Resolution enhancement for light sheet microscopy systems and methods

Inventors

Shroff, Hari • Wu, Yicong • Abrahamsson, Sara

Assignees

US Department of Health and Human Services

Abstract

Embodiments of a resolution enhancement technique for a light sheet microscopy system having a three objective lens arrangement in which one objective lens illuminates a sample and the second and third objective lenses collect the fluorescence emissions emitted by the sample are disclosed. The second objective lens focuses a first portion of the fluorescence emissions for detection by a second detection component, while the third objective lens focuses a second portion of the fluorescence emissions through a diffractive or refractive optic component for detection by a first detector component. A processor combines the images resulting from the first and second portions of the fluorescence emissions for generating composite images with increased axial and lateral resolution.

Core Innovation

This patent discloses embodiments of a resolution enhancement technique for light sheet microscopy systems that use a three objective lens arrangement. In this arrangement, one objective lens illuminates a sample with a light sheet, while two other objective lenses collect fluorescence emissions from the sample. One of these collecting lenses focuses a first portion of the fluorescence emissions for detection by a first detector, and the other collects a second portion of the fluorescence emissions, which is processed through a diffractive or refractive optical component to extend axial resolution and preserve lateral resolution before detection by a second detector. A processor then combines the images from both detections to generate composite images with increased axial and lateral resolution compared to conventional light sheet microscopy.

The problem addressed is that conventional light sheet microscopy systems, which use perpendicular geometry between illumination and detection with relatively low numerical aperture objectives, have lower spatial resolution than conventional microscopy systems such as confocal microscopy. Attempts using multi-view acquisition or structured illumination for resolution improvement come with drawbacks including increased illumination exposure, larger image capture requirements, reduced signal or photon budget, and slower imaging speed. Additionally, conventional dual-objective light sheet microscopy discards a significant portion of fluorescence emissions and suffers from limited depth of field and resolution.

The disclosed solution employs a third objective lens equipped with an extended depth-of-focus optical system, such as a diffractive cubic phase mask or an incoherent refractive “layer cake” optical arrangement, to capture additional fluorescence emissions with preserved lateral resolution and extended axial depth of field. This third lens's images are combined with those from the second objective lens, which preserves axial resolution. The combination via a processing system yields composite images with enhanced spatial resolution in both lateral and axial dimensions, overcoming the limitations of low numerical aperture and inefficient fluorescence collection inherent in conventional light sheet microscopy.

Claims Coverage

The patent includes three independent claims that define inventive features related to a light sheet microscopy system with resolution enhancement, and a method for operating such a system.

Together, these inventive features provide a light sheet microscopy system and method that resolve the low spatial resolution and inefficient fluorescence collection problems of conventional systems by employing a three-lens arrangement, optical components that extend axial depth of field while preserving lateral resolution, and image combination processing to produce composite images with improved spatial resolution.

Stated Advantages

Documented Applications