Two-Photon Fluorescent Probes for Acidic Vesicle Imaging
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Solution Overview
Problem
Conventional two-photon fluorescent probes have limited two-photon action cross sections and fail to selectively visualize acidic vesicles in live cells and tissues due to shallow penetration depth and mistargeting issues, as they stain both cytosol and membranes.
Innovation Solution
Development of two-photon fluorescent probes with a specific chemical structure, such as those represented by Formula 1, which have high two-photon action cross sections, are selectively soluble in water, and exhibit increased fluorescence intensity under acidic conditions, allowing for deep tissue imaging without mistargeting by preferentially staining vesicles in the cytosol.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Temperature
If conventional two-photon fluorescent probes are used, then the probes can penetrate tissue to some extent, but the penetration depth is limited and the probes fail to selectively visualize acidic vesicles
Solution Approach 1:
The patent modifies the chemical structure of two-photon fluorescent probes by introducing specific functional groups (carboxyl, hydroxyl, or amine groups) that confer pH responsiveness. This parameter change enables the probes to selectively respond to acidic conditions (pH 4.0-5.0) in lysosomes while maintaining deep tissue penetration capability through two-photon excitation at near-infrared wavelengths.
Solution Approach 2:
The patent creates local quality differentiation by designing probes that exhibit different fluorescence properties in different pH environments. The probes are engineered to have enhanced fluorescence intensity specifically in acidic compartments (lysosomes) compared to neutral cytosol, enabling selective visualization of acidic vesicles without requiring separate staining procedures.
2Measurement precision
If conventional fluorescent probes are used for one-photon microscopy, then the probes can stain vesicles, but the excitation light causes shallow penetration depth and damage to biomolecules
Solution Approach 1:
The patent replaces one-photon excitation with two-photon excitation mechanism. Instead of using high-energy UV or blue light that penetrates shallowly and damages biomolecules, the system uses two lower-energy near-infrared photons that collectively provide the necessary excitation energy. This substitution enables deep tissue penetration while reducing photodamage to living cells.
3Temperature
If two-photon fluorescent probes with high penetration depth are used, then deep tissue imaging is enabled, but the probes stain both cytosol and membranes causing mistargeting
Solution Approach 1:
The patent introduces pH responsiveness as a key parameter to differentiate between cytosol (neutral pH) and lysosomal compartments (acidic pH). By incorporating pH-sensitive functional groups, the probes undergo conformational changes or fluorescence intensity changes specifically in acidic environments, enabling selective staining of lysosomes over cytosol and membranes.
Solution Approach 2:
The patent uses pH as an intermediary parameter to achieve selective targeting. The acidic environment of lysosomes acts as a mediator that triggers the probe to accumulate or activate specifically in this compartment. The pH gradient serves as a natural mediator that directs the probe from the cytosol into acidic vesicles, enabling indirect but specific targeting without requiring direct membrane interaction.
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 probes achieve high fluorescence intensity and deep tissue penetration, enabling clear visualization of acidic vesicles with minimal background interference, thereby facilitating accurate real-time imaging of acidic compartments in live cells and tissues.
Implementation Method 1
Two-photon excitation is referred to as a phenomenon in which two photons are simultaneously absorbed in the same fluorophore having a sufficiently high photon density per unit volume and time by irradiation with a strong light source
Implementation Method 2
The photons in the excited state transit to the ground state and emit energy as fluorescence corresponding to the bandgap energy. This energy emission is called 'two-photon fluorescence'
Implementation Method 3
the fluorescence intensity of the probe increases with decreasing pH of the cell to be visualized
Data Source
AI summary
Provided are two-photon fluorescent probes for imaging acidic vesicles in live cells and tissue. The probes are represented byThe probes can selectively bind to vesicles in cytosol to emit two-photon excited fluorescence with high intensity. Therefore, the use of the probes enables effective imaging of acidic vesicles. Further provided is a method for imaging acidic vesicles in live cells and tissue using the probes.