Optimized photon collection for light-sheet microscopy
Inventors
Truong, Thai • Keomanee-Dizon, Kevin • Fraser, Scott E.
Assignees
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Publication Number
US-12411088-B2
Publication Date
2025-09-09
Expiration Date
Abstract
Systems and methods are provided for increasing photon collection during imaging with a light-sheet fluorescence microscope. In one example, one or more adaptive optical elements may be positioned in a detection light path between a detection objective and an imaging sensor. A depth of field of the detection objective is adjusted as a function of a thickness of an excitation light-sheet used to illuminate a sample during imaging. As a result, the detection objective captures more fluorescence photons generated by light-sheet excitation.
Core Innovation
The invention relates to light-sheet fluorescence microscopy, including single/selective plane illumination microscopy, where a sample is illuminated with an excitation light-sheet and fluorescence is received by a detection objective. The approach improves photon collection by extending the depth of field (DOF) of a high numerical aperture detection objective so that it matches the excitation light-sheet thickness.
The excitation light-sheet thickness is measured, for example using full-width at half-maximum (FWHM), and the detection objective DOF is adjusted based on that measured thickness. Adaptive optical elements are placed in the detection-path conjugate/pupil planes, and are configured to adjust the detection DOF so that it is equal to or greater than the excitation light-sheet thickness, thereby capturing fluorescence across the entire light-sheet in one shot.
The document further describes microscope system implementations and imaging modes that use adaptive optics to maintain coverage while performing multi-color adjustment and intensity/threshold-based switching, as well as static versus dynamic DOF adjustment when the light-sheet thickness varies. Optional axial scanning via remote refocusing is also described, and experimental results are provided using larval zebrafish calcium functional imaging with an annular phase mask DOF extension.
Claims Coverage
The partial content identifies three independent claims: a method (clm-00001), a microscope system (clm-00017), and a light-sheet microscope system (clm-00026). Across these, the claims share the same core inventive feature: adjusting a detection-objective depth of field according to a measured excitation light-sheet thickness using adaptive optical elements, including specified quantitative relationships in some dependent claim refinements.
Depth of field adjusted according to excitation light-sheet thickness
Illuminating a sample with an excitation light-sheet; adjusting a depth of field of a detection objective according to a measure of a thickness of the excitation light-sheet; receiving, at an imaging sensor, fluorescence signal from the sample via the detection objective; and generating the image of the sample according to the fluorescence signal.
Adaptive optics adjusting detection depth of field according to excitation light-sheet thickness
A microscope system comprising an illumination system for generating an excitation light-sheet; a detection objective positioned to receive photons; at least one adaptive optical element for adjusting a depth of field of the detection objective; and a controller configured to adjust the depth of field via the adaptive optical element according to a thickness of the excitation light-sheet.
Detection system with adaptive optical elements for depth-of-field adjustment based on excitation light-sheet thickness
A light-sheet microscope system comprising at least one detection system including a detection objective, one or more optical elements, and one or more adaptive optical elements; at least one illumination system for generating an excitation light-sheet; and an imaging sensor acquiring imaging data via the detection system; wherein the adaptive optical elements are configured to adjust a depth of field of the objective according to a thickness of the excitation light-sheet.
All independent claims are directed to light-sheet microscopy where fluorescence is detected while a detection objective’s depth of field is adjusted based on the excitation light-sheet thickness, enabled by adaptive optical elements under control or as part of the detection system.
Stated Advantages
Improved photon collection by extending the depth of field of a high-NA detection objective to match the excitation light-sheet thickness.
Capturing fluorescence across the entire light-sheet in one shot.
Higher signal-to-noise ratio and contrast.
Faster acquisition with shorter exposures.
Reduced photobleaching and photodamage.
Demonstrated increased number of active neurons captured (e.g., 46% more active neurons captured with annular phase mask DOF extension).
Documented Applications
Light-sheet fluorescence microscopy for calcium functional imaging in larval zebrafish, including capturing active neurons using adaptive DOF extension.
Multi-color imaging adjustment using adaptive optics to maintain detection DOF coverage.
Axial scanning/volumetric imaging by optional axial scanning via remote refocusing while maintaining the adjusted depth of field.
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