Sandip Patel and Renato V Iozzo

1. Introduction

Ca2+ is a crucial biological messenger involved in a host of diverse cellular processes (1). Many hormones, growth factors, and neurotransmitters raise cytosolic Ca2+ levels through activation of phospholipase C, which catalyzes the production of the second messenger, inositol 1,4,5-trisphosphate (IP3) (2). G-protein-linked receptors and receptor tyrosine kinases couple to distinct isoforms of phospholipase C (3). Once generated, IP3raises cytosolic Ca2+ levels by activating Ca2+ channels located on the membranes of intracellular Ca2+ stores (4,5).

Extracellular matrix constituents are also known to induce Ca2+ signals through activation of integrins (6). More recently, we have demonstrated that decorin, a member of the small leucine-rich proteoglycan family (7), mediates cytosolic Ca2+ increases through a novel action on the epidermal growth factor (EGF) receptor (Fig. 1) (8). Indeed, interaction of the extracellular matrix with cell surface receptor tyrosine kinases may be more widespread than previously thought (9-11). Changes in cytosolic [Ca2+] are therefore likely to be important in mediating the effects of the extracellular matrix on cell function. In this chapter, the principles of measuring single cell cytosolic [Ca2+] are discussed.

Measuring cytosolic [Ca2+] at the single cell level using imaging approaches has several advantages over monitoring [Ca2+] in populations of cells. In many cells, submaximal hormone stimulation induces complex changes in cytosolic [Ca2+], including Ca2+ oscillations (12), which in population studies are "averaged" out and remain undetected. Such temporal averaging also distorts the kinetics of even relatively simple changes in cytosolic [Ca2+]. For example, if all cells do not respond under a particular condition or do so with differing latencies, the average population response will underestimate the peak and rate of rise in cytosolic [Ca2+] of the responsive cells. In addition, population studies provide no spatial information concerning the Ca2+

From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Fig. 1. Decorin mediates increases in cytosolic [Ca2+] of single A431 cells. Images of single fura-2-loaded A431 cells before (top left) and after (at the times indicated) stimulation with decorin (100 ^g/mL). The overlaid red image is proportional to changes in [Ca2+]. Bottom right-hand panel shows a typical [Ca2+] response of a single cell.

Fig. 1. Decorin mediates increases in cytosolic [Ca2+] of single A431 cells. Images of single fura-2-loaded A431 cells before (top left) and after (at the times indicated) stimulation with decorin (100 ^g/mL). The overlaid red image is proportional to changes in [Ca2+]. Bottom right-hand panel shows a typical [Ca2+] response of a single cell.

increase within the cell. Indeed, Ca2+ signals often originate from a specific cellular locus and travel throughout the cytosol as planar or even spiral "waves" (12). Finally, population measurements do not distinguish signals from damaged or contaminating cells.

1.1. Fluorescent Ca2+-Sensitive Indicators

The use of fluorescent Ca2+-sensitive indicators is currently the most popular method for monitoring cytosolic [Ca2+]. Fluorescence is the process whereby a molecule in an excited state dissipates some of its energy prior to relaxation, upon absorbing a photon of light, such that the emitted light is of lower energy and thus longer wavelength than that of the exciting light. Fluorescent Ca2+ indicators are molecules that change their fluorescence properties upon binding Ca2+, most of which are based on the Ca2+ chelators, ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) and 1,2-bis(2-Aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA). Many Ca2+ indicators are commercially available for measuring [Ca2+] [see ref. (13)]. Factors to be considered when choosing a dye are its molar extinction coefficient and quantum yield—that is, the efficiency of light absorption and the amount of light emitted relative to what was absorbed. In order to maximize fluorescence signals in response to Ca2+, a dye with an affinity appropriate to the range of anticipated [Ca2+] changes should be chosen. Upon stimulation, cytosolic [Ca2+] can be elevated several fold from resting levels of < 200 nM.

