What's A Ratiometric Indicator

Ions play an intricate role in many physiologically processes. For example, intracellular calcium ions are essential in signal transduction pathways promoting the release of neurotransmitters from neurons, and involved in the mechanisms required for the contraction of all muscle cells. Cellular ionic concentrations are regulated by passive and active ion channels and pumps. Malfunctions in ionic channels and pumps can result in the improper regulation of ionic concentrations creating adverse conditions non-conducive for normal cell functionality.

To investigate cellular ion fluxes, ionic imaging techniques are used in conjunction with fluorescence ionic indicators. Fluorescence ion indicators fall under two categories: a single-wavelength indicator or a ratiometric indicator. For qualitative analysis, single-wavelength indicators are recommended. Their large dynamic range makes them sensitive at detecting modest and transient ionic changes. Unfortunately, drawbacks such as variable dye loading and extrusion, associated with the usage of single-wavelength indicators makes it difficult to accurately determine cellular ionic concentrations. For these reasons, ratiometric indicators are recommended for the quantitative measurements of ionic concentrations.

Ratiometric, or dual-wavelength, ion indicators are a subcategory of fluorescent dyes utilized for their ability to quantitatively measure intracellular ion concentrations. Compared to single-wavelength indicators, dual-wavelength indicators possess unique spectral properties which are triggered in response to binding its target ion. In the case of ratiometric calcium indicators, such dyes undergo a shift in either their optimum absorption or emission wavelength intensities when binding to free Ca²⁺. For example, dual-excitation Ca²⁺ indicators exhibit two peak excitation wavelengths when either bound or free of Ca²⁺. An increase in Ca²⁺ concentration initiates an increase and decrease in the fluorescence emission intensities of the dual-excitation indicator when excited at the Ca²⁺-bound and Ca²⁺-free peak excitation wavelengths, respectively. Using ratios derived from the photometric data gathered, researchers can accurately determine intracellular Ca²⁺ concentrations. Ratioing techniques are advantageous because it reduces effects indicative of uneven dye loading, poor dye retention and photobleaching. Since their introduction in 1985 by Tsien and collaborators, ratiometric indicators have been cited in countless scientific papers and have aided in advances in investigating the role of calcium in cellular regulation (Grynkiewicz et al. 1985).

Calcium Indicator	Tools	Catalog #	Excitation Source	Advantage	Applications
Single-Wavelength	Calbryte-520	20650, 20656	Visible Light	Large dynamic range excellent for detecting modest and transient Ca2+ fluxes	Qualitative Data Analysis Fluorescence Microscopy Flow Cytometry Calcium Imaging
	Fluo-8	21080, 21086, 21087
	Rhod-2	21060
	Rhod-4	21120
Dual-Wavelength	Fura-2	21020, 21025, 21026	Ultra Violet Light	Accurately measures Ca2+ concentrations. Avoids artifacts associated with variable dye distribution, photobleaching, and indicator leakage.	Quantitative Data Analysis Fluorescence Microscopy Flow Cytometry Calcium Imaging
	Indo-1	21030
	Fura-8	21055
	Fura-Red	21045, 21046	Visible Light

Fura-2 & Indo-1

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Ratiometric indicators are powerful tools for detecting and imaging fluxes in target ion concentrations. Calcium indicators, such as fura-2 and indo-1, are UV-excitable fluorescent molecules utilized for investigating the regulatory roles of calcium at a cellular level. Depending on the binding status of fura-2 or indo-1's calcium chelating moieties, these ratiometric indicators will experience a shift in their maximum excitation or emission wavelengths. Fura-2 and its analogs are excitation-ratioable indicators for measuring intracellular Ca²⁺ concentrations. Upon binding to free Ca²⁺, these dual excitation indicators experience an absorption shift towards the blue spectrum while maintaining a fixed emission wavelength at ~510 nm. Fura-2 indicators with Ca²⁺-free chelators exhibit a maximum excitation wavelength at ~363 nm, while fura-2 indicators with Ca²⁺-bound chelators excite at ~335 nm.

