Two-Photon Fluorescent Dye for Deep Tissue Sulfide Imaging
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Solution Overview
Problem
Current methods for detecting hydrogen sulfide (H2S) in living cells or tissues are invasive and lack non-destructive techniques, with one-photon fluorescent dyes experiencing issues like low penetration depth and photobleaching, and there is no method to detect hydrogen sulfide without damaging cells or tissues.
Innovation Solution
Development of a ratiometric two-photon fluorescent dye capable of detecting hydrosulfide ion and total sulfide in vivo, using a compound with specific chemical structures that can be excited by a femtosecond pulse at 700-800 nm, allowing imaging at depths of 100 μm or greater with high photostability and minimal interference from pH or cytotoxicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If one-photon fluorescent dyes are used for detecting hydrogen sulfide, then fluorescence imaging can be performed, but penetration depth is limited and photobleaching occurs
Solution Approach 1:
The patent changes the excitation parameter from one-photon (single high-energy photon) to two-photon (two low-energy photons) excitation. This parameter change enables deeper tissue penetration because the two-photon excitation uses lower energy photons that can penetrate deeper into biological tissues, while still achieving the desired fluorescence detection capability through simultaneous absorption of two photons.
Solution Approach 2:
The patent employs pulsed laser excitation for two-photon microscopy, using periodic short pulses to accumulate sufficient photon density for two-photon absorption while minimizing photodamage and photobleaching between pulses. This periodic action allows deep tissue imaging with reduced phototoxicity compared to continuous one-photon excitation.
2Measurement precision
If one-photon fluorescent dyes are used, then fluorescence imaging can be performed, but photobleaching occurs reducing imaging duration
Solution Approach 1:
The patent uses pulsed laser excitation with periodic timing, where short high-intensity pulses provide sufficient two-photon excitation followed by relaxation periods. This periodic action reduces cumulative photodamage and photobleaching compared to continuous one-photon excitation, thereby extending the usable imaging duration while maintaining detection precision.
Solution Approach 2:
The patent substitutes the one-photon excitation mechanism with a two-photon excitation mechanism. This substitution fundamentally changes the interaction between light and fluorophore, reducing photobleaching because the lower energy per photon and more localized excitation volume in two-photon microscopy cause less chemical degradation of the fluorophore over time.
3Measurement precision
If one-photon fluorescent dyes are used, then fluorescence imaging can be performed, but cellular autofluorescence interferes with detection
Solution Approach 1:
The patent changes the excitation wavelength parameter by using two-photon excitation with infrared wavelengths (typically 700-900 nm) instead of visible light. This parameter change shifts the excitation to a region where cellular autofluorescence is minimal, while the two-photon process still efficiently excites the fluorophore, thereby improving signal-to-noise ratio by reducing autofluorescence interference.
Solution Approach 2:
The patent employs pulsed excitation in the infrared region where cellular autofluorescence is naturally low. The periodic pulsing at these wavelengths further minimizes background interference while maintaining sufficient excitation intensity for detection, effectively reducing the harmful autofluorescence effect.
4Measurement precision
If current hydrogen sulfide detection methods are used, then detection can be performed, but tissue and cells must be processed and damaged
Solution Approach 1:
The patent substitutes invasive chemical and mechanical processing methods with non-invasive optical detection using two-photon microscopy. This substitution allows direct imaging of hydrogen sulfide in living tissues through the endogenous fluorophore or exogenous probes, eliminating the need for tissue fixation, extraction, or other damaging processing steps while maintaining detection precision.
Solution Approach 2:
The patent uses fluorescent probes or endogenous fluorophores as intermediaries to detect hydrogen sulfide. These intermediaries allow indirect detection of hydrogen sulfide through fluorescence signal changes, enabling non-invasive measurement in living systems without direct chemical processing that would damage the tissue.
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 two-photon fluorescent dye enables accurate imaging and quantitative estimation of total sulfide concentration in vivo with enhanced penetration depth and stability, providing a non-destructive method for visualizing total sulfide in living cells and tissues.
Implementation Method 1
two-photon microscopy (TPM) uses a two-photon probe capable of detecting the target substance deep in the living cells and tissues
Implementation Method 2
two-photon fluorescent dye for imaging total sulfide in vivo, which is capable of detecting hydrosulfide ion and imaging total sulfide in vivo
Implementation Method 3
using a compound with specific chemical structures that can be excited by a femtosecond pulse at 700-800 nm
Data Source
AI summary
The present disclosure relates to a two-photon fluorescent dye for imaging total sulfide in vivo and a method for quantitatively estimating total sulfide concentration in vivo using the same. The two-photon fluorescent dye, which includes a compound of [Chemical Formula 1], can detect hydrosulfide ion and image total sulfide and allows quantitative estimation of total sulfide concentration in vivo.