Two-Photon Probe for Intracellular Calcium Imaging
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Solution Overview
Problem
Current two-photon probes for intracellular calcium imaging suffer from low two-photon action cross sections, requiring high concentrations and laser power, and experience photobleaching and mistargeting issues, limiting their ability to visualize calcium waves deep within tissues.
Innovation Solution
A two-photon probe with a significant two-photon cross section, using 2-acetyl-6-(dimethylamino)naphthalene as the chromophore and O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid (BAPTA) as the Ca2+ ion chelator, which offers high selectivity and photostability, allowing for bright imaging at low concentrations and discrimination between cytosolic and membrane-bound probes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional two-photon probes are used, then imaging can be performed, but the two-photon action cross section is low requiring high probe concentration and laser power
Solution Approach 1:
The patent modifies the molecular structure of the probe by changing parameters such as introducing a naphthalene chromophore with high two-photon absorption cross section and adjusting the chelator structure. This structural parameter change results in a 44-fold increase in two-photon action cross section, enabling imaging at low probe concentrations without requiring high laser power
2Duration of action of stationary object
If conventional probes are used, then calcium imaging can be performed, but photobleaching occurs limiting imaging duration
Solution Approach 1:
The patent creates a composite probe structure combining a naphthalene chromophore with a BAPTA chelator moiety. This composite molecular design provides both high two-photon absorption cross section and enhanced photostability, allowing prolonged imaging sessions without significant photobleaching while maintaining signal intensity
3Measurement precision
If membrane-bound probes are used, then calcium detection is possible, but fluorescence quantum yield is higher in membrane causing mistargeting errors
Solution Approach 1:
The patent introduces a hydrophilic character to the probe through the BAPTA chelator structure and solvent-exposed chromophore orientation, creating local quality differentiation that favors cytosolic localization over membrane binding. This ensures the probe resides in the cytosol where it can accurately detect free calcium without the spurious signals from membrane-bound probes
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 probe achieves 44-fold two-photon excited fluorescence enhancement in response to Ca2+, enabling selective detection of intracellular free Ca2+ without interference from other metal ions or membrane-bound probes, with a dissociation constant of 0.25 ± 0.03 µM, and can monitor calcium waves at depths of 100-300 µm for over 1,100 seconds without photobleaching.
Implementation Method 1
TPM employs two lower energy, near-infrared photons to produce an excited fluorescent substance
Implementation Method 2
fluorescence imaging with fluorescent probes such as Oregon Green 488 BAPTA-1 (OG1) and fura-2 have most often been used
Data Source
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AI summary
A two-photon probe for real-time monitoring of intracellular calcium ions is provided. The two-photon probe is very suitable for real-time imaging of intracellular calcium ions, shows 20-50-fold TPEF enhancement in response to Ca2+, has a dissociation constant (KdTP) of 0.14 ± 0.02 to 0.25 ± 0.03 µ M, and emits 5-fold stronger TPEF than currently available one-photon fluorescent Ca2+ probes. Unlike the previously available probes, the two-photon probe can selectively detect dynamic levels of intracellular free Ca2+ in live cells and living tissues without interference from other metal ions and from the membrane-bound probes. Moreover, the two-photon probe is capable of monitoring the calcium waves at a depth of 100-300 µm in live tissues for 1,100-4,000 s using two-photon microscopy (TPM) with no artifacts of photo-bleaching. Further provided are a method for preparing the two-photon probe and a method for real-time monitoring of intracellular calcium ions using the two-photon probe.