Fluorescence Measurement Using IR Excitation and Triplet-State Control
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Solution Overview
Problem
Existing fluorescence measurement techniques require high peak powers from femtosecond lasers and point scanning systems, limiting throughput and accessibility, and the mechanisms of photobleaching and phototoxicity in fluorophores are not fully understood.
Innovation Solution
Utilizing azimuthally polarized beams to access higher order triplet states through magnetic dipole transitions, combining IR light beams with different wavelengths to generate and measure fluorescence, reducing power requirements while enabling precise excitation and imaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If femtosecond lasers with high peak power are used for fluorescence measurement, then fluorescence signal strength is improved, but device complexity and cost increase
Solution Approach 1:
The patent changes the wavelength parameter from visible to infrared, enabling two-photon excitation at lower peak powers. This parameter change allows the use of more accessible laser systems while maintaining fluorescence signal strength through the nonlinear optical process.
Solution Approach 2:
The patent employs pulsed laser operation with specific duty cycles, using periodic high-intensity bursts for excitation followed by lower power intervals. This periodic action enables sufficient fluorescence signal generation while reducing average power requirements and heat management complexity.
2Measurement precision
If point scanning systems are used for fluorescence imaging, then measurement precision is improved, but productivity decreases
Solution Approach 1:
The patent transitions from point-by-point scanning to volumetric excitation by focusing infrared beams through the entire sample depth simultaneously. This dimensional change from 1D scanning to 3D parallel excitation maintains spatial resolution while dramatically increasing imaging throughput.
Solution Approach 2:
The patent divides the excitation process into multiple infrared wavelength components that can be independently tuned and combined. This segmentation allows simultaneous excitation of different fluorophores at different depths, enabling parallel information acquisition from multiple spatial zones.
3Illumination intensity
If higher light power is applied to fluorophores, then fluorescence intensity is improved, but photobleaching and phototoxicity increase
Solution Approach 1:
The patent changes the excitation wavelength to the infrared region, which has lower photon energy per quantum. This parameter change reduces the energy deposited into each fluorophore molecule, decreasing the probability of photodamaging reactions while still achieving fluorescence through two-photon absorption.
Solution Approach 2:
The patent replaces single-photon excitation mechanics with two-photon excitation mechanics. This substitution allows the use of lower peak power infrared light that penetrates deeper with less scattering and causes less photodamage, while the nonlinear optical process concentrates excitation only at the focal volume.
4Illumination intensity
If visible light sources are used for excitation, then fluorescence generation is achieved, but penetration depth in complex samples is limited
Solution Approach 1:
The patent changes the excitation wavelength from visible to infrared, exploiting the optical window in biological tissues where absorption and scattering are minimized. This parameter change enables deeper penetration into complex samples while maintaining the ability to generate fluorescence through two-photon excitation at the focal point.
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 efficient fluorescence generation and measurement with reduced power requirements, facilitating large-volume and complex sample analysis, and providing insights into photobleaching mechanisms.
Implementation Method 1
configured to produce a light beam, e.g., one or more light beams, having a wavelength range to produce a population of excited singlet states derived from relaxation of a population of higher order triplet states in the sample
Implementation Method 2
a first objective constructed and arranged to irradiate the sample with the light beam
Implementation Method 3
a detector constructed and arranged to receive the signal representative of fluorescence in the sample and to provide an output representative thereof
Data Source
AI summary
Systems for irradiating a sample are disclosed. The system includes a first light source configured to produce a visible light beam having a visible wavelength range to produce a population of triplet states in the sample. The system further includes an optical assembly to direct light beam. The system additionally includes an objective that directs the light beam onto the sample. Methods of irradiating a sample are also disclosed.