deep imaging where resolution along the axial direction remains unchanged, and are not suitable for fast imaging or thick samples due to sensitivity to refractive index discrepancies and optical aberrations.
Innovation Solution
The method involves using two depletion beams with angularly inclined propagation axes intersecting at the focal point of the objective lens, allowing for effective fluorescence volume reduction in all three dimensions, achieved by adjusting the distance between the depletion beams and the objective lens, and using donut-shaped depletion beams to de-energize fluorophores through stimulated emission or triplet state relaxation, enabling high-resolution imaging beyond the diffraction limit in all spatial dimensions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of stationary object
If traditional SR microscopy techniques are used for deep imaging in thick samples, then imaging depth is increased, but resolution along the axial direction remains unchanged and sensitivity to optical aberrations increases
Solution Approach 1:
The patent divides the single depletion beam into two separate depletion beams with different propagation axes. Each beam independently contributes to axial resolution improvement, and their combined effect achieves superior 3D resolution throughout the sample depth without increasing sensitivity to optical aberrations
Solution Approach 2:
The patent introduces a second depletion beam with an angularly inclined propagation axis, adding a new dimensional component to the depletion process. This dual-beam configuration enables effective axial resolution enhancement while maintaining robustness against optical aberrations in thick samples
2Length of stationary object
If traditional SR microscopy techniques are used for deep imaging in thick samples, then imaging depth is increased, but sensitivity to optical aberrations increases
Solution Approach 1:
The patent creates different local depletion conditions by using two beams with distinct propagation axes. The angular inclination allows each beam to independently address local optical variations, reducing cumulative sensitivity to aberrations while maintaining effective depletion throughout the sample depth
Solution Approach 2:
The patent converts the potential harmful effect of optical aberrations in thick samples into a benefit by using the angularly inclined dual-beam configuration. The separated propagation paths allow the system to tolerate and compensate for aberrations that would otherwise degrade image quality in traditional single-beam approaches
3Device complexity
If single depletion beam is used, then device complexity is reduced, but 3D resolution enhancement is insufficient
Solution Approach 1:
The patent segments the depletion function into two separate beams, each with its own propagation axis. This segmentation enables comprehensive 3D resolution enhancement by addressing both lateral and axial dimensions effectively, while the modular beam configuration allows for relatively simple implementation using standard optical components
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 enhances optical resolution both laterally and axially, reduces sensitivity to optical aberrations, and allows for deep imaging in thick, optically inhomogeneous samples, facilitating easy implementation of 3D SR microscopy without requiring additional mirrors or sequential image acquisition.
Implementation Method 1
de-energize fluorophores through stimulated emission or triplet state relaxation
Implementation Method 2
donut-shaped depletion beams to de-energize fluorophores
Data Source
Figure 1~2
Figure 3~4
Figure 5a~5b
AI summary
Lightening method of at least one biological sample (S), in which said at least one biological sample includes at least one or more fluorophores, at the focal point (F) of at least one objective lens (L) having a main optical axis (z-z), the method comprising the following operational steps:- lightening (step 10) said at least one biological sample (S) with at least one excitation beam (EB), which propagates between said at least one objective lens (L) and said at least one biological sample (S) along at least one first propagation axis (a-a);- lightening (step 20) said at least one biological sample (S) with at least two depletion beams (DB, DB'), which propagate between said at least one objective lens (L) and said at least one biological sample (S) along the respective second propagation axes (b-b, b'-b'), said depletion beams being donut-shaped, each one in a plane orthogonal to the respective second propagation axis (b-b, b'-b'); whereby said at least one first propagation axis (a-a) and said at least second propagation axes (b-b, b'-b') are angularly inclined with each other, and said at least one first propagation axis (a-a) and said second propagation axes (b-b, b'-b') intersect on said at least one biological sample (s) only at the focal point (F) of said at least one objective lens (L), so that an effective fluorescence volume (FV) is generated in said at least one biological sample (S) which is limited both orthogonally and axially with respect to said main optical axis (z-z).