Ca2+-sensitive indicators can be classified according to the nature of the fluorescence change that Ca2+ binding induces. Ratiometric (dual-wavelength) indicators undergo a shift in their fluorescence spectra (excitation or emission) upon binding Ca2+, whereas intensiometric (single wavelength) dyes do not. Fura-2 (14) is a ratiometric indicator that is currently the most commonly used Ca2+-sensitive dye in imaging studies (Fig. 2A). Important properties of this dye include the following:

1. In the absence of Ca2+, the excitation and emission peaks are 360 and 510 nm, respectively.

2. In the presence of saturating Ca2+ levels, the excitation peak is shifted to 340 nm with no appreciable change in the emission spectra.

3. The isosbestic (Ca2+-independent) wavelength is 360 nm.

4. Reciprocal changes in the fluorescence of the dye occur upon Ca2+ binding when excited either side of the isosbestic wavelength.

5. At 340-nm excitation, fluorescence intensity increase as the [Ca2+] increases.

6. At 380-nm excitation, fluorescence intensity decreases as the [Ca2+] increases.

7. The ratio of the two intensities at 340 and 380 nm is proportional to [Ca2+].

Fluo-3 is an example of an intensiometric indicator. This visible wavelength dye has excitation and emission peaks of ~500 and ~525 nm, respectively. This dye is particular useful in that in it undergoes a large increase in fluorescence upon binding Ca2+. Additionally, the absolute florescence of the Ca2+-free dye is extremely low, thus further improving the signal-to-noise ratio. Ratiometric dyes, however, are advantageous over intensiometric dyes in several respects. For intensiometric indicators, fluorescence is proportional to the concentration of both Ca2+ and the dye. Since changes in cellular dye concentration can occur during experimentation, such

Fig. 2. (A) Excitation spectra of fura-2 in the presence of zero and saturating Ca2+. The graph shows fluorescence (F) of the dye measured at 510 nm at a range of excitation wavelengths. Binding of Ca2+ reduces the excitation peak from 360 to 340 nm. Fluorescence of the dye therefore increases at 340 nm and decreases at 380 nm in response to Ca2+. No change in fluorescence occurs at 360 nm. (B) Ratio measurements cancel out nonreciprocal changes in fura-2 fluorescence. A schematic fura-2 timecourse recording showing a change in [Ca2+] manifest as a transient increase and decrease in fluorescence at excitation wavelengths of 340 and 380 nm, respectively (left). The response is superimposed on a progressive slow decrease in fluorescence at both wavelengths. These non-reciprocal (Ca2+-independent) changes in fluorescence are eliminated after calculation of the fluorescence ratio (340/380, right).

Fig. 2. (A) Excitation spectra of fura-2 in the presence of zero and saturating Ca2+. The graph shows fluorescence (F) of the dye measured at 510 nm at a range of excitation wavelengths. Binding of Ca2+ reduces the excitation peak from 360 to 340 nm. Fluorescence of the dye therefore increases at 340 nm and decreases at 380 nm in response to Ca2+. No change in fluorescence occurs at 360 nm. (B) Ratio measurements cancel out nonreciprocal changes in fura-2 fluorescence. A schematic fura-2 timecourse recording showing a change in [Ca2+] manifest as a transient increase and decrease in fluorescence at excitation wavelengths of 340 and 380 nm, respectively (left). The response is superimposed on a progressive slow decrease in fluorescence at both wavelengths. These non-reciprocal (Ca2+-independent) changes in fluorescence are eliminated after calculation of the fluorescence ratio (340/380, right).

changes are difficult to distinguish from actual changes in [Ca2+] when using intensiometric indicators. For example, in many cells, dye can be actively extruded from the cytosol or undergo photobleaching (see Note 1), which results in a decrease in fluorescence intensity. This decrease in fluorescence would appear as a decrease in [Ca2+] when measured at a single wavelength. For ratiometric dyes such as fura-2, however, nonreciprocal changes in the fluorescence intensities are cancelled out after calculation of the fluorescence ratio (Fig. 2B). Ratiometric recording is also less prone to artefacts arising from cell movement, uneven dye distribution, or inhomogeneities in cell thickness. Fura-2, then is the dye of choice for single cell Ca2+ imaging.

