cd - (1990) 11, -7

0LongmmnGrcuplJKUd1990

Quantitative intracellular calcium imaging with laser-scanning confocal microscopy

D.A WILLIAMS

Department of Physiofogy, The University of Melbourne, Pa&i/k, Victoria, Australia

Abstraq - Laser-scanning confocai micro-scopy hae been used to visuaiise the fluorescence of a visible wavelength Ca2+sensitive fluorophore, Fiuo4 in isolated wrdiac myocytes. A protocol for the derivation of quantitative information from this single waveiength indicator is presented. This paradigm invoives co-loading wiis with two Ca2+-sensitive fiuoreacent indicators, Fiuo-3 and Furs-2. Wide-fieid ratiometric measurements of Fura- fluorescence provided a baseiine [Ca2’] upon which changee in Fiuo43 fiuorescence could be directly expreued as [Ca2q changes. The Ca2’ changes occurring in spontaneously active wrdiac ceils are presented ae an example of the method. Although fluorescence energy transfer between Fur& and Fiuo-3 was detectable in some in vitro mixturea of the two fluorophoree, this process was not evident in co-loaded wrdiac ceils under the loading conditions-employed.

With their recent commercial availability, confocal microscopes are rapidly becoming an invaluable tool in the investigation of cell structure and function [l, 21. There are a number of potential advantages of confocal over conventional wide-field microscopy including significant increases in both the x-y plane and z depth resolution of an optical cell section, as well as rejection of the vast amount of out-of-focus information in any Zdimensional view obtained with wide-field microscopy [3]. Such improvements may potentially allow for precise localization of ion concentrations within isolated cells particularly when coupled with highly fluorescent visible wavelength cation indicators.

A new visible wavelength indicator for C.k?‘, FM-3 [4], has a number of features which differ from those of its main predecessors Fura- and Indo-l [5]. Ca2+ binding to Fluo-3 results in a large enhancement in fluorescence emission which could serve to potentially amplify cellular Ca’+ changes.

Fluo-3 exhibits a lower Ca2+-binding affinity (2-fold) than Fura- allowin$for greater resolution of elevated (micromolar) Ca + levels. In addition, the esterified form of this dye exhibits negligible fluorescence until internalised and fully hydrolysed by cellular esterases, reducing the requirement for repeated cell washing following loading and allowing dye loading to be observed while in progress. Peak excitation occurs at 506 MI which obviates the requirement for quark microscope optics and high transmission objectives and filters. These spectral characteristics make Fluo-3 the best available fluorescent Ca2’ probe for use with commercially available laser-scanning confocal microscopes which utilise an argon-ion laser. However, the major limitation of this combination results from the negligible wavelength shift in either excitation or emission spectra with Ca2+-binding to Fluo-3. This precludes the use of the well established techniques of ratiometric recording of

589

5!3cl CELL CALCIUM

fluorescence (imaging or other modes) and potentially requires a return to the use of destructive ionophores/detergents and Ca2+ free/saturated solutions for calibration of signals [6].

However, this paper describes several nondestructive paradigms for deriving quantitative fluorescence information with single wavelength indicators, such as Fluo-3 and the confocal microscope. One method involves the co-loading within single cells of Fluo-3 and a relatively low level of Fura-2. The standard ratiometric calibration [7] of the Fura- signal provides a framework within which absolute changes in Fluo-3 fluorescence can be readily calibrated. In addition, the same estimation protocols for stimulated Ca2+ levels could be made by adopting an assumed or literature value for resting Ca2+ levels, but this imposed more assumptions on the calculation. These strategies have allowed quantitative analysis of Ca2+ changes (waves and transients) occurring in isolated CXldiU myocytes exhibiting spontaneous contractility, examples of which are presented here. They should be equally useful in other isolated cell or tissue preparations where the single wavelength Ca2+-indicators (Flue-3 & Rhod-2) are used, especially in conjunction with laser scanning confocal microscopy, to investigate stimulated or spontaneous Ca2+ fluctuations.

