Immersion Microscope Objective for Multiphoton Excitation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Multiphoton excitation microscopy faces challenges in achieving bright fluorescence and high-resolution images due to the need for high photon density, aberration correction, and efficient detection of weak fluorescent light, particularly in deep tissue samples where infrared light is used and scattered.
Innovation Solution
An immersion microscope objective with a specific lens group configuration, including a first lens group with positive refractive power, a second lens group with low refractive power, a third lens group that moves to correct aberrations, and a fifth lens group with positive refractive power, optimized for multiphoton excitation to enhance fluorescence collection and image resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a microscope objective lens with large numerical aperture is used to achieve high photon density for multiphoton excitation, then fluorescence excitation efficiency is improved, but aberration correction becomes more difficult and complex
Solution Approach 1:
The objective lens is divided into multiple lens groups (first through fifth lens groups) with different refractive powers and functions. Each group addresses specific optical requirements: the first group (positive power) collects light at large angles, the second group (low positive power) controls spherical aberration, the third group (movable) corrects depth-dependent aberrations, the fourth group (negative power) corrects coma and astigmatism, and the fifth group (positive power) focuses light while maintaining a sharp leading end. This segmentation allows high numerical aperture (0.85-1.15) while systematically correcting aberrations.
Solution Approach 2:
The third lens group is designed to move along the optical axis to dynamically correct aberrations that vary with observation depth in the sample. This dynamic adjustment capability allows the lens to maintain optimal performance when observing fluorescent structures at different depths, addressing the aberration correction challenge associated with large numerical aperture lenses.
2Loss of energy
If the number of lens elements is reduced to minimize loss of fluorescent light, then detection efficiency is improved, but aberration correction capability deteriorates
Solution Approach 1:
Each lens group is assigned specific local optical functions tailored to its position in the optical path. The first group handles light collection at large angles, the second group addresses spherical aberration, the third group provides dynamic depth correction, the fourth group corrects off-axis aberrations, and the fifth group maintains focus with a sharp leading end. This localized functional assignment achieves comprehensive aberration correction with only 5 groups, minimizing light loss while maintaining correction capability.
Solution Approach 2:
The lens groups utilize different optical materials with specific refractive indices and dispersion properties optimized for their functions. The first group uses materials suitable for high-angle light collection, the fourth group uses materials optimized for correcting coma and astigmatism, and the fifth group uses materials that maintain sharp focusing. This composite material approach enables effective aberration correction with fewer elements.
3Ease of operation
If a large working distance is provided to accommodate patch clamping technique, then accessibility for cellular biology studies is improved, but numerical aperture and resolution are reduced
Solution Approach 1:
The lens design separates the working distance dimension from the numerical aperture dimension. By optimizing the lens group configurations and spacing, the lens achieves a large working distance (enabling patch clamping access) while maintaining high numerical aperture (0.85-1.15) through the sharp leading end design of the fifth lens group and the overall optical path optimization. This resolves the traditional trade-off between working distance and resolution.
4Length of stationary object
If infrared light is used for multiphoton excitation to reduce scattering and photo toxicity, then penetration depth is improved, but aberration correction for infrared wavelengths becomes challenging
Solution Approach 1:
The lens groups are designed with specific refractive power distributions and material selections optimized for infrared wavelengths (used in multiphoton excitation). The second lens group's low positive refractive power and the fourth lens group's negative refractive power are specifically tuned to correct spherical aberration and coma/astigmatism for infrared light, enabling deep tissue observation while maintaining image quality.
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
The solution ensures bright fluorescence and high-resolution images by correcting aberrations and optimizing the optical path for infrared light, allowing for deeper tissue observation with minimal damage and improved fluorescence detection.
Implementation Method 1
During multiphoton excitation, a fluorescent object is illuminated with a light beam having wavelengths of integral multiples of an inherent absorption wavelength so as to cause excitation nearly equivalent to that caused by light having a wavelength equal to the inherent absorption wavelength. Multiphoton excitation is a non-linear phenomenon.
Implementation Method 2
For observation of the deep parts of a sample, aberrations resulting from the refractive index of the sample cannot be ignored.
Implementation Method 3
Generally speaking, light having longer wavelengths tends to scatter less (i.e., Rayleigh scattering). On the other hand, infrared excitation light reaches deep inside scattering samples such as living samples.
Implementation Method 4
a fluorescent object is illuminated with a light beam having wavelengths of integral multiples of an inherent absorption wavelength so as to cause excitation nearly equivalent to that caused by light having a wavelength equal to the inherent absorption wavelength
Data Source
Figure 1
Figure 2
Figure 3
AI summary
An immersion microscope objective formed of thirteen or fewer lens elements includes, in order from the object side, first and second lens groups (G1,G2) of positive refractive power, a third lens group (G3), a fourth lens group (G4) having negative refractive power with its image-side surface being concave, and a fifth lens group (G5) having positive refractive power with its object-side surface being concave. The first lens group (G1) includes, in order from the object side, a lens component that consists of a lens element of positive refractive power (when computed as being in air) and a meniscus lens element having its concave surface on the object side. Various conditions are satisfied to ensure that images of fluorescence, obtained when the immersion microscope objective is used in a laser scanning microscope that employs multiphoton excitation to observe a specimen, are bright and of high resolution. Various laser scanning microscopes are also disclosed.