1.2. Loading Fluorescent Ca2+ Indicators Into Cells

Most Ca2+ indicators are available as acetoxymethyl (AM) esters, rendering them cell permeable (15). Once inside the cell, endogenous esterases cleave the ester bonds, releasing the negatively charged dye and essentially trapping it within the cell. Thus, simple incubation of the cells in a physiological medium supplemented with the AM

Fig. 3. Schematic representation of a typical imaging system. Light from an arc lamp (1) is attenuated by a neutral density filter (2) and passed through a shutter (3) to a filter wheel (4) that selects the appropriate excitation wavelength. The excitation light (Ex) then enters the microscope and is reflected by a dichroic mirror (5) up into the objective (6) to the sample (7) loaded with the fluorescent dye. The emitted fluorescence (Em) is then collected by the objective and directed back to the dichroic mirror, where it is transmitted to a second mirror (8) that reflects the light to the camera (9). The shutter, filter wheel, and camera are computer-controlled.

Fig. 3. Schematic representation of a typical imaging system. Light from an arc lamp (1) is attenuated by a neutral density filter (2) and passed through a shutter (3) to a filter wheel (4) that selects the appropriate excitation wavelength. The excitation light (Ex) then enters the microscope and is reflected by a dichroic mirror (5) up into the objective (6) to the sample (7) loaded with the fluorescent dye. The emitted fluorescence (Em) is then collected by the objective and directed back to the dichroic mirror, where it is transmitted to a second mirror (8) that reflects the light to the camera (9). The shutter, filter wheel, and camera are computer-controlled.

ester for a defined period of time results in significant accumulation of the dye within the cell (see Subheading 3.1.). The ease by which these dyes can be introduced into cells is a major advantage of using these dyes for monitoring [Ca2+].

1.3. Instrumentation

Central to an imaging system (Fig. 3) is the epifluorescence microscope that houses appropriate optics to allow delivery of the exciting light and isolation of the emitted fluorescence. Briefly, cells grown on a glass cover slip are loaded with a fluorescent indicator and placed into a holder on the stage of the microscope. Excitation light of the appropriate wavelength for the indicator is selected (usually with a filter) and directed to the cells via the objective lens through which the cells are imaged. Part of the resulting emitted fluorescence is collected by the objective and discriminated from the excitation light by a dichroic mirror. The light is then focused onto a camera, digi tized, and stored to a computer. By periodically illuminating the cells through the use of a computer-controlled shutter, a series of images is collected. Regions of interest, corresponding to individual cells (or subcellular regions) are selected by the user, and data are extracted from the image at each time point. Fluorescence intensities/ratios can then be calibrated to [Ca2+] for individual cells in the entire field. A brief description of the principle components of an imaging system is given in Table 1.

Many imaging systems are now available commercially. Normally, an inverted as opposed to an upright microscope configuration is adopted, since access to the sample (for perfusion or microinjection) is unhindered. The choice of camera is one of the most important considerations in low-light-level studies. Cameras can be broadly classified according to their output, which may in the form of a standard video signal (e.g., silicon-intensified target cameras) that is later digitized or direct digital readout (e.g., cooled charged-coupled-device cameras). The latter provide better signal-to-noise ratios but are inherently slower. High-sensitivity cameras allow illumination intensity to be reduced without compromising signal, thus reducing photobleaching of the dyett. Imaging formats such as 1024 by 1024 pixels are typical. The range of digitisation (8-16 bit) determines the dynamic range (256-65536 intensity levels per pixel) (see ref. 16 for further details). 1.4 Calibration of Fluorescence Data

Once fluorescence data are acquired, it is relatively straightforward to convert them to [Ca2+]. Data should first be corrected for background fluorescence that is, cell-independent ("instrument") noise. For intensiometric indicators, fluorescence intensity (F) is related to [Ca2+] by the following equation:

where Fmin and Fmax are the fluorescence intensities of the Ca2+-free and Ca2+-satu-rated dye, respectively, and Kd is the dissociation constant of the dye for Ca2+. With these indicators, an in situ calibration is necessary since measured fluorescence is dependent on dye concentration (see Subheading 3.4.1.). This involves exposing cells to a Ca2+ ionophore such as ionomycin in order to equilibrate Ca2+ across the plasma membrane, and then setting the extracellular [Ca2+] to zero and saturating levels to derive Fmin and Fmax, respectively. The parameters are thus determined under similar dye loading levels as those during experimentation.