Materials and Methods

Cell preparation and dye loading

Isolated ventricular cardiac myocytes were prepared from adult rats with a standard enzymatic technique described elsewhere [8]. Cell suspensions (cell density 105 cells/ml) were incubated with e&rifted fluorescent Ca2+-indicators in a HEPES-buffered ionically balanced solution at 30°C for 30 min. Fluo-3 (2 @4) was included at the initiation of the incubation period while Fura- (05 @I) was added 10 mm from its completion. These concentrations were chosen retrospectively, following determination of the levels of intracellular Flue-3 and Fura- resulting from a wide range of loading concentrations. Determinations of intracellular fluorophore concentrations were made with a

spectrofluorimeter (Hitachi 2000) following lysis of each loaded cell suspension. A 20 mm post-incubation period followed the co-loading period which allowed for complete hydrolysis of both intemalised dyes [9]. A small aliquot of cell suspension was diluted (1OO:l) with fresh medium and cells were allowed to settle and attach to protamine sulphate-coated coverglass. The levels of fluorescent dyes within the individual cell used for experiments were calculated with the method recently described by Moore et al., [lo]. In these experiments Flue-3 and Fura- levels for individual cells ranged between 42-175 PM and 13-27 @i, respectively. All experiments were conducted at room temperature (2&22’C).

Confocal microscope - conventional wide field microscope

Cells were viewed with the combined laser scanning co&&-conventional widefield microscope system as previously described [ll]. Briefly, a Lasersharp MRC-500 confocal microscope was coupled to a standard Olympus IMP-2 inverted microscope through a side access MTV video port. The 488 mn output of an argon ion laser was scamred through galvanometric mirrors to excite the Fluo-3 fluorescence of myocytes. The emitted fluorescence, after passing through a long-pass filter (cut-on 515 nm), was collected after retracing its entry path, by a photomultiplier tube under manual gain and black level control. The effective thickness of the confocal optical section was controlled by varying the opening of a pinbob aperture in the detection path. Each section acquired with this objective represented a limiting theoretical minimum thickness of 0.26 pm although in reality limiting Z-plane resolution was probably around 0.5 pm [12]. Coufocal optical sections, estimated to be of l-l.5 pm thickness through comparison with fluorescent beads of known size, were used in these experiments. A glycerine immersion Nikon CF-W F x40 objective proved to be the ideal lens for these experiments. This oil-immersion lens has a large numerical aperture @.a. 1.3) allowing for high x-y and z-depth discrimination, and high transmission at the excitation wavelengths used with Fura- (340 and 380 run; see [lo]).

CALCIUM IMAGING WlTH LASER-SCANNING CONFOCAL MICROSCOPY 591

A number of different scanning modalities were used to collect confocal images of cardiac myocytes. Images were generally collected as either (i) single frame, 768 pixel x 128 line images repeated every 250 ms (designated ‘fast-scan’ images); or (ii) ‘line scan’ images which represented multiple versions (512) of a single scan line (orientated along the major cell axis). This latter option effectively provided a plot of cell length (and fluorescence intensity) as a function of time (6 r&line). Where such high temporal resolution was not required, non-weighted averaged images of 8-16 frames (designated ‘slow-scan’ images; 768 pixel x 512 lines resolution) were collected.

The Fura- fluorescence of the same co-loaded cells was excited through narrow band pass Elters (11 nm) with a Xenon lamp source attached to the same inverted microscope. Resultant images were captured with a silicon-intensified-target (SIT) camera, and frame-averaged (8 frames) and background corrected with the frame-grabber and other hardware of the Lasersharp MRC-500. Ratio images of resting Fura- fluorescence were constructed, corrected and calibrated into cellular Ca2+ levels with the techniques previously described in detail [lo, 13, 141.

Properties of intracellular Flu~3 and Fura-

The dissociation constants (Kd’s) for Flue-3 and Fura- were determined by exposing dye-loaded cardiac cells to solutions buffered to accurately defined [Ca2’] in the presence of an ionophore WA23187 (described in detail elsewhere, [14]), and were found to be 422 nM and 236 &I, respectively.

The degree of enhancement of fluorescence intensity with Ca2’ binding to Flue3 was defined as Fmax/Fmin, where Fmax and Fmin represent the absolute fluorescence intensities in the presence of saturating Ca2+ (mM) and Ca2+ free (< 10s9 M) conditions, respectively. In vitro estimates, utilising Ca2+-buffered solutions and monochromator-based detection systems, have indicated a 38-fold increase in Euoreacence intensity of Flue-3 with Ca2+-binding [4]. However, FmuJFmti (a function of the quantum yield and extinction coefficient of the respective bound and free forms of Fluo-3) is affected by the wavelengths used to excite and

record emission for the fluorophore. As the fluorescence detection system of the BioRad (MRC-500) employed long-pass filters which integrate the fluorescence emission of Flue-3 over a wide range of wavelengths, it was necessary to determine Fmax/Fmin for this experimental system.

