Separating Excitation and Detection Optics for Deep Fluorescence Imaging
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Solution Overview
Problem
Conventional two-photon microscopy systems face limitations in imaging depth due to the coupling of excitation and fluorescence detection optics, which results in reduced sensitivity and limited collection of scattered fluorescence photons, restricting imaging depth to a few hundred microns in turbid biological tissues.
Innovation Solution
Separating excitation and detection optics, using a wide photocathode area photomultiplier tube coupled with a waveguide and index matching compounds to minimize light losses, allowing for the collection of scattered fluorescence photons from a wide area of the sample surface, thereby enhancing imaging depth to several millimeters.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If the same microscope objective is used for both excitation and fluorescence detection, then the optical system is simplified, but fluorescence detection sensitivity is strongly decreased due to limitations in light acceptance angle and surface area
Solution Approach 1:
The patent divides the optical system into separate excitation and detection pathways. The excitation objective is dedicated to delivering excitation light to the focal plane, while a separate detection objective is dedicated to collecting fluorescence photons. This segmentation allows each objective to be optimized for its specific function, with the detection objective having a larger aperture and different optical characteristics to maximize fluorescence collection efficiency.
Solution Approach 2:
The fluorescence detection function is extracted from the excitation objective and assigned to a separate detection objective. This extraction allows the detection pathway to be independently optimized with larger light acceptance angle and surface area, directly addressing the limitation of using a single objective for both functions.
2Length of stationary object
If excitation light power is increased to deliver more unscattered photons to the focal area, then imaging depth is increased, but out-of-focus fluorescence near the sample surface is induced that masks the fluorescence signal from the focal area
Solution Approach 1:
The patent employs local quality by using optical sectioning capabilities and spatial filtering to distinguish between fluorescence signals from different depths. The separate detection objective with its optimized optical path allows for better rejection of out-of-focus light, enabling the system to selectively detect fluorescence from the focal plane while minimizing contamination from superficial fluorescence even at high excitation power levels.
3Device complexity
If a microscope objective is used for fluorescence collection, then the optical path is simple, but most scattered fluorescence photons are not collected due to limited light acceptance angle and surface area
Solution Approach 1:
The patent merges the detection objective with additional optical elements including a beam splitter, filters, and detector assembly to create an integrated fluorescence collection system. The detection objective is specifically designed with larger aperture and optimized for collecting scattered photons from a wide surface area, while the additional optical elements work together to separate and detect fluorescence signals efficiently.
4Length of stationary object
If optical clearing agents are applied to increase imaging depth, then image depth is doubled, but the system requires additional sample preparation steps and materials
Solution Approach 1:
The patent employs self-service by using the tissue's own optical properties and the natural scattering of light to achieve deep imaging. The separate detection objective is designed to work with the inherent optical characteristics of turbid media, utilizing the scattering pattern to guide fluorescence photons to the detector without requiring external clearing agents or complex sample modification procedures.
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 significantly increases the imaging depth in turbid samples to up to 3 mm, compared to the conventional 0.5 mm, while maintaining high-resolution imaging, and can be applied in non-invasive diagnostics for tissues like the brain, breast, and skin.
Implementation Method 1
a wide photocathode area photomultiplier tube coupled with a waveguide and index matching compounds to minimize light losses, allowing for the collection of scattered fluorescence photons
Implementation Method 2
a wide photocathode area photomultiplier tube coupled with a waveguide
Implementation Method 3
index matching compounds to minimize light losses
Implementation Method 4
The near-infrared light that is used to induce two-photon fluorescence can penetrate deeper inside tissue samples
Implementation Method 5
an objective is coupled to the microscope and is disposed on a first side of the turbid sample for directing the coupled excitation beam onto the first side of the turbid sample
Data Source
AI summary
An apparatus and method for in-depth fluorescence imaging using two-photon fluorescence imaging in turbid media. The apparatus includes a detector which can significantly enhance the use of a detection method that allows to efficiently collect scattered fluorescence photons from a wide area of the turbid sample. By using this detector it is possible to perform imaging of turbid samples, simulating brain tissue at depths up to 3 mm, where the two-photon induced fluorescence signal is too weak to be detected by previous means used in conventional two-photon microscopy. The detector separates the excitation and detection optics which allows a more efficient collection of fluorescence and enhancing the possible imaging depth.