For ratiometric indicators, the fluorescence ratio, R (Ca2+-bound fluorescence/Ca2+-free fluorescence) is related to [Ca2+] by the following equation:

where Rmin and Rmax are the fluorescence ratios of the Ca2+-free and Ca2+-saturated dye, respectively, Kd is the dissociation constant of the dye for Ca2+, and Sf2/Sb2 is the ratio of fluorescence values for the Ca2+-free and Ca2+ saturated dye at the wavelength used to monitor the Ca2+-free indicator. For fura-2, R is the fluorescence of the dye at

340 nm excitation/fluorescence of the dye at 380 nm excitation and Sf2 /Sb2 is determined at 380 nm. At 37°C, the Kd of fura-2 is 224 nM.

Fig. 4. In vitro calibration of ratiometric fluorescence data. (A) A cytosol-like calibration medium (initially Ca2+-free) is used to determine calibration parameters (see Table 2). For fura-2, fluorescence of the medium at 340-nm (solid line) and 380-nm (dotted line) excitation is acquired first in the absence of dye to determine background fluorescence. Fura-2 free acid (1 ^M) and then CaCl2 (500 ^M) are added to determine ^mjn, Rmax, and Sf2/Sb2 as shown. These parameters are used to convert acquired ratio values to [Ca2+] using Eq. (2). (B) Autofluorescence (AF) is measured with either MnCl2 or digitonin (see text) and subtracted from all experimental data prior to calculation of the fluorescence ratio.

Fig. 4. In vitro calibration of ratiometric fluorescence data. (A) A cytosol-like calibration medium (initially Ca2+-free) is used to determine calibration parameters (see Table 2). For fura-2, fluorescence of the medium at 340-nm (solid line) and 380-nm (dotted line) excitation is acquired first in the absence of dye to determine background fluorescence. Fura-2 free acid (1 ^M) and then CaCl2 (500 ^M) are added to determine ^mjn, Rmax, and Sf2/Sb2 as shown. These parameters are used to convert acquired ratio values to [Ca2+] using Eq. (2). (B) Autofluorescence (AF) is measured with either MnCl2 or digitonin (see text) and subtracted from all experimental data prior to calculation of the fluorescence ratio.

Rmin and Rmax (from which Sf2/Sb2 is calculated) can be determined in situ with iono-phore as with intensiometric indicators. However, as ratios are independent of dye concentration, calibration parameters can also be obtained (for the particular instrument configuration employed) in vitro that is, in the absence of cells (see Fig. 4A; Subheading 3.3.2.). This is achieved using an intracellular-like solution supplemented with the free (de-esterified) form of the dye in the presence of BAPTA and excess Ca2+ to determine the fluorescence of the Ca2+-free and Ca2+-bound dye, respectively. Unlike in situ calibrations, which need to be performed at the end of each experimental run, in vitro calibrations need only be performed once on the day of experimentation. Furthermore, it is often difficult to completely equilibrate Ca2+ across the plasma membrane in in situ calibrations. However, a major assumption in in vitro calibrations is that the conditions mimic the environment of the dye within the cell, which may not necessarily be the case.

Table 1

Components of a Single-Cell Imaging System

Component_Function_

Arc Lamp Provide illumination

Neutral-density Filter Attenuate light source Computer-controlled Provide illumination only shutter

Excitation selector Provide appropriate wavelength of light for optimal excitation of the fluorophore

Dichroic mirror

Reflect excitation light to, and isolate emitted fluorescence from sample

Objective

To deliver excitation light and collect emitted fluorescence

Sample chamber

Chamber that fits onto microscope stage and houses the cover slip containing adherent cells

Notes

Xenon arc lamps are preferred over mercury lamps, because they provide a more even illumination over the range of wavelengths required for excitation commonly used Ca2+ indicators. Attenuates light evenly at all wavelengths.

