Two-Photon Fluorescence Lifetime Imaging for Ocular Fluorophores
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Solution Overview
Problem
Existing methods for in vivo eye measurements using two-photon excitation microscopy face challenges such as low signal levels, photobleaching, phototoxicity, and the inability to differentiate between fluorophores with similar spectral bandwidths, which limits their effectiveness for accurate diagnosis and monitoring of ocular diseases.
Innovation Solution
A two-photon fluorescence lifetime imaging system is developed, comprising a laser, scanner, dichroic mirror, detector, and a set of spectral filters including a sine-wave, cosine-wave, and bandpass filter. This system allows for phasor analysis, enabling the separation and quantification of multiple fluorophores without direct spectral measurements, thus overcoming the limitations of existing methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If the illuminating light intensity is increased to improve signal level, then the signal detection capability is improved, but photochemical damage to the retina and phototoxicity increase
Solution Approach 1:
The patent changes the fundamental parameter of light-matter interaction from single-photon to two-photon excitation. This nonlinear optical process allows excitation at longer wavelengths (lower energy per photon) while achieving the same fluorescence excitation effect, thereby improving signal detection without increasing photodamage risk
Solution Approach 2:
The system uses pulsed laser illumination with specific duty cycles to deliver excitation light in periodic bursts. This allows sufficient signal accumulation during pulse windows while providing rest periods that reduce cumulative phototoxicity and photochemical damage to ocular tissues
2Measurement precision
If prolonged exposure times are used to accumulate sufficient photons, then the signal level is improved, but motion artifacts and patient discomfort increase
Solution Approach 1:
Two-photon excitation provides significantly higher signal intensity per unit time compared to single-photon methods due to the nonlinear excitation process. This enables sufficient photon accumulation within brief measurement windows, eliminating motion artifacts and patient discomfort associated with prolonged exposure
Solution Approach 2:
The enhanced signal generation from two-photon excitation allows the system to rapidly acquire sufficient data in a single brief measurement pass, skipping the need for prolonged cumulative exposure that would cause patient discomfort and motion artifacts
3Measurement precision
If spectral measurements are performed to differentiate fluorophores, then the identification accuracy is improved, but the measurement time and signal acquisition complexity increase
Solution Approach 1:
The patent introduces fluorescence lifetime as an intermediary parameter for fluorophore differentiation. Instead of directly measuring spectra, the system measures the temporal decay characteristics of fluorescence, which provides fluorophore identification capability without requiring complex spectral decomposition or extended measurement times
Solution Approach 2:
The system replaces complex spectral measurement mechanisms with a simpler time-resolved detection approach. By substituting spectral analysis with fluorescence lifetime measurement, the patent achieves fluorophore differentiation with reduced measurement complexity and faster acquisition time
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 system achieves effective in vivo imaging of fluorophores in the eye by enhancing signal detection, reducing measurement time, and minimizing phototoxicity, while allowing for precise identification and quantification of multiple fluorophores, thereby improving diagnostic accuracy and comfort for patients.
Implementation Method 1
two-photon excitation microscopy
Implementation Method 2
fluorophores that play critical roles in physiological and pathological processes
Implementation Method 3
a dichroic mirror positioned in the optical path between the laser and the measured object, whereby the dichroic mirror reflects the excitation light and transmits the emission light
Implementation Method 4
The set of spectral filters comprises a first filter having sine-wave spectral transmission characteristics, a second filter having cosine-wave spectral transmission characteristics
Data Source
Figure 1~2
Figure 3~4
Figure 5A~5B
AI summary
Human eye contains a variety of endogenous fluorophores. The precise identification, quantification, and spatial distribution of these fluorophores are essential for understanding eye health. In general, known approaches to measurements of fluorophores are complicated and lengthy, and thus not suitable for the use in in-vivo human eye measurement. The present invention provides a two-photon fluorescence lifetime imaging system for imaging fluorophores in a living eye, comprising a laser for providing excitation light; a scanner; a detector for collecting the emitted light; and a dichroic mirror positioned between the laser and the measured object. The system further comprises a set of spectral filters positioned on the optical path between the dichroic mirror and the detector. The set of spectral filters comprises a first filter having sine-wave spectral transmission characteristics, a second filter having cosine-wave spectral transmission characteristics, and a third filter being a bandpass filter. The transmission range of the third filter matches the emission spectrum of the fluorophore of interest. The data obtained using the set of spectral filters is suitable for performing phasor analysis. According to the second aspect, the invention provides a method for two-photon fluorescence lifetime imaging of fluorophores in a living eye, performed in the system as described above. In this method, the emission light is captured in at least one frame series comprising sequences captured using a set of three spectral filters. Said frame series comprises capturing a sequence in which the emission light passes through a first filter having sine-wave spectral transmission characteristics; a sequence in which the emission light passes through a second filter having cosine-wave spectral transmission characteristics; and a sequence in which the emission light passes through a third filter being a bandpass filter.