Individual Fluo-3 loaded cardiac cells were exposed to saturating and Ca2’-free conditions in the presence of Br-A23187 (5 PM per 5 x lo5 cells) to determine the limiting fluorescence levels. Fmax/Fmin was found to vary little between individual cells with a mean (* SEM) of 4.95 * 0.04 (n = 14). This value is close to that expected from the emission filter characteristics and is similar to in vitro estimates obtained with Flue-3 acid in Ca2’-containing solutions (mean t SD, 5.10 a.17, n = 3). Fluo-3 liberated from these cells, following lysis with Triton X-100, had the characteristics of the fully ionised acidic indicator [4].

Results and Discussion

Effects of co-loading on cell contractility

The combination of Fura- and Fluo-3 within individual cardiac myocytes had no apparent deleterious affects on cell function. The combined intracellular dye concentrations (100-200 @I, Fura=2 plus Flue-3) were in the concentration range generally used in physiological experiments. At these internal dye levels the isotonic contractile parameters (rate and extent of shortening and relaxation) were identical to those of unloaded cells measured with a rapid cell-length imaging technique [S], following electrical stimulation. Moreover, the incidence of spontaneous contractility and the proportion of morphologically rod-shaped cells was similar in co-loaded and unloaded cell suspensions.

Fluorescence energy transfer

The presence within cardiac cells of two distinct Euorophores with some overlap between their spectral characteristica presented the potential for fluorescence energy transfer [15], between Fura- (potential donor) and Fluo-3 (potential acceptor). In vitro spectral analysis of varied proportional

592

mixtures of the two fluorophores showed energy transfer could occur over certain concentration ranges of each fluorescent species. Examples of this are shown in Figure 1A which illustrates the emission spectra of two mixtures of Fura- with Fluo-3 (A - 0.5 @l/OS @k B - 0.5 t&4/2.5 PM) excited in a spectrofluorimeter at 340 nm. This wavelength is well below the range (450 to 550 mn) capable of directly exciting Fluo-3. The emission spectrum of the equimolar mix (curve a) gives evidence of a bell-shaped curve with a single peak at approximately 502 nm, similar to that characteristic of Fura- emission in this fluorimeter. However, two distinct peaks are apparent in the spectrum for the second mixture (curve b). This curve clearly represents the summed emission of both Fura- and Fluo-3 (peak around 527 nm) and the bimodal shape strongly suggests that, in this case, photons from the fluorescence emission of Fura- were able to excite neighbouring Fluo-3 molecules.

Although fluorescence energy transfer could readily be demonstrated in vitro, there was little evidence of this process in the co-loaded cells used in these experiments. The emission spectrum for a suspension of cardiac cells co-loaded (as described in Materials and Methods) with Fura- and Fluo-3 is shown in Figure 1. The negligible fluorescence contribution of esterified Fluo-3 (Flue-3/AM) allowed for the collection of emission spectra from this mixture as the second (Flue-3) stage of the loading procedure progressed. Therefore, it was possible to investigate potential fluorescence energy transfer between an initial fixed intracellular [Fura-2] and an intracellular Fluo-3 level progressively increasing with time. The emission spectrum at a period 60 min after introduction of Flue-3 (Fig. 1B) clearly shows that a second peak characteristic of Fluo-3 emission is not present, even though Fluo-3 fluorescence could be recorded when cells were illuminated at wavelengths (505-510 nm) known to directly excite this fluorophore (results not shown). At this time point, the average cellular Fura- and Flue-3 concentrations were determined to be 25 and 115 @v& respectively.

As fhrorescence energy transfer is heavily denendent on the distance (to the sixth power)