Prevents premature burning out of filters shutter during acquisition and unnecessary photobleaching.

Normally achieved with a barrier filter. For dual-excitation studies (as with fura-2), it is necessary to have a device for the rapid changing of the excitation wavelength. This can be achieved with a computer-controlled rotating filter wheel, which houses the appropriate excitation filters (18,19). Alternatively, a monochromator system can be employed, which is more flexible in that the number of available wavelengths is essentially unlimited. Designed to reflect a specific range of wavelengths while allowing light of other wavelengths to pass through. Chosen such that the cutoff for passing light is greater than the wavelength of exciting light. Excitation light is therefore reflected up to the sample, whereas the resulting emitted light (of longer wavelength) passes through. For fura-2, a dichroic mirror with a cutoff of 400 nm is appropriate to separate excitation light (340/380 nm) and fluorescence emission (peak 510 nm).

16 x or 20 x magnification is sufficient to accurately resolve most single cells. Higher magnification (40 x /63 x) is required for subcellular recording. Must efficiently transmit light at wavelengths to be used. Fura-2, for example requires quartz or Fluor objectives in order to pass light of 340 nm. Lenses with high numerical apertures (NA), an index of light-collecting capacity, are preferred.

In an inverted microscope configuration, the cover slip itself forms the base of the chamber. It is usually thermostatted to physiological temperatures.

Emission selector Select appropriate emission wavelengths

Camera Capture emitted fluorescence

A filter designed to pass a broad range of wavelengths (long-pass) is normally used, in order to maximize the fluorescence signal, but sufficiently removed from the cutoff wavelength of the dichroic mirror in order to filter out stray excitation light. For Fura-2, a 420- to 600-nm filter can be used with a 400-nm cutoff dichroic mirror.

For dual-emission dyes such as indo-1, a filter system similar in design to that used to select alternate excitation wavelengths can be fitted to the exit port of the microscope.

Silicon-intensified target (SIT) cameras or cooled charged-coupled-device (CCD) cameras are commonly used.

Before calculation of fluorescence ratios in dye-loaded cells, it is important to subtract any fluorescence not emanating from Ca2+-sensitive dye from the measured signal. Such autofluorescence can derive from many sources, including flavoproteins and reduced adenine nucleotides. Autofluorescence is determined by first eliminating the fluorescence due to the dye and then subtracting the residual fluorescence from the data. For fura-2, this can be achieved by introducing Mn2+ into the cell (using iono-phore), which quenches dye fluorescence (see Fig. 4B, Subheading 3.4.1.). Note that with intensiometric indicators, autofluorescence is cancelled out in both the numerator and denominator of Eq. (1), and thus does not need to be determined separately.

In some cells, AM loading can result in accumulation of the dye into noncytosolic compartments such as the endoplasmic reticulum. Since Ca2+ levels in the endoplas-mic reticulum are much higher than in the cytosol, compartmentalized dye will, under normal conditions, remain saturated, thereby contributing to background fluorescence. In this case, the Mn2+ quench method for determining autofluorescence is unsuitable, because ionomycin will equilibrate Mn2+ into most cellular compartments. An alternative method to determine autofluorescence in cells where there is significant compart-mentalization of dye (or when using dyes, such as fluo-3, that are not completely quenched by Mn2+) is to permeabilize selectively using, the plasma membrane with detergents such as digitonin (see Fig. 4B, Subheading 3.4.2.). This method effects release of just the cytosolic dye from the cell. More problematic is compartmentaliza-tion of dye into organelles such as mitochondria, where indicators used for measuring cytosolic Ca2+ will also report mitochondrial Ca2+ changes. In such cases "mixed" signals will result, since cytosolic and mitochondrial Ca2+ changes may occur asynchronously (see Note 2).

