TEFLabs is a global leader in ion indicators owing to the unique expertise of its founder, Dr. Akwasi Minta. Dr. Minta was a member of Dr. Roger Tsien’s small team of pioneers in the early 1980s that introduced the fluorescent ion indicators Fura -2, Rhod-2, Indo-1, SBFI, PBFI, Fluo-2 and Fluo-3. Dr. Minta commercialized these indicators at Molecular Probes and at TEFLabs, and expanded the repertoire by introducing indicators for zinc, chloride, and other analytes. In 2006, TEFLabs set a focus on producing a new generation of enhanced ion indicators. The exceptional fruits of this effort are presented in this handbook. TEFLabs is wholly dedicated to the continued creation of the best fluorescent ion indicators.
We provide most of our indicators in two forms:
The acetoxymethyl (AM) ester form, first introduced in 1981 by Dr. Tsien is non-invasive and is the most popular method for loading fluorescent ion indicators into cells. The phenolic and carboxylic acid functions of the molecule are derivatised as AM esters. These esters make the molecule hydrophobic enough to be membrane permeant. Once inside the cell, non-specific esterases, found in almost all cell types, hydrolyze the esters back to the polyanionic form necessary for water-solubility; retention in the cell; and, in some cases, for sensing ions.
The water-soluble salt (e.g., K+ or TMA+) is the active form of the indicator. It is available for calibration purposes or for invasive loading, such as microinjection into cells or loading through a whole-cell patch electrode.
TEFLabs sells AM forms of dyes in aliquots of 1 mg, 500 µg, or 50 µg. For testing purposes we offer 2 x 50 µg units. We also offer the AM forms in dry dimethylsulfoxide (DMSO) solution (sealed under argon) and provide Pluronic F127, a surfactant that aids in dispersing the dye in the aqueous loading buffer.
AM Ester Loading Guidelines
When cells are incubated with the AM ester of an indicator, the AM ester permeates into the cell and is hydrolyzed by intracellular esterases to yield the active form of the indicator, which becomes trapped and accumulated in the cell. For loading, a 1-10 mM stock solution of the AM ester is prepared using anhydrous DMSO and stored at -20ºC. In order to prevent the deterioration of the AM esters that occurs with repeated thawing and freezing, we recommend that 1 mg or 500 µg quantities be divided into aliquots containing 50 µg each. Loading is usually performed in a serum-free culture medium with the AM ester at a final concentration ranging from 1-10 µM. Pluronic F-127 may be added to the loading medium to aid dispersal of the AM esters; it is typically used at a concentration of < 0.1% (wt/vol). For ease of use, a stock solution of Pluronic F-127 should be made in dry DMSO at a concentration of 20% (wt/vol). The required volumes of the DMSO stock solutions of the AM ester and of the Pluronic should be premixed and then dispersed into aqueous medium for loading. The cells can be incubated at room temperature or 37ºC (although quality of loading is often better at room temperature), and the time of incubation typically ranges from 30 to 60 minutes. After loading, the cells should be washed at least once with fresh serum-free culture medium to minimize extracellular background fluorescence.
1) If the loading medium is buffered with bicarbonate, then loading should be done under a 5% CO2 atmosphere to prevent alkalinization of the medium through loss of CO2;
2) if serum-containing medium is used for loading, then the loading concentration of AM ester may need to be increased to compensate for binding of AM esters to serum proteins;
3) in some cases (particularly when the AM ester has a molecular weight near or exceeding 1000), the loaded cells may require a further incubation in medium without AM ester for 20 – 60 minutes to allow complete processing of the AM ester by intracellular esterases;
4) there are also procedures for performing “no wash” assays; and
5) incubation conditions can vary among cell types and among indicators and ideally should be optimized for each cell type.
Fluorescence and Ratiometry
Two types of spectral response are possible when an ion binds to a fluorescent indicator. First, the fluorescence intensity changes with essentially no significant change in the shape of the fluorescence spectrum. (Figure. 1.1) Second, the shape of the emission (Figure 1.2) or excitation (Figure 1.3) spectrum changes so that the optimal wavelength of excitation or emission changes. Spectral changes as a function of ion concentration can be analyzed by means of a Hill plot or by non-linear regression to obtain the dissociation constant, Kd, of the indicator.
FIG. 1.1 Fluorescence emission traces of a titration of Asante Calcium Red (excitation at 540 nm) showing a calcium-dependent enhancement of fluorescence but not a shift of wavelength.
FIG. 1.2 Emission spectra of Asante Calcium Red (excitation at 488 nm) acquired at zero (Ca2+-free) and saturating (Ca2+-bound) concentrations of Ca2+. The spectral change can be used for emission ratiometry. [J. Kao, Univ of Maryland]
FIG. 1.3 Fluorescence excitation traces of a titration of Fura-2 showing calcium-dependent ratiometry.
When there is a ion-dependent change in the wavelength of maximal emission or excitation, the ion’s concentration can be determined from a ratio of the fluorescence intensities acquired at two distinct wavelengths. The advantage of such ratiometric measurements is that the ratioing of intensities eliminates potential artifacts due to variable degree of cell loading, loss of intracellular indicator by leakage or photobleaching, changes of cell thickness during an experiment, as well as certain changes in detector sensitivity.
The remarkable benefits of ratiometry make it an attractive goal in the design of an indicator. One of our new products is a novel ratiometric dye, Asante Calcium Red (ACR, Figure 1.2). ACR is the first true emission ratiometric indicator that is excited at visible wavelengths (488 nm). By exciting at 540 nm, ACR can also be used non-ratiometrically (Figure 1.1).
For an indicator that is not ratiometric, a large fluorescence dynamic range is vital, i.e., the difference between the minimal fluorescence (Fmin, at zero ion concentration) and the maximal fluorescence (Fmax, at saturating ion concentration) should be as large as possible. This ensures high detection sensitivity so that even small changes in ion concentration translate into fluorescence changes that are easily measurable. We are excited to introduce Asante Calcium Green (ACG), with the largest dynamic range measured to date (Fmax/Fmin = 220).(FIG. 1.4)
FIG. 1.4 Fluorescence titration of Asante Calcium Green, showing an exceptional dynamic range (Fmax/Fmin = 220). [J. Kao, Univ. of Maryland]
Kd and sensitivity
Calibration is necessary to obtain the dissociation constant (Kd) of each indicator dye. Physical conditions such as temperature, pH, ionic strength, and solution viscosity, as well as interactions of the dye with cellular constituents such as proteins, all affect Kd. In practice, the Kd in the cell is typically higher than the value obtained in vitro. Therefore, if precise calibration is required, then the Kd should be determined in the cells under study.
The Kd’s of our ion indicators vary, so it is important to select the indicator based on the ion concentration range expected from an experiment. In general, the Kd is the midpoint of the concentration range to which the indicator is sensitive. A practical rule-of-thumb is that indicators are typically responsive to ion concentrations ranging from Kd/30 to 30×Kd (or, in logarithmic terms, pKd ± 1. 5).