In contrast to fura-2, which exhibits a shift in absorption upon binding to Ca²⁺, indo-1 and its analogs are emission-ratioable indicators for measuring intracellular Ca²⁺ concentrations. In Ca²⁺ free environments, indo-1 exhibits a maximum emission wavelength at ~475 nm. Upon binding to free Ca²⁺, indo-1's emission shifts toward the blue spectrum emitting at ~400 nm. Regardless of calciums presence, indo-1 maintains a maximum absorption at ~350 nm. This feature makes indo-1 well-suited for flow cytometry applications where it is easier to change emission filters rather than excitation sources.

Preparing Sample with Fura-2

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Sample protocol for preparing cells with Fura-2, AM ester ratiometric indicators

Materials & Reagents:

• Fura-2 AM (Cat# 21020)
• HHBS (Cat# 20011)
• 04% Pluronic^® F-127 (Cat# 20052)

Labeling Live Cells:

1. Prepare a 2 to 5 mM AM ester stock solution by dissolving fura-2, AM in high-quality, anhydrous DMSO.
   1. For future intended use, store DMSO stock solutions desiccated at -20 °C and protected from light.
2. Prepare a working solution of 2 to 20 µM in HHBS with 0.04% Pluronic^® F-127.
   1. For most cell lines we recommend a final concentration of Ca²⁺indicators to be 4-5 µM. To avoid any artifacts caused by overloading and potential dye toxicity, use the minimal probe concentration necessary to obtain an adequate signal, typically as low as 0.1 µM.
3. For samples containing cells with organic anion-transports, such as CHO cells, probenecid (2-5 mM, Cat#20060) may be added to the dye working solution to reduce leakage of de-esterified indicators.
   1. Final in well concentration will be 1-2.5 mM for probenecid.
4. Add equal volume of the dye working solution (from Step 2 or 3) into your cell plate.
5. Incubate at room temperature or 37 °C for 1 – 2.5 hours.
6. Replace the dye working solution with HHBS to remove excess probes.
7. Run the desired experiment at Ex/Em wavelengths appropriate for fura-2.
   1. Ca²⁺-bound peak excitation wavelength at ~340 nm.
   2. Ca²⁺-free peak excitation wavelength at ~380 nm.
   3. Peak emission wavelength ~510 nm

Fluorescence Detection of Fura-2 & Indo-1

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Monitoring the absorption and emission spectrum of ratiometric indicators under appropriate conditions is an excellent technique for observing the wavelength shifts of indicators in response to free intracellular Ca²⁺. The absorption shift of fura-2 can be observed by scanning the excitation spectrum between 300 nm and 400 nm with a fixed emission wavelength at ~510 nm. Likewise, indo-1 can be well excited by the argon-ion laser (~350 nm) of flow cytometers. When bound to free intracellular Ca²⁺, indo-1's emission shifts from a wavelength of ~475 nm to ~400 nm. A key feature of fura-2 and indo-1's fluorescence emissions is the presence of an isosbetic point at ~360 nm and ~460 nm, respectively (Figure 1). At this point, both ratiometric indicators' fluorescence intensity is insensitive to the concentration of free Ca²⁺. Additionally, the presence of an isosbetic point is indicative of good laboratory practices, as its absence is associated with either contamination by other ions or sloppy pipetting.

Fura-2 & Indo-1 Derivatives

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AM Esters: Live Cell Loading of Ratiometric Indicators

Fura-2 and indo-1 ratiometric indicators are available in AM ester form for live cell loading. Labeling ratiometric indicators with AM esters facilitates their passive diffusion across intact cell membranes of live cells. Once inside the cell, non-specific intracellular esterases hydrolyze the AM esters which activate the Ca²⁺ sensitive indicator.

