Ratio-based Indicators for Cytosolic Ca with Visible Light Excitation

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2025 Feb 12

PMID 39937863

Authors

Xinqi Zhou

Kayla J Belavek

Marisol X Navarro

Kayli N Martinez

Abigail Hinojosa

Evan W Miller

Affiliations

Soon will be listed here.

Abstract

Calcium ions (Ca) play central roles in cellular physiology. Fluorescent indicators for Ca ions revolutionized our ability to make rapid, accurate, and highly parallel measurement of Ca concentrations in living cells. The use of ratio-based imaging with one particular indicator, fura-2, allowed practitioners to correct for a number of experimental confounds, including dye bleaching, variations in sample thickness, and fluctuations in illumination intensity. Ratio-based imaging with fura-2 was the most accurate and reliable method for measuring Ca concentrations. Two drawbacks to fura-2 exist. First, it requires ultraviolet (UV) excitation, which is more toxic to living cells than visible light. Second, our ability to use fura-2 for accurate, stable, ratio-based determinations of Ca concentration in living cells is fast becoming a method of the past. This is due, in part, because modern microscopes are phasing out the use of mercury arc lamps that provide the UV excitation needed for fura-2 imaging. To address this problem, we describe the design, synthesis, and cellular application of benzo[]phosphole-based fluorescent Ca indicators for ratio-based imaging of Ca in living cells that can be used with modern light emitting diode (LED)-equipped fluorescence microscopes. We report isoCaRed-1Me, a Ca indicator that enables ratio-based imaging in immortalized cell lines, primary mammalian hippocampal neurons, and human-induced pluripotent stem cell-derived cardiomyocytes. These data show that isoCaRed-1Me will be useful for ratio-based Ca imaging using modern microscopes.

References

Dulhunty A . Excitation-contraction coupling from the 1950s into the new millennium. Clin Exp Pharmacol Physiol. 2006; 33(9):763-72. DOI: 10.1111/j.1440-1681.2006.04441.x. View

Ortiz G, Liu P, Naing S, Muller V, Miller E . Synthesis of Sulfonated Carbofluoresceins for Voltage Imaging. J Am Chem Soc. 2019; 141(16):6631-6638. PMC: 6546115. DOI: 10.1021/jacs.9b01261. View

Iatridou H, Foukaraki E, Kuhn M, MARCUS E, Haugland R, Katerinopoulos H . The development of a new family of intracellular calcium probes. Cell Calcium. 1994; 15(2):190-8. DOI: 10.1016/0143-4160(94)90058-2. View

Wang C, Fukazawa A, Taki M, Sato Y, Higashiyama T, Yamaguchi S . A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy. Angew Chem Int Ed Engl. 2015; 54(50):15213-7. DOI: 10.1002/anie.201507939. View

Oheim M, van t Hoff M, Feltz A, Zamaleeva A, Mallet J, Collot M . New red-fluorescent calcium indicators for optogenetics, photoactivation and multi-color imaging. Biochim Biophys Acta. 2014; 1843(10):2284-306. DOI: 10.1016/j.bbamcr.2014.03.010. View

Mathur A, Loskill P, Shao K, Huebsch N, Hong S, Marcus S . Human iPSC-based cardiac microphysiological system for drug screening applications. Sci Rep. 2015; 5:8883. PMC: 4352848. DOI: 10.1038/srep08883. View

Muranaka A, Yasuike S, Liu C, Kurita J, Kakusawa N, Tsuchiya T . Effect of periodic replacement of the heteroatom on the spectroscopic properties of indole and benzofuran derivatives. J Phys Chem A. 2008; 113(2):464-73. DOI: 10.1021/jp8079843. View

Zhang X, Chen L, Huang Z, Ling N, Xiao Y . Cyclo-Ketal Xanthene Dyes: A New Class of Near-Infrared Fluorophores for Super-Resolution Imaging of Live Cells. Chemistry. 2020; 27(11):3688-3693. DOI: 10.1002/chem.202005296. View

Fu M, Xiao Y, Qian X, Zhao D, Xu Y . A design concept of long-wavelength fluorescent analogs of rhodamine dyes: replacement of oxygen with silicon atom. Chem Commun (Camb). 2008; (15):1780-2. DOI: 10.1039/b718544h. View

10.

Li C, Wang T, Li N, Li M, Li Y, Sun Y . Hydrogen-bonding-induced bathochromic effect of Si-coumarin and its use in monitoring adipogenic differentiation. Chem Commun (Camb). 2019; 55(78):11802-11805. DOI: 10.1039/c9cc05895h. View

11.

Fukazawa A, Suda S, Taki M, Yamaguchi E, Grzybowski M, Sato Y . Phospha-fluorescein: a red-emissive fluorescein analogue with high photobleaching resistance. Chem Commun (Camb). 2015; 52(6):1120-3. DOI: 10.1039/c5cc09345g. View

12.

Liu J, Sun Y, Zhang H, Shi H, Shi Y, Guo W . Sulfone-Rhodamines: A New Class of Near-Infrared Fluorescent Dyes for Bioimaging. ACS Appl Mater Interfaces. 2016; 8(35):22953-62. DOI: 10.1021/acsami.6b08338. View

13.

Ando N, Soutome H, Yamaguchi S . Near-infrared fluorescein dyes containing a tricoordinate boron atom. Chem Sci. 2019; 10(33):7816-7821. PMC: 6764465. DOI: 10.1039/c9sc02314c. View

14.

Jackson A, Timmerman M, Bagshaw C, Ashley C . The kinetics of calcium binding to fura-2 and indo-1. FEBS Lett. 1987; 216(1):35-9. DOI: 10.1016/0014-5793(87)80752-4. View

15.

Zhou X, Belavek K, Miller E . Origins of Ca Imaging with Fluorescent Indicators. Biochemistry. 2021; 60(46):3547-3554. PMC: 8612960. DOI: 10.1021/acs.biochem.1c00350. View

16.

Choi A, Miller S . Silicon Substitution in Oxazine Dyes Yields Near-Infrared Azasiline Fluorophores That Absorb and Emit beyond 700 nm. Org Lett. 2018; 20(15):4482-4485. PMC: 6524783. DOI: 10.1021/acs.orglett.8b01786. View

17.

Shimomura N, Egawa Y, Miki R, Fujihara T, Ishimaru Y, Seki T . A red fluorophore comprising a borinate-containing xanthene analogue as a polyol sensor. Org Biomol Chem. 2016; 14(42):10031-10036. DOI: 10.1039/c6ob01695b. View

18.

Neher E, Sakaba T . Multiple roles of calcium ions in the regulation of neurotransmitter release. Neuron. 2008; 59(6):861-72. DOI: 10.1016/j.neuron.2008.08.019. View

19.

Gonzalez M, Walker A, Cao K, Lazzari-Dean J, Settineri N, Kong E . Voltage Imaging with a NIR-Absorbing Phosphine Oxide Rhodamine Voltage Reporter. J Am Chem Soc. 2021; 143(5):2304-2314. PMC: 7986050. DOI: 10.1021/jacs.0c11382. View

20.

Grimm J, Gruber T, Ortiz G, Brown T, Lavis L . Virginia Orange: A Versatile, Red-Shifted Fluorescein Scaffold for Single- and Dual-Input Fluorogenic Probes. Bioconjug Chem. 2015; 27(2):474-80. DOI: 10.1021/acs.bioconjchem.5b00566. View