Systems and methods for multi-view nonlinear optical imaging for improved signal-to-noise ratio and resolution in point scanning multi-photon microscopy
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-11041807-B2
Publication Date
2021-06-22
Expiration Date
2036-08-22
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Abstract
Various embodiments of a multi-photon microscopy system that uses sequential excitation of a sample through three or more objective lenses oriented at different axes intersecting the sample are disclosed. Each objective lens is capable of focused sequential excitation of the sample that elicits fluorescence emissions from the excited sample, which is then simultaneously detected by each respective objective lens along a respective longitudinal axis every time the sample is illuminated through only a single objective lens.
Core Innovation
The invention relates to a multi-photon microscopy system and method that uses sequential excitation of a sample through three or more objective lenses arranged at different longitudinal axes intersecting the sample. Each objective lens delivers a focused sequential excitation beam to the sample, which then elicits fluorescence emissions. These emissions are simultaneously detected through each respective objective lens every time the sample is illuminated through only a single objective lens.
The system includes at least three objective lenses oriented along different axes intersecting the sample, laser sources, and scanners associated with these lenses for sequential illumination. The fluorescence emissions detected from each illumination event are processed by a processor, which combines and registers image data from multiple views to improve imaging quality. Additionally, the processor may apply joint deconvolution methods to enhance resolution and signal-to-noise ratio.
The problem addressed is the inherent limitation in nonlinear optical imaging caused by scattering, which reduces penetration depth, signal-to-noise ratio, and spatial resolution especially when imaging deep into tissue. Excitation and emission side scattering destroy the ability to form a sharp focus and cause lost fluorescent signals, resulting in poor image resolution and signal quality. There is a need for nonlinear optical imaging improvements that mitigate scattering effects to achieve better image quality at depth.
Claims Coverage
The patent includes two independent claims directed to a multi-photon microscopy system and a method for multi-view nonlinear imaging, featuring multiple objective lenses and sequential illumination coupled with simultaneous fluorescence detection.
Multi-photon microscopy system with multiple objective lenses and sequential illumination
A system comprising at least three objective lenses oriented along different longitudinal axes intersecting a sample, at least one laser source and scanner for sequentially transmitting a laser beam through each objective lens to illuminate the sample at focused points, and simultaneous detection of fluorescence emissions from the sample through all objective lenses from a detection volume within the working distance of at least one objective lens.
Optical arrangements with mirrors and detectors for fluorescence emission detection
First, second, and third optical arrangements operatively associated with each objective lens, each comprising a mirror, such as a dichroic mirror, for transmitting fluorescence emissions to respective point detectors (e.g., photomultiplier tubes) for detection of fluorescence emissions through each objective lens.
Processor for image data combination, registration, and deconvolution
A processor in communication with detectors that processes image data derived from fluorescence emissions produced when only one objective lens illuminates the sample. The processor generates images from each lens's detected emissions, combines and registers these images relative to each other, and may apply deconvolution algorithms such as Richardson-Lucy to enhance resolution and signal-to-noise ratio.
Method for multi-view nonlinear imaging using sequential illumination and simultaneous detection
A method involving positioning at least three objective lenses oriented along intersecting longitudinal axes; associating laser sources with objective lenses for sequential illumination of the sample; sequentially illuminating the sample through each objective lens; and simultaneously detecting fluorescence emissions generated by illuminations through all objective lenses from a detection volume within working distance of at least one lens. The method includes sequential illumination sequences with simultaneous detection after each illumination, and the use of mirrors and detectors for fluorescence transmission and detection.
The independent claims cover a multi-photon microscopy system and a method featuring multiple objective lenses oriented at different angles intersecting the sample, sequential illumination through each lens, simultaneous multi-lens fluorescence emission detection, optical arrangements employing mirrors and point detectors, and processing of image data through combination, registration, and deconvolution to improve signal-to-noise and resolution.
Stated Advantages
Improvement in signal-to-noise ratio in nonlinear optical imaging.
Improvement in spatial resolution by employing joint deconvolution processing.
Mitigation of scattering effects that limit penetration depth and fluorescence signal quality in deep-tissue imaging.
Documented Applications
Deep-tissue nonlinear optical imaging utilizing multi-photon microscopy systems.
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