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2.3: Calcium imaging experiments
It is widely accepted that calcium signalling plays an important role in the majority of physiological pathways such as exocrine secretion, muscular and non-muscular motility, and the activity and regulation of several metabolic pathways (Alasmar et al,2013, Bezprozvanny, 2012 and Supnet and Bezprozvanny 2011). Calcium acts as a universal second messenger in a variety of cells (Berridge et al, 2000). Critical evaluation of the role of calcium as an intracellular messenger requires measurement of cytosolic free calcium concentrations, and comparison with varied stimuli and cell responses. Numerous techniques and methods for analyzing the mechanisms of cellular Calcium activity have been established and the uses of fluorescent dyes have become the most popular.
Since the 1920s, scientists have attempted to measure Ca+2, but few were successful due to limited availability of Ca+2 probes. The first reliable measurements of Ca+2 were performed by Ridgway and Ashley in 1967 by injecting the photoprotein aequorin into the giant muscle fibre of the barnacle and measuring the resultant changes in fluorescence to applied calcium agonists. Subsequently, in the 1980s, Tsien and colleagues produced a variety of fluorescent indicators, which are currently still regarded as the most reliable. Since the development of these Ca+2 probes, investigations of Ca+2 -related intracellular phenomena have skyrocketed.
Calcium Imaging is a technique which typically combines microscopy with the use of ion sensitive fluorescent dyes in order to measure and visualize intracellular calcium concentrations. Ion sensitive dyes are fluorescent molecules which reversibly bind to specific ions such as calcium and produce large increases in fluorescence. These dyes are very sensitive to any change in calcium concentration (Bootman, 2013 and Tsien, 1989). Different side groups at the molecule may in fluence the affinity of the dye to calcium. Binding of the calcium causes conformational changes in the dye altering its fluorescence excitation and emission properties when they are exposed to ultra violet light which can be used to report the concentration (Grynkiewicz et al., 1985).
There are several different fluorescent Ca+2 indicators, and they can be divided into various groups based on several different criteria. One way of dividing these probes is to separate them based on whether they are ratiometric and nonratiometric indicators (Paredes et al, 2008 ,Takahashi et al, 1999 and Tsien 1989). A ratiometric dye is excited to fluoresce by two excitation wavelengths whereas a non-ratiometretic dye can be excited to fluoresce only by one excitation wavelength. This means that ratiometric fluorescent dyes allow the measurement of fluorescence intensities as a result of changes in calcium concentration at two excitation wavelengths whereas a non-ratiometretic dye allow the measurement of fluorescence intensities at only one excitation wavelength (Grynkiewicz et al., 1985;Tsien et al., 1985). Non-ratiometric fluorescent dyes such as quin-2,are known as first generation indicators as they were the first type to be used widely (Tsien et al., 1982a). However, quin-2 had many known disadvantages and limitations which reduced the use of this dye . The fluorescence of quin-2 is not exceptionally bright, so that intracellular loading of several hundred micromolar is usually required for the quin- 2 signal to dominate cellular autofluorescenc, but high concentrations are known to buffer [Ca2+]; (Tsien and Pozzan 1989). The short excitation wavelength (339 nm) of quin-2 , causes significant autofluorescence and also penetrates the optics of the microscope inefficiently. Quin-2 signals changes in Ca+2 by increasing fluorescence intensity but has little effect on either the excitation or emission wavelengths. Because fluorescence intensity is not only affected by changes in calcium levels, it can be altered by other factors such as illumination intensity, dye concentration, cell thickness and dye leakage, the method was unreliable (Tsien and Pozzan 1989). Ratiometric indicators permit a very accurate quantification of the Ca+2 concentrations that is corrected for uneven dye loading, dye leakage, photo bleaching and changes in cell volume. Quin-2 is non-selective as it also bound to magnesium Mg+2 and gave falsely low readings of calcium concentration when high levels of exchangeable heavy metals were present.It was necessary to address these problems, a new generation of calcium indicators was designed by Roger Tsien and collaborators to replace quin-2 (Grynkiewicz et al, 1985;Tsien et al, 1985).
