Two-Photon Light-Sheet Autofluorescence Imaging for Low-Photodamage Embryos
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Solution Overview
Problem
Current microscopy techniques face limitations in deep tissue penetration, photodamage, and image blurriness due to out-of-focus excitation, especially in biological samples, which affect the assessment of biological samples like embryos.
Innovation Solution
A device utilizing two-photon excitation with a light-sheet or virtual light-sheet configuration and orthogonal objectives to minimize photodamage, combined with hyperspectral imaging to classify biological samples by their intrinsic autofluorescence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If single photon excitation is used to excite fluorophores, then excitation can occur throughout the sample, but this causes out-of-focus excitation leading to photobleaching, phototoxicity, and image blurriness
Solution Approach 1:
The patent applies two-photon excitation which concentrates excitation energy locally at the focal plane where photon density is highest. This creates a localized excitation zone that eliminates out-of-focus photodamage while maintaining effective excitation coverage. The non-linear two-photon absorption process inherently provides spatial selectivity, exciting fluorophores only where the laser focus concentrates sufficient photon density.
Solution Approach 2:
The patent employs pulsed laser excitation with femtosecond-duration pulses at high repetition rates. This periodic pulsed action delivers intense excitation energy in brief intervals, allowing the focal region to accumulate sufficient photon density for two-photon excitation while minimizing cumulative photodamage to out-of-focus regions between pulses.
2Use of energy by moving object
If visible light wavelengths (300-600 nm) are used to excite fluorophores, then excitation energy is sufficient, but light scatters heavily in biological tissue limiting penetration depth
Solution Approach 1:
The patent changes the excitation wavelength parameter from visible light (300-600 nm) to near-infrared light (700-1100 nm). This parameter change exploits the optical window in biological tissue where infrared light experiences minimal scattering and absorption, enabling penetration depths of 0.5-1 mm while maintaining sufficient photon energy for two-photon excitation of fluorophores.
Solution Approach 2:
The patent transitions from single-photon linear absorption to two-photon non-linear absorption, effectively adding a dimensional aspect to the excitation process. By requiring simultaneous absorption of two lower-energy infrared photons instead of one higher-energy visible photon, the system achieves both deep tissue penetration and sufficient excitation energy through a different physical mechanism.
3Length of stationary object
If two-photon excitation is used with focused laser, then deep penetration and sharp focusing are achieved, but requires very fast light pulses with high power
Solution Approach 1:
The patent uses high-repetition-rate pulsed laser operation (e.g., 80 MHz repetition rate) with femtosecond pulse durations. This periodic pulsed regime allows the average power to be distributed across many pulses, reducing peak power requirements while accumulating sufficient energy delivery to achieve deep penetration and effective two-photon excitation at the focal plane.
Solution Approach 2:
The patent employs continuous-wave or quasi-continuous pulsed laser operation at high repetition rates, maintaining continuous useful action at the focal plane. The high pulse repetition rate ensures that the focal region receives continuous excitation energy delivery, eliminating gaps between pulses and maintaining steady-state two-photon excitation conditions for deep tissue imaging.
4Productivity
If conventional microscopy is used for biological imaging, then sample exposure to light occurs, but this increases phototoxicity and reduces imaging speed
Solution Approach 1:
The patent implements selective plane illumination where only the focal plane receives excitation light through two-photon excitation. This local excitation approach illuminates only the region being imaged at any given time, dramatically reducing total light exposure to the sample while maintaining high imaging speed through efficient focal plane excitation and detection.
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
Enables non-invasive, real-time classification of biological samples, particularly embryos, with reduced photodamage and fast processing, providing interpretable images and spectral data for quality assessment.
Implementation Method 1
The two-photon absorption effect (TPA) would overcome the aforementioned problems: deep penetrance, sharp focusing, reduced photodamage. TPA is a non-linear effect where two photons of identical or different frequencies can excite electrons in a molecule from one energy state (usually the ground state) to a higher energy state in a single quantum event.
Implementation Method 2
A key benefit of two-photon microscopy is its ability to restrict excitation to a thin focal volume in thick samples. The objective focal point is the only space with a high enough photon density to ensure simultaneous presentation of two photons to the fluorophore.
Implementation Method 3
After excitation, the electrons will drop back down to its stable state and during this process it releases a photon of light that can be collected and detected by a detector.
Data Source
AI summary
A device for measuring intrinsic autofluorescence of molecules present in a biological sample wherein the device includes: a) a light-sheet, virtual light-sheet or light line delivery pathway module including a laser generator capable of generating ultrashort pulses with a duration between 1 microsecond and 1 attosecond, and optical elements shaping the laser light into a light sheet, virtual light-sheet or light-line; b) an imaging chamber including an imaging dish inside of which the biological sample is placed, a translational stage, an illumination objective and a collection objective both immersed in an immersion liquid in a sealed immersion chamber, wherein both objectives are positioned below the imaging dish, wherein the immersion chamber is placed inside an incubation chamber, allowing environmental control; and c) a fluorescence detection pathway including optical elements, light-splitting elements such as diffractive elements or dichroic mirrors, an array detector, and a device control and signal processing unit.