2. Materials

1. Loading of the cells with Ca2+ indicator and data acquisition are performed in an extracellular-like, imaging medium (see Table 2 for composition), which should be prepared fresh on the day of experimentation. Alternatively, the medium can be prepared in bulk, sterile filtered, aliquoted, and stored at 4°C.

2. Autofluorescence and calibration media (see Table 2) are made in bulk and stored at 4°C.

3. AM esters of Ca2+ indicators (1 mM) should be reconstituted in dimethylsulfoxide (DMSO), aliquoted into single-use vials, and stored at -20°C.

4. Free acids of Ca2+ indicators (1 mM) should be reconstituted in H2O, aliquoted into single-use vials, and stored at -20°C.

5. Ionomycin (2 mM) should be prepared in DMSO, aliquoted into single-use vials, and stored at -20°C.

6. Digitonin (10 mg/mL) and MnCl2 (1 M) stock solutions should be prepared in H2O and stored at room temperature.

3. Methods

3.1. Dye Loading

1. Cells should be plated onto glass (no. 1 thickness) cover slips and cultured until 70-80% confluent in the appropriate tissue culture medium.

2. Remove tissue culture medium and rinse cover slips twice with 2-3 mL of prewarmed imaging medium.

Table 2

Solutions for Single Cell Ca2+ Imaging

Table 2

Solutions for Single Cell Ca2+ Imaging

Imaging medium

Autofluorescence medium

Calibration medium

NaCl

121 mM

10 mM

10 mM

KCl

4.7 mM

120 mM

120 mM

MgCl2

1.2 mM

2 mM

2 mM

CaCl2

2 mM

150-300 ^M

BAPTA

500 ^M

500 ^M

KH2PO4

1.2 mM

NaHCO3

5 mM

Glucose

10 mM

BSA

0.25%

HEPES

20 mM

20 mM

20 mM

pH 7.4 @ 37°C

pH 7.2 @ 37°C

pH 7.2 @ 37°C

3. Incubate cover slip with fresh imaging medium supplemented with the appropriate concentration of Ca2+ indicator and incubate with gentle agitation (see Note 3).

4. Remove medium and rinse cover slips twice with 2-3 mL of imaging medium.

5. Transfer cover slip to incubation chamber.

6. Leave for sufficient time (e.g., 10 min) to effect complete de-esterification of the dye.

3.2. Data Acquisition

1. Experiments can be performed either in a static chamber or by continual perfusion of the cells. The former approach is recommended, because much smaller quantities of (usually precious) materials are required. Indeed, experiments can be performed in volumes as small as 0.3 mL.

2. Capture images for at least 60 s prior to stimulation of the cells in order to obtain an accurate measure of the basal [Ca2+] and to characterize any possible spontaneous changes in [Ca2+] (see Note 4).

3. For static chambers additions can be made by complete removal (by pipet) of the medium and addition of fresh medium supplemented with the test agent. Alternatively, a small aliquot of the medium can be removed, mixed with the appropriate volume of a concentrated stock solution of the test agent, and the entire volume added back. This method is less prone to addition artefacts than complete exchange of the medium.

3.3. Calibration of Fluorescence Data

3.3.1. In Situ Calibration

1. This method can be used to calibrate fluorescence data from both intensiometric and ratiometric indicators. For ratiometric indicators, autofluorescence (see below) must be subtracted prior to calculation of the ratio.

2. At the end of the experimental run, rinse cells into imaging medium (without added Ca2+) supplemented with 2 mM BAPTA and add 2 ^M ionomycin to equilibrate Ca2+ across the cell membrane.

3. Reinitiate data acquisition and monitor until fluorescence intensity reaches a stable plateau to obtain Fmin (for intensiometric indicators) or Rmin (for ratiometric indicators).

4. Add 10 mM CaCl2 to saturate the dye with Ca2+.

5. Reinitiate data acquisition and monitor until fluorescence intensity reaches a stable plateau to obtain Fmax (for intensiometric indicators) or Rmax (for ratiometric indicators).

6. Use eq. (1) (for intensiometric) or eq. (2) (for ratiometric indicators) to calculate [Ca2+].

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