Although visible-wavelength ratiometry has been sought for decades, the most effective calcium ion fluorescent indicators to date have been either ratiometric UV-excited (Fura-2) or non-ratiometric visible-wavelength (Fluo).
Since his post-graduate work at UC Berkeley in 1982, Dr. Akwasi Minta has been committed to improving ion indicators. His new indicator family - Asante Calcium RedTM - provides a fully visible-wavelength ratiometric response and other advantages over current optical tools.
In his effort to create an indicator for the calcium ion (1980 publication), Roger Tsien, PhD and Chemistry Nobel Laureate 2008 synthesized BAPTA (BisAminoPhenyl TetraAcetic acid). He next derived from BAPTA the first fluorescent calcium indicator Quin-2. Henceforth, a myriad of fluorescent calcium indicators have been developed.
The first effort at improving on Quin-2 led to many visible wavelength indicators which shift wavelength upon binding calcium. Unfortunately, all of these indicators had flaws rendering them ineffective in the cell such as low quantum yield, inappropriate calcium dissociation constants (Kd) and acid dissociation constants (pKa), or strong susceptibility to photobleaching. After this first effort, Roger’s team of chemists, physicists, and biologist at UC Berkeley in the early 1980’s produced today’s array of fluorescent calcium indicators Fura-2; Indo-1; Fluo’s 2, 3, and 4; and Rhod-2. Dr. Minta is a co-inventor of Fluo-2, Fluo-3, Fluo-4, and Rhod-2.
The team was disappointed when the Fluo and Rhod series did not shift wavelength upon binding calcium, merely producing a non-ratiometric enhancement instead. Ratiometry was considered to be an essential property of a fluorescent indicator because ratiometry allows for the true quantitative measurement of calcium by canceling variations in loading, optical path, and instrumentation. He therefore promoted Fura-2, with its excitation spectrum significantly shifting wavelength upon binding calcium, as the dye of choice and even helped develop the instrumentation for dual excitation measurements of this near-UV dye.
Indo-1 also gained favor because it is emission ratiometric, so that no excitation choppers were required in measurements. Fura-2 however remained more popular due to Indo-1’s tendency to photobleach at more rapid rates.
Roger favored ratiometry over the visible wavelength excitation and emission of the Fluo and Rhod dyes, despite the simpler optical equipment for visible wavelengths. Due to the lack of ratiometry in these dyes, complicated protocols were developed to attempt to calibrate them. Currently, the popularity of the Fluo and Rhod dyes attests to the efforts of scientists to take advantage of visible light fluorescence, surfeit of instruments ready for measurement and feasible with standard lasers.
Other visible dyes
There have been other poorly designed dyes in the Fluo and Rhod models that simply link a whole fluorescein or rhodamine to BAPTA through an amide, not through the carbon to carbon linkage that optimizes conjugation between chelator and fluorophore. Of course, these visible dyes are also non-ratiometric.
Efforts to make visible wavelength ratiometric dyes have not encountered much success. Two results of these efforts are BTC, suffering from an inappropriate Kd and blue and green responses to calcium, and Fura-Red, not only exhibiting violet and blue responses to calcium but also poor fluorescence in water.
Design and synthesis
The design and synthesis of Asante Calcium Red required three years of research to make a dual emission ratiometric visible wavelength dye. As shown in the graph below, in a ratiometric mode the emission intensity increases with increasing calcium at 650 nm and decreases with increasing calcium at 525 nm when exciting at 488 nm. This response provides a dual emission ratiometry in the visible spectrum.
In a non-ratiometric mode, the dye is excited at 540 nm and has a strong red spectrum response at 650 nm.
An ideal calcium indicator would provide a long wavelength response with accuracy and brightness. When shifting the emission response to longer wavelength in the red spectrum, however, there is typically a diminished quantum efficiency. While Asante Calcium Red is not as bright as Fluo dyes, it is more than twice as bright as Fura-Red.
The dye has the appropriate Kd, a low pKa, and comes in the easily loadable acetoxymethyl (AM) ester form. Fully visible excitation, dual emission ratiometry, and a strong response to calcium at 650 nm for a large dynamic range, all make this an interesting indicator for many calcium applications. Its visible character enables experiments prohibited by the UV character of Fura-2 and Indo-1. Its ratiometric response will clarify previous Fluo and Rhod experimental approximations.
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The table below summarizes key properties of Asante Calcium Red and existing calcium dyes.
Asante Calcium Red Dye Characteristic Comparison