Multiphoton Microscope Collector Lens Design
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Solution Overview
Problem
Multiphoton-excitation microscopes face challenges in efficiently detecting fluorescence due to limited numerical aperture of the objective lens, which restricts the collection of emitted fluorescence, especially in thick specimens and electrophysiological experiments where the tip angle of the objective lens cannot be increased.
Innovation Solution
A multiphoton-excitation laser scanning microscope design that includes a collector lens with a higher numerical aperture and larger field number than the objective lens, along with a light detector configured to efficiently collect and detect fluorescence, allowing for the use of ultrashort pulsed laser light and excitation-light cutting filters to prevent laser light leakage, and an optical-path splitter for wavelength-based detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the numerical aperture of the objective lens is increased to improve fluorescence collection efficiency, then the fluorescence detection efficiency is improved, but the objective lens interferes with the needle used in electrophysiological experiments
Solution Approach 1:
The fluorescence collection function is separated from the objective lens and assigned to a dedicated collector lens. The objective lens focuses excitation light while the collector lens collects fluorescence, dividing the optical functions to resolve the conflict between high numerical aperture requirements and experimental accessibility.
Solution Approach 2:
The collector lens acts as an intermediary component between the specimen and the light detector. It receives fluorescence emitted from the specimen and directs it to the detector, enabling efficient fluorescence collection without requiring the objective lens to have high numerical aperture.
2Illumination intensity
If the objective lens has high numerical aperture to collect more fluorescence, then the fluorescence signal intensity is improved, but the tip angle of the objective lens cannot be increased due to interference with the needle
Solution Approach 1:
The optical system is segmented into excitation path (objective lens) and emission path (collector lens). The collector lens is positioned to collect fluorescence without interfering with needle access to the specimen, separating the requirements for signal intensity from operational accessibility.
Solution Approach 2:
The fluorescence collection is achieved by utilizing a different spatial dimension - the collector lens is positioned at an angle to collect fluorescence emitted from the specimen, rather than requiring the objective lens to have high collection angle in the same dimension where needle access is needed.
3Measurement precision
If ultrashort pulsed laser light is used for multiphoton excitation, then high resolution deep imaging is achieved, but intense light may damage the light detector
Solution Approach 1:
The collector lens serves as an intermediary that can be designed with appropriate optical density or filtering characteristics to attenuate the intense ultrashort pulsed laser light while transmitting the fluorescence signal, protecting the light detector from damage.
Solution Approach 2:
The harmful intense laser light is extracted or separated from the optical path before reaching the detector, either through filtering in the collector lens or through wavelength-specific detection, allowing the useful fluorescence signal to be detected while blocking the damaging laser light.
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 configuration enables the efficient collection and detection of fluorescence, resulting in a brighter multiphoton-excitation fluorescence image, particularly effective in thick specimens and allowing for simultaneous examination with multiple fluorescent dyes, while preventing damage to the light detector from intense light.
Implementation Method 1
induces multiphoton excitation at the focus position of the ultrashort pulsed laser light, thus causing fluorescence to be emitted
Implementation Method 2
a collector lens disposed opposite the objective lens, with the specimen disposed therebetween, to collect fluorescence emitted from the specimen
Implementation Method 3
an optical-path splitter configured to split the fluorescence collected by the collector lens based on wavelength
Implementation Method 4
excitation-light cutting filters disposed between the collector lens and the light detector
Data Source
AI summary
A multiphoton-excitation laser scanning microscope capable of efficiently collecting fluorescence emitted from a specimen to acquire a brighter multiphoton-excitation fluorescence image is provided. This multiphoton-excitation laser scanning microscope includes a multiphoton-excitation laser light source for emitting ultrashort pulsed laser light, a light-scanning unit configured to scan a specimen with the ultrashort pulsed laser light emitted from the multiphoton-excitation laser light source in two dimensions, an objective lens configured to focus the ultrashort pulsed laser light scanned by the light-scanning unit on the specimen, a collector lens disposed opposite the objective lens, with the light-scanning unit disposed therebetween, to collect fluorescence emitted from the specimen, and a light detector configured to detect the fluorescence collected by the collector lens. The collector lens has a higher numerical aperture and a larger field number than the objective lens.