Fura-2 & Indo-1: Salt Derivatives

Fura-2 and indo-1 are also available as cell-impermeant salt derivatives for determining the concentration of free Ca²⁺ in the cytosol, using the following equation: [Ca²⁺]_free = K_d[F - F_min]/[F_max - F]

Where:

• K_d is the Ca²⁺ dissociation constant of the indicator
• F is the fluorescence value of the indicator at experimental Ca²⁺ levels
• F_min is the observed fluorescence value of the indicator in the absence of Ca²⁺
• F_max is the observed fluorescence value of a Ca²⁺-saturated indicator.

The Ca²⁺-binding and spectroscopic properties of ratiometric indicators vary significantly in cellular environments compared to calibration solutions. In situ calibrations of intracellular indicators typically yield K_d values significantly higher than in vitro determinations. In situ calibrations are performed by exposing loaded cells to controlled Ca²⁺ buffers in the presence of ionophores such as A-23187 which are used to raise intracellular Ca²⁺ levels. Alternatively, cell permeabilization agents such as digitonin can be used to expose the indicator to the controlled Ca²⁺ levels of the extracellular medium. The K_d values of some calcium ratiometric indicators are listed in Table 1.
Table 1. Spectral and Ca²⁺-binding properties of ratiometric calcium indicators.

Ca2+ Indicator	Catalog Number		Excitation	Emission	Kd of Ca2+-Binding
	AM Ester	Salt
Fura-2	21020, 21021 21022, 21023	21025, 21026	340/380 nm	510 nm	140 nM
Fura FF	21027	21028	340/380 nm	510 nm	5.5 µM
Fura-8™	21055, 21056	21057, 21058	354/415 nm	524 nm	260 nM
Fura-8™ FF	20620	20621	354/415 nm	524 nm	6 µM
Fura Red	21046, 21048	21045, 21047	436/471 nm	630/652 nm	400 nM
Indo-1	21030, 21032 21033, 21036	21040, 21044	355 nm	400/475 nm	230 nM

Fura-8 and Its Derivatives

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AAT Bioquest has developed fura-8™ ratiometric indicators available in AM ester and salt derivative forms for an improved and sensitive response to measuring intracellular calcium concentrations. Compared to fura-2, fura-8™ has a higher signal-to-background ratio while maintaining a similar affinity for Ca²⁺ (Figure 2). The significant advantage to fura-8™ is a shift of its absorption and emission wavelengths in the direction of the red spectrum. Fura-8™ can be excited at ~365 nm (Ca²⁺-bound) and 410 nm (Ca²⁺-free) wavelengths with and emission wavelength ~ 525 nm. Such spectral properties makes fura-8™ excitable by relatively inexpensive high power LEDs and compatible with common emission filter sets such as FITC. Additionally, fura-8™'s shift in excitation wavelengths makes it acceptable to use with organic compounds that tend to fluoresce when excited below 360 nm, which could lead to a false signal. When using fura-8™ we recommend using Ex/Em = 354/530 nm and 415/530 nm for ratio measurements.

 

References

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1. Belz, Mathias, et al. "Fiber optic biofluorometer for physiological research on muscle slices."; Search the world's largest collection of optics and photonics applied research, International Society for Optics and Photonics, 17 Mar. 2016, www.spiedigitallibrary.org/conference-proceedings-of spie/9702/1/Fiber-optic-biofluorometer-for-physiological-research-on-muscle-slices/10.1117/12.2220291.full.
2. Grynkiewicz, G, et al. "A new generation of Ca2 indicators with greatly improved fluorescence properties."; The Journal of biological chemistry., U.S. National Library of Medicine, 25 Mar. 1985, www.ncbi.nlm.nih.gov/pubmed/3838314.
3. Pritchard, K., and C.c. Ashley. "Na /Ca2 Exchange in isolated smooth muscle cells de monstrated by the fluorescent calcium indicator fura-2."; FEBS Letters,vol. 195, no. 1-2, 1986, pp. 23–27., doi:10.1016/0014-5793(86)80122-3.