Fura-2 has made a major contribution to advances in the understanding of the role of calcium in cellular regulation. The ability to determine ratio measurements, makes fura-2 an important indicator (Robinson et al, 2004). At low concentrations of the indicator, use of the 340/380 nm excitation ratio for fura-2 allows accurate measurements of the intracellular Ca+2 concentration (Grynkiewicz et al, 1985, Hurley et al, 1992 and Cobbold and Rink 1987). Rationing considerably reduces the effects of uneven dye loading, leakage of dye, and photo bleaching, as well as problems associated with measuring Ca+2 in cells of unequal thickness. Measurements of fura-2 fluorescence can usually be made over a period of an hour without significant loss of fluorescence resulting from either leakage or bleaching. In addition, fura-2 is bright enough to permit measurements at intracellular concentrations of dye unlikely to cause significant Ca+2 buffering or damping of Ca+2 transients.
Fura-2, like most modern second-generation fluorescent indicators, is cell impermeant and in order to load these dyes into cells the indicators are derivatised with an acetoxymethyl ester (AM), which is cell permeable (Tsien,1981). This form of the dye is free to diffuse passively across cell membranes and once the dye enters the cells, the ester group is cleaved by intracellular esterases, trapping the dye in the cells. The chemical structure of fura-2 has a tetracarboxylate chelating site (Figure 3.1) which binds calcium (Grynkiewicz et al, 1985). The maximal absorption peaks for fura-2 are 335 and 363 nm at maximal and minimal [Ca2+], respectively, and the emission peak of both the calcium-bound and calcium-free forms of fura-2 is 500 nm (Bootman et al, 2013). The wavelengths that are usually preferred are 350nm and 380nm, because the absorption peak of free fura-2 is close to its isosbestic point (the point at which the amount of free fura-2 and bound fura-2 are the same) and when exposed to light at 370 nm, free fura-2 fluoresces greater than bound (Nakamura et al, 1996). The fluorescence excitation spectra for fura-2 shift to shorter wavelengths as [Ca2+]i levels increase.
Figure 3.1. Chemical structure of Fura-2 (Grynkiewicz et al, 1985).
2.3.3: Fura-2AM loading
Cells were seeded on to 25mm glass coverslips (approx. 1000 cells per coverslip) in 35mm petri dishes and 2ml of 5% William’s Medium E was added to maintain the cells. After 2-4 days, coverslips showing healthy cell growth were incubated with 3µM/ml Fura-2AM (Calbiochem, Merck Biosciences, UK) and 0.8nM F127 Pluronic Acid (Sigma, UK), (figure 3.2) added directly into the culture medium, for 45 minutes in the dark at 37°C. Pluronic F-127 is a nonionic dispersing agent that helps solubilize large dye molecules in physiological media (Cobbold and Rink 1987) . Coverslips were washed using Margo’s ringer solution then stored in the dark until use. Glass coverslips were then mounted in a specialised chamber attached to the stage of a Nikon Diaphot inverted microscope (Figure 3.3) and to a peristaltic pump for continuous perfusion, which was attached to the microspectrofluorimetry setup (Figure 3.2) . Regions of 3-6 cells were selected to be recorded by the Meta Fluor programme (Figure 3.5). For each experiment, cells were perfused with Margo’s ringer solution for 2-5 minutes to establish the baseline fluorescence for cells. Each agonist, prepared in the ringer solution, was superfused onto the cells then washed off (Figure 3.4). An increase in the fluorescence ratio indicates an increase in intracellular calcium which was analysed using the Meta Fluor analysis.
Figure 3.2: Fluorescent image of NCL –SG3 cells were loaded with fura-2-AM (X40)
Figure 3.3: Nikon Diaphot inverted microscope connected with the computer.
2.3.4: Measurement of intracellular calcium
The light from a Nikon xenon arc lamp (figure 3.3) passed through the filter wheel within the microscope containing filters with 350nm and 380nm excitation wavelengths. The filter wheel, which is controlled by the computer, switches between the two filters- and provides excitation at these two wavelengths. Images of fura-2 fluorescence are detected at 500 nm emissions. The ratio is calculated by dividing the number of photons of light detected at the 350 nm wavelength by the number detected at the 380nm wavelength. Readings were taking from the resulting graphs by subtracting the baseline ratio, prior to addition of agonist, from the peak of the calcium response (figure 3.5) . Agonists were used purinergic agonists (Sigma, UK) and Thapsigargin (TG) (Tocris, UK).
Figure 3.5: Graph traces the transient change in calcium in individual cells over time after adding agonists.