Structured Plane Illumination Microscopy Using Bessel Beams
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Solution Overview
Problem
Current microscopy techniques, such as widefield and confocal microscopy, face limitations in confining excitation to the focal plane, leading to photodamage and photobleaching, while also being wasteful of fluorescence and introducing out-of-focus background noise, which hinders high-resolution imaging of thick specimens.
Innovation Solution
The use of Bessel-like beams in structured plane illumination microscopy, which generate a coherent light sheet that is decoupled from its thickness, allowing for efficient illumination of large fields of view with minimal photodamage and background noise by creating a thin sheet of excitation light that is confined to the focal plane.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If widefield or confocal microscopy is used to illuminate the entire specimen, then fluorescence excitation is achieved across all planes, but photodamage and photobleaching increase and out-of-focus background noise is introduced
Solution Approach 1:
The illumination is segmented into multiple Bessel-like beams arranged in a periodic pattern, where each beam independently illuminates a specific region of the specimen. This segmentation allows the excitation to be confined to the focal plane while maintaining widefield detection capabilities, thereby reducing photodamage and photobleaching compared to uniform illumination of the entire specimen.
Solution Approach 2:
The patent applies local quality by creating structured illumination patterns where different regions of the specimen receive tailored illumination. The Bessel-like beams provide localized excitation with extended depth of field, concentrating the excitation energy where needed while leaving other regions unilluminated, thus reducing overall photodamage while maintaining high-resolution imaging in the focal plane.
2Area of stationary object
If conventional light sheets are used for illumination, then out-of-focus background is reduced, but the light sheet thickness is coupled to its length, limiting the field of view
Solution Approach 1:
The patent transitions from conventional light sheets to Bessel-like beams, which fundamentally changes the illumination geometry. Bessel-like beams propagate over extended distances while maintaining a constant transverse profile, effectively decoupling the illumination length from the beam width. This dimensional transformation allows for a large field of view without compromising light sheet thickness control.
Solution Approach 2:
The patent changes the fundamental parameters of the illumination by using Bessel-like beams instead of conventional Gaussian light sheets. The Bessel beam profile, characterized by its non-diffracting nature and extended depth of field, allows independent control of beam width and propagation length through parameter adjustment, enabling both large field of view and precise thickness control simultaneously.
3Measurement precision
If two-photon fluorescence excitation is used to restrict excitation to the focal plane, then photodamage is reduced, but high intensities required cause nonlinear photodamage mechanisms
Solution Approach 1:
The patent substitutes the nonlinear two-photon excitation mechanism with linear fluorescence excitation using Bessel-like beams. By replacing the intensity-dependent nonlinear process with a linear excitation process, the system achieves focal plane confinement through the extended depth of field of Bessel beams without requiring high intensities, thereby avoiding nonlinear photodamage mechanisms while maintaining excitation confinement.
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 high-resolution imaging with reduced photobleaching and phototoxicity, allowing for extended observations of living cells with isotropic resolution and high volumetric frame rates, while maintaining efficient use of the photon budget.
Implementation Method 1
beam-forming optics configured for receiving the generated light beam and for generating a plurality of substantially parallel Bessel-like beams directed into a sample
Implementation Method 2
a) confine excitation predominantly to the focal plane of imaging optics, to reduce photodamage and photobleaching
Data Source
AI summary
An apparatus includes a light source configured for generating a coherent light beam having a wavelength, λ, a light detector, and beam-forming optics configured for receiving the generated light beam and for generating a plurality of substantially parallel Bessel-like beams directed into a sample in a first direction. Each of the Bessel-like beams has a fixed phase relative to the other Bessel-like beams. Imaging optics are configured for receiving light from a position within the sample that is illuminated by the Bessel-like beams and for imaging the received light onto the detector. The imaging optics include a detection objective having an axis oriented in a second direction that is non-parallel to the first direction, where the detector is configured for detecting light received by the imaging optics. A processor configured to generate an image of the sample based on the detected light.