Wavefront Matching Optic for Inverted Light Sheet Microscopy
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Solution Overview
Problem
High scattering in dense tissue limits the quality of fluorescence images obtained through microscopy, particularly due to lateral aberrations and reduced spatial resolution, which existing confocal techniques attempt to mitigate at the cost of signal intensity and increased image collection times.
Innovation Solution
A system employing structured illumination with a spatially modulated imaging field, adjusted frequency, and a wavefront matching optic to reduce scattering interference and enhance resolution, utilizing a spatial light modulator and optical elements to separate and combine beams, and a sample interface for inverted light sheet microscopy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If confocal fluorescence techniques are used to reduce scattering interference, then image quality is improved, but signal intensity is reduced
Solution Approach 1:
A wavefront matching optic is introduced as an intermediary element between the illumination objective and the sample. This optic has a center of curvature positioned beyond the flat side, creating a curved wavefront that matches the curvature of the sample interface. This matching eliminates refraction-induced aberrations at the interface, allowing high-NA illumination without the lateral aberrations that normally reduce signal intensity at image edges.
Solution Approach 2:
The system changes the wavefront curvature parameter by using a wavefront matching optic with a specific center of curvature position. This parameter change allows the illumination to maintain high intensity across the entire field of view by compensating for the curvature mismatch that would otherwise cause signal loss at the edges of the imaging field.
2Manufacturing precision
If high numerical aperture (NA) is used in high magnification systems, then spatial resolution is improved, but lateral aberrations increase at the edge of images
Solution Approach 1:
The wavefront matching optic serves as a mediator that corrects the mismatch between the planar wavefront from the objective and the curved sample interface. By introducing this intermediary element with appropriate curvature, the system maintains high NA illumination without the lateral aberrations that would otherwise degrade image quality at the edges.
3Area of stationary object
If conventional microscopy is used for deep tissue imaging, then field of view is reduced due to scattering, but image collection time increases when using compensation mechanisms
Solution Approach 1:
The invention replaces mechanical scanning compensation mechanisms with an optical solution. Instead of using moving parts to correct lateral aberrations, the wavefront matching optic provides static optical correction, eliminating the need for convolved scanning mechanisms and reducing image collection time while maintaining a large field of view.
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 improves image resolution and contrast by effectively reducing scattered light and out-of-focus signals, enabling high-speed acquisition of detailed images with lower photo-bleaching, suitable for thick specimens and volumetric reconstructions.
Implementation Method 1
wavefront matching optic having a center of curvature beyond a plane forming a flat side of the wavefront matching optic
Implementation Method 2
The wavefront matching optic has a center of curvature beyond the flat side forming a plane of the wavefront matching optic
Implementation Method 3
an illumination objective configured to form an illumination field on a sample by directing two optical beams through the wavefront matching optic
Implementation Method 4
the illumination field includes a structured illumination field having a spatial frequency on the incident plane
Implementation Method 5
a microscope objective configured to collect a fluorescent signal excited in the sample by the illumination field along a direction perpendicular to the incident plane
Implementation Method 6
spatial frequency modulation stage to adjust the frequency of the spatially modulated imaging field
Data Source
AI summary
A system, including a structured illumination stage to provide a spatially modulated imaging field is provided. The system further includes a spatial frequency modulation stage to adjust the frequency of the spatially modulated imaging field, a sample interface stage to direct the spatially modulated imaging field to a sample, and a sensor configured to receive a plurality of fluorescence emission signals from the sample. The system also includes a processor configured to reduce a sample scattering signal and to provide a fluorescence emission signal from a portion of the sample including the spatially modulated imaging field. A method for using the above system to form an image of the sample is also provided.