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400 450

Emkaim Weidmgtb (tm)

b

E

400 4Kl 500 5n 6110

Fig. IA Fluorescence emission spectra of mixtures of Fura- and

Flue-3 in a medium with similar composition to the intracellular

environment (mkl): K’, 100, Na+, 20, MgG, 1; HEPFS 10; total

EGTA 10; pH 7.10. The ratio of Flu+3 to Fura- was (a) 1:l

and (b) 5:l with similar relative curves obtained over the

concentration ranges l-10 @4 for fluorophores and 15 nM to 20

@vf for Ca z+

Fig. 1B Emission spectrum for a suspension of cardiac cells

(density lo4 cells/ml) containing average Fur-2 and Rue-3

concentrations of 25 amd 115 pM, respectively

Excitation wavelength (A & B); 340 nm

CALCIUM IMAGING WITH LASF&SCANNING CONFOCAL. MICROSCOPY 593

between potential donor and acceptor fluorophores [15], these results suggest that the individual Ca2+ fluorophores must stay well separated over this concentration range in co-loaded cardiac cells. Repulsive interactions between the two populations of highly negatively charged molecules may influence this distribution. Alternatively, the two fluorophores may occupy distinctly different compartments within cardiac CellS, although preliminary results on dye localisations suggest that

Pig. 2 Fluorescence images from a single cardiac cell loaded

with Fura- and Fh-3. (A, top left) Frame-averaged (8 frames)

‘slow-scan’ and single ‘fast-scan (B, top right) coufocal image

of Fluo-3 fluorescence. (C, bottom left) Ratio (344X380 nm)

image of Fura- fluorescence for the same cell. Au images were

collected within a 2 min period Cell length: 110 pm

this is unlikely ([16]; Williams, Delbridge, Cody & Harris, unpublished).

Calcium in cardiac cells

Figure 2 shows three images of a single, quiescent, cardiac myocyte, co-loaded with Fluo-3 and Fura-2. Confocal ‘slow-scan’ (A) and ‘fast-scan’ (B) images of the Fluo-3 fluorescence are shown in comparison with a ratio image (340/3t30 mn) of the wide-field fluorescence of Fura- of the same cell (C). The ‘slow-scan’ image (8 frames averaged) gives evidence for a marked heterogeneity of intracellular Fluo-3 fluorescence, similar to that described in a recent confocal investigation of cardiac cells [16]. There were broad longitudinal bands of fluorescence with clearly distinguishable cross-striations. These cross-striations were also evident in confocal images of the cell autofluorescence (frame-averaged ‘slow-scan’) viewed at 1000 times higher gain (not shown).

In contrast, the ‘fast-scan’ single frame, confocal image (Fig. 2B), indicates a more homogeneous fluorescence distribution with one area of higher

Fig. 3 Line-scan images of the cardiac cell shown in Figure 2 (A, left) A single diagonal fluorez+cxnce band which originates at the

right cell edge terminates at the left cell margin. The slope of this band indicates propagath of Ca% through the cytosol at 87 p&s.

(B, right) Ca% wave which originates from a central location of the cell. Thii wave pqagatea towards both cell ends at a rate of 115

pm/s. Initial cell length (L,); 110 pm. Maximum cell shortening: (A) 5 pm (4.5% L), (B) 12 pm (11% 14)

intensity. Interestingly the Fura- fluorescence ratio image (350/380 nm), obtained with wide-field microscopy for the same myocyte (Fig. 2C), and contrast scaled to emphasize any apparent heterogeneity, gives evidence of a [Ca2T distribution which is similar to the distribution of the Flue-3 fluorescence within this cell. The Fura- ratio image for this cell represents an average resting Ca2+ level of 238 r&f with the area of highest intensity averaging 265 nM.

Fluorescence changes in spontaneously contracting cells

A small percentage of individual cardiac cells from enzymatically dispersed suspensions of rat myocardium elicits spontaneous contractions which are believed to be the result of propagation of a wave of Ca2+ by a cyclical calcium-induced calcium-release process (for review, see [17]). The cell, depicted in Figure 2, exhibited these spontaneous contractile events at a frequency of 0.06 Hz. Two line scan images of this cell are shown in Figure 3 and each image portrays the fluorescence intensity distribution of the central long axis of the cell for a period of about 3 s. Concomitant with each spontaneous contraction, which can be seen as a narrowing of the fluorescence plot (indicative of cell shortening), a band of high fluorescence intensity originating at the

left ccl1 edge (A) or at a central cell point (B), could be seen to propagate along the length of the cell. These fluorescence changes represent the passage of a wave of calcium-induced, calcium-release generating spontaneous contractions in these cells, as has been shown elsewhere [18]. The slope of the diagonal bands in each image indicates the velocity of the spread of Ca2’ release/resequestration through the cell, which in these line-scan images pig. 3A 8c 3BJ was 87 and 115 ws, respectively. The higher Ca + propagation rate shown in Figure 3B, coupled with the presence of two coincident bands of propagating, elevated [Ca2+], lead to a greater degree of contraction (cell shortening) as a direct result of a larger number of sarcomeres subjected to elevated Ca2+ at any given time.

