STED Microscope Using First-Order Bessel Beam for Deep Imaging
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Solution Overview
Problem
Conventional STED microscopy experiences a rapid decrease in resolution at deep image depths due to spherical aberration and scattering distortions, requiring complex operations or expensive optics for correction.
Innovation Solution
Employing a first-order Bessel beam as the depletion beam, which maintains its shape and resolution due to its anti-diffracting and self-healing properties, combined with a confocal system and precise beam overlapping techniques, to achieve consistent super-resolution imaging deep within specimens.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a conventional Gaussian depletion beam is used in STED microscopy, then the system structure remains simple, but the imaging resolution rapidly decreases at deep image depths due to spherical aberration and scattering distortions
Solution Approach 1:
The patent transforms the conventional Gaussian beam profile into a Bessel beam profile by modifying the phase distribution and spatial characteristics of the depletion beam. This parameter change in beam shape enables the depletion beam to maintain its focal spot integrity at deep imaging depths, resolving the contradiction between simple system structure and stable beam shape.
2Measurement precision
If correction collar lens adjustment is used to improve deep imaging resolution, then resolution stability at 80-100 micron depth is achieved, but the operation becomes complex and unsuitable for highly scattering samples
Solution Approach 1:
The Bessel beam depletion mode inherently provides self-correcting properties for spherical aberration and scattering distortions through its mathematical structure and propagation characteristics. The system achieves deep imaging resolution without requiring manual correction collar adjustments, making the operation simple and suitable for highly scattering samples.
3Measurement precision
If self-adaptive optics system is used to compensate spherical aberration, then imaging resolution is improved, but the system becomes expensive and operationally complex
Solution Approach 1:
The Bessel beam depletion mode provides intrinsic compensation for spherical aberration through its mathematical structure, eliminating the need for expensive self-adaptive optics systems. The beam's self-healing property and extended depth of focus are achieved through simple optical elements, resolving the contradiction between resolution improvement and system complexity.
4Measurement precision
If a first-order Bessel beam is used as depletion beam, then constant imaging resolution is maintained deep in scattering specimens, but the system requires precise beam overlapping adjustment
Solution Approach 1:
The patent employs feedback mechanisms through beam combining optics and alignment procedures that allow precise overlapping of the Bessel beam depletion mode with the excitation beam. The confocal arrangement and beam combining system provide the necessary feedback for achieving and maintaining precise beam alignment, enabling constant resolution at depth while managing the operational complexity.
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
The method provides a simple, cost-effective solution for maintaining high resolution up to 155 μm deep, outperforming conventional STED microscopy by maintaining nearly constant resolution without the need for active adjustments or complex optics.
Implementation Method 1
The first-order Bessel beam through the Bessel generating system passes through the depletion beam focusing lens and the objective lens to produce a line-shaped focal spot along optical axial
Implementation Method 2
The other beam, called depletion beam, is a Gaussian beam modulated by 0-2π vortex phase-plate
Implementation Method 3
The depletion beam focusing lens and the objective lens satisfy the confocal condition
Implementation Method 4
It can generate a nearly Airy spot after the focus by the objective lens, which can produce fluorescent signals by a fluorescent material at the focus area
Implementation Method 5
In the ring area of high depletion intensity, the excited molecules jump back to the ground state via stimulated emission instead of spontaneous radiation
Data Source
AI summary
A stimulated-emission-depletion (STED) super-resolution microscope includes an excitation light source, a depletion light source, an excitation light expanded beam alignment system, a spiral-shaped phase plate, a Bessel beam generating system, a depletion light focus lens, a beam combination system, an objective lens, a piezoelectric scanning system, a filter, a signal collection system, and a single-photon detector. The depletion light can be a first-order Bessel beam. The depletion light has anti-scattering and self-healing characteristics, and is capable of keeping the spot shape at a deeper position of a sample, thereby improving image resolution in the deep region of the sample. Compared to conventional STED super resolution microscope of deep-layer imaging using an adjustable correction collar, the present invention is simpler in experimental operations and does not require active adjustments. Compared to adaptive optical systems, the present experimental apparatus is simpler and less expensive.