Phosphorylated Fluorescent Dyes for STED Microscopy
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Solution Overview
Problem
Current fluorescent dyes used in microscopy, such as coumarin and rhodamine dyes, face challenges with photostability, solubility in aqueous solutions, and fluorescence quantum yield degradation upon bioconjugation, limiting their effectiveness in high-intensity imaging techniques like STED microscopy and FCS.
Innovation Solution
Development of novel fluorescent dyes with phosphorylated hydroxymethyl groups, specifically coumarins and rhodamines, that maintain high fluorescence quantum yields in aqueous solutions and after bioconjugation, offering improved photostability and solubility, suitable for applications like STED microscopy and FCS.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If conventional fluorescent dyes (coumarin and rhodamine) are used in high-intensity imaging techniques like STED microscopy, then the imaging resolution and intensity can be achieved, but photobleaching occurs and photostability deteriorates
Solution Approach 1:
The patent modifies the molecular structure of fluorescent dyes by introducing phosphorylated hydroxymethyl groups, which changes the chemical parameters of the dye molecules. This structural modification enhances photostability and reduces triplet state formation, allowing the dyes to withstand high-intensity illumination in STED microscopy without rapid photobleaching
Solution Approach 2:
The patent creates composite fluorescent dye structures by combining coumarin or rhodamine core structures with phosphorylated hydroxymethyl substituents. These composite molecular structures integrate the beneficial properties of both the parent dyes (brightness, quantum yield) and the phosphorylated groups (enhanced photostability, water solubility)
2Quantity of substance
If fluorescent dyes are used in aqueous solutions for biological applications, then solubility is improved, but aggregation and non-specific binding increase
Solution Approach 1:
The phosphorylation of hydroxymethyl groups introduces charged phosphate moieties to the fluorescent dye molecules, fundamentally changing their solubility parameters. This modification enables the dyes to dissolve in aqueous buffers without requiring organic co-solvents, while the charged phosphate groups provide electrostatic repulsion that prevents aggregation
Solution Approach 2:
The phosphorylated hydroxymethyl groups act as intermediary structures that mediate between the hydrophobic aromatic core of the fluorescent dye and the aqueous biological environment. These groups provide hydrophilic, charged interfaces that enable water solubility while the spacer hydroxymethyl linkage maintains appropriate spacing to prevent non-specific binding
3Measurement precision
If fluorescent dyes are conjugated to biomolecules for labeling, then specific imaging of biological structures is achieved, but fluorescence quantum yield decreases
Solution Approach 1:
The patent modifies the molecular parameters of fluorescent dyes by adding phosphorylated hydroxymethyl groups at specific positions on the coumarin or rhodamine core structures. This structural parameter change reduces steric hindrance and electronic perturbation during bioconjugation, thereby maintaining high fluorescence quantum yields even after attachment to biomolecules
Solution Approach 2:
The patent segments the fluorescent dye molecule into distinct functional regions: a core chromophore (coumarin or rhodamine) responsible for fluorescence, phosphorylated hydroxymethyl groups providing solubility and reduced steric bulk, and reactive groups for bioconjugation. This segmentation allows each part to optimize its function independently, maintaining quantum yield during conjugation
4Quantity of substance
If organic solvents are used to dissolve fluorescent dyes for bioconjugation, then dye solubility is improved, but protein denaturation occurs
Solution Approach 1:
The phosphorylation of hydroxymethyl groups fundamentally changes the solubility parameters of the fluorescent dyes from hydrophobic to hydrophilic. This parameter change enables the dyes to dissolve in aqueous buffers without requiring organic co-solvents, thereby eliminating the harmful effect of protein denaturation while maintaining adequate dye solubility for bioconjugation reactions
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 phosphorylated dyes provide enhanced photostability and fluorescence quantum yields, enabling high-intensity imaging with reduced photobleaching and improved resolution, particularly in STED microscopy and FCS, while maintaining solubility in aqueous buffers.
Implementation Method 1
Water-soluble fluorescent markers are advantageous in this regard, because they do not require any organic solvents at all. Moreover, hydrophilic labels are less prone to aggregation and to non-specific binding with biological objects
Implementation Method 2
Coumarin and rhodamine dyes are known as bright fluorescent labels with large absorption coefficients, high fluorescent quantum yields
Implementation Method 3
Biological applications require fluorescent dyes absorbing and emitting in the red spectral region, because the excitation in this area reduces the background originating from autofluorescence of the cells
Implementation Method 4
a red-shifted doughnut-shaped STED beam switches off the fluorescence of the excited molecules by stimulated emission (S1 → S0) everywhere, except in the very center of the doughnut, where the quenching intensity is zero
Implementation Method 5
Other important qualities of the STED and common fluorescent dyes are high fluorescent quantum yields (Φfl) and oscillator strengths (high absorption coefficients, ε), low rate of triplet state formation
Data Source
Figure 1A~1B
Figure 2~3B
Figure 4~5
AI summary
The invention relates to novel fluorescent dyes with phosphorylated hydroxymethyl groups, a method for preparing the same as well as to their use in imaging techniques. Said fluorescent dyes are coumarin, rhodamine or BODIPY dyes having of one of the following general formulae (I-III) wherein W = OP(0)Y1Y2 or P(0)Upsilon1Upsilon2, where Y1 and Y2 independently denote any of the following residues: OH, O(-), ORa and ORb, NHRa and NHRb, NRaRb and NRcRd, ORa and NHRb, ORa and NRbRc, NHRa and NRbRc; and any salt thereof.