A vertical transect through a line scan image illustrates the change in fluorescence intensity at a fixed location (volume element - voxel) within the cell as a function of time. The fluorescence intensity plot for the arbitrarily selected transect highlighted within Figure 3B is illustrated in Figure 4. Also shown is the plot of cell length at the same time points of the transect.

Qua&&ing jluorescence levels

Changes in fluorescence intensity at a single wavelength, as displayed for the Ca2+-sensitive fluorophore Fluo-3, are not readily interpreted in

CALCIUM IMAGING WITH LASEB-SCANNING CONFOCAL. MICROSCOPY 59s

E

d s

terms of actual [Ca2+j changes. However, the fluorescence emission from Fluo-3 in conjunction with confocal microscopy, is derived from almost fixed and well defined cell volumes, and is relatively unaffected by cell contraction [18]. As such, the predetermined fluorescence characteristics of Fluo-3 (i.e. Fmax, Fmo 8z Kd) could be directly employed to estimate [C’a ‘] changes in these cells.

Fluorescence increased from an initial resting level (RL - resting level fluorescence) of 122 arbitrary grey scale units (scale &255; 8 bit grey scale resolution), to an intensity of 170 units (SL -

A. Fluorescmce intensity profde for the cell location depicted

(black line) in Figure 3A

B. Conversion of intensity data to Chh concentrations (for details

see text)

C. The change in cell length (calculated from the width of the

fluorescace profile, Fig. 3A) for the same time period

stimulated level) at the peak of the transect of the fluorescence band which traversed this cell (Fig. 4). To convert changes in Flue-3 fluorescence to changes in [Ca2+j, the resting [Ca2+J of 238 nM determined from the Fura- ratio image of this cell (see Fig 2C), was equated with the resting level fluorescence (RL) of the line-scan transect (Fig. 4). The fluorescence increase (SIJRL) occurring in this cell is a direct function of the change in Ca2+-saturation of the total intracellular Fluo-3 pool, and the resultant fluorescence enhancement (Fmax/F,in) with each additional Ca2’ bound to Fhlo-3. As the - C!a2+ saturation fraction ([CaFluo-3]/~luo-3]~t) is a function of the equilibrium equation where:

[Ca2+] = (Kdcar+o-3 l [QFhro-3])/ [Flu*3]free ..,........... Eqnl

[CaFluo-31 t [Fluo-3]rree = [Fhlo-3]total . . . . . . . . . . . . . .Eqn2

these equations can be solved simultaneously for

5% CELL CALCIUM

any @‘I. For the resting [Ca”‘] determined in using a similar fast Fura- technique has reported Figures2&3 Ca2+-fluctuations of similar magnitude in isolated

[CaFluo-3]/[Fluo_3]total= 0.36 guinea-pig cardiac myocytes [20]. Secondly, an alternative visible wavelength fluorescent Ca2’

and, therefore, probe, Rhod-2 [4], which has a much higher Kd

[Fluo-3]fn&Fluo-3]total = 0.64

The resting level fluorescence intensity (EL) is, therefore, determined as the sum of the fluorescence of the Ca2+-bound and Ca2+-free pools of Fluo-3:

RL = &ax ([QFhm-3]/~uo-3)tota1) + Fm1n ([~u*3]freB/[~u+3]tota1)

. . . . . . . . . . . . . . Eqn 3 = 0.36 x 4.95 t 0.64 x 1.00 = 2.42 intensity units

At the peak of the intensity transect through the cell, fluorescence increases by a factor of 1.40 (SW) and hence SL = 1.40 x 2.42 = 3.39 fluorescence units.

This can simply be deconvolved given Equations 1, 2 & 3, such that SL represents a saturation fraction flCaFluo-3l/[Flu~3]t1) of 0.61. From Equation 1, the [Ca2’] at this point was calculated to be 660 nM. This transformation of Fluo-3 fluorescence intensities to [Ca”T was performed for the complete fluorescence transect and is displayed in Figure 4. Cell shortening reached a maximum of 12 l.4m (representing 11% of initial cell length) within 50 ms of the peak Ca2’ level of 660 nM. This extent of cell shortening compams favourably with that in the same cell type following electrical field stimulation [18]. [Ca2T remained elevated in the area of the measurement transect for 800 ms before