Two-Photon Light Sheet Microscopy for Deep Fast Live Imaging
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Solution Overview
Problem
Conventional microscopy techniques face a trade-off between depth penetration, acquisition speed, and phototoxicity, with two-photon laser scanning microscopy (2p-LSM) excelling in depth but limited by slow acquisition, and one-photon light sheet (1p-LISH) offering high speed but limited depth due to scattering and phototoxicity.
Innovation Solution
A multiple-photon excitation light sheet microscope that uses pulsed lasers for high-intensity excitation, employing orthogonal geometry and nonlinear excitation processes to achieve high depth penetration, fast acquisition, and low phototoxicity, utilizing focal volume engineering and adjustable numerical apertures to optimize signal generation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of stationary object
If two-photon laser scanning microscopy (2p-LSM) is used, then depth penetration is improved, but acquisition speed deteriorates
Solution Approach 1:
The patent merges the depth penetration advantage of two-photon excitation with the acquisition speed advantage of light sheet illumination by implementing a two-photon excited light sheet illumination microscope. This combines the nonlinear excitation mechanism of 2p-LSM with the parallel imaging capability of light sheet microscopy, allowing simultaneous achievement of deep tissue penetration and high-speed acquisition.
Solution Approach 2:
The patent employs continuous light sheet illumination across the entire field of view rather than sequential point scanning. The light sheet continuously illuminates the sample plane, enabling parallel detection of fluorescence signals across thousands of pixels simultaneously, thus maintaining continuous useful action and achieving high acquisition speed while preserving depth penetration through two-photon excitation.
2Productivity
If one-photon light sheet (1p-LISH) microscopy is used, then acquisition speed is improved, but depth penetration deteriorates
Solution Approach 1:
The patent changes the excitation parameter from one-photon to two-photon excitation. This parameter change allows the use of near-infrared wavelengths that penetrate deeper into scattering biological tissues while maintaining the light sheet illumination geometry that enables high acquisition speed. The two-photon excitation process overcomes the depth limitation of 1p-LISH by utilizing the nonlinear optical effect that is less sensitive to scattering.
3Productivity
If one-photon light sheet (1p-LISH) microscopy is used, then acquisition speed is improved, but phototoxicity increases
Solution Approach 1:
The patent changes the excitation wavelength parameter to near-infrared for two-photon excitation. This parameter change reduces phototoxicity because NIR light has lower energy per photon and causes less photo-damage to biological samples. Additionally, the two-photon excitation process is highly localized to the focal plane where photon density is sufficient, reducing out-of-focus phototoxicity while maintaining high acquisition speed through parallel imaging.
4Length of stationary object
If two-photon laser scanning microscopy (2p-LSM) is used, then depth penetration is improved, but phototoxicity decreases
Solution Approach 1:
The patent merges the low phototoxicity advantage of two-photon excitation with the high acquisition speed of light sheet illumination. By implementing two-photon excited light sheet illumination, it maintains the localized excitation benefit of 2p-LSM (reducing phototoxicity) while eliminating the slow acquisition speed through parallel imaging across the entire light sheet.
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 MP-LISH microscope achieves a balance between depth penetration and acquisition speed while minimizing phototoxicity, providing superior imaging capabilities with improved signal uniformity and resolution, especially in scattering biological samples.
Implementation Method 1
the detected signal contrast can be two-photon-excited fluorescence, second harmonic generation, third harmonic generation, sum frequency generation, and stimulated Raman scattering
Implementation Method 2
Signal and spatial resolution are maintained significantly deeper into scattering samples compared with modalities that use 1 - photon excitation
Implementation Method 3
a planar sheet of light is used to illuminate the sample, generating fluorescence signal over a thin optical section of the sample
Implementation Method 4
both ballistic and scattered fluorescence photons contribute to the signal
Data Source
Figure 1A~1B
Figure 1C~1D
Figure 1E
AI summary
An apparatus for and method of performing multi-photon light sheet microscopy (MP-LISH), combining multi-photon excited fluorescence with the orthogonal illumination of light sheet microscopy are provided. With live imaging of whole Drosophila and zebrafish embryos, the high performance of MP-LISH compared to current state-of-the-art imaging techniques in maintaining good signal and high spatial resolution deep inside biological tissues (two times deeper than one-photon light sheet microscopy), in acquisition speed (more than one order of magnitude faster than conventional two-photon laser scanning microscopy), and in low phototoxicity are demonstrated. The inherent multi-modality of this new imaging technique is also demonstrated second harmonic generation light sheet microscopy to detect collagen in mouse tail tissue. Together, these properties create the potential for a wide range of applications for MP-LISH in 4D imaging of live biological systems.