Two-photon probe for real-time monitoring of intracellular calcium ions, method for preparing the probe and method for real-time monitoring of intracellular calcium ions using the probe
View Patent ↗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 Ca 2+ , has a dissociation constant (K d TP ) of 0.14±0.02 to 0.25±0.03 μM, and emits 5-fold stronger TPEF than currently available one-photon fluorescent Ca 2+ probes. Unlike the previously available probes, the two-photon probe can selectively detect dynamic levels of intracellular free Ca 2+ 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.
1. A two-photon probe for real-time monitoring of intracellular calcium ions, said probe being represented by Formula 1:
wherein R 1 is CH 3 or H,
R 2 is
and R 3 is H or CH 2 OCOCH 3 .
2. The two-photon probe according to claim 1 , wherein R 3 is CH 2 OCOCH 3 .
3. A method for preparing a two-photon probe for real-time monitoring of intracellular calcium ions, said probe being represented by Formula 1:
wherein R 1 is CH 3 or H, and R 2 is
and R 3 is H or CH 2 OCOCH 3 ,
the method comprising reacting a compound of Formula 2:
wherein R 4 is NH 2 or CHO and R 5 is H or CH 3 , with a compound of Formula 3:
wherein R 6 is NH 2 or
4. The method according to claim 3 , wherein the hydrogen atoms in R 3 are replaced with CH 2 OCOCH 3 by reacting bromomethyl acetate and triethylamine with the compound of Formula 1 (R 3 ═H).
5. A method for real-time monitoring of intracellular calcium ions, the method comprising introducing the two-photon probe according to claim 1 into cells of interest and imaging two-photon excited fluorescence emitted from the two-photon probe.
6. The method according to claim 5 , comprising quantitatively determining intracellular calcium ion concentration using Equation 1:
[Ca 2+ ]=K d [( F−F min )/( F max −F )] (1)
where K d is the dissociation constant of the two-photon probe for Ca 2+ , F is the observed two-photon fluorescence intensity, F min is the minimum fluorescence intensity, and F max is the maximum fluorescence intensity.
7. The method according to claim 5 , wherein the two-photon excited fluorescence imaging is at wavelengths ranging from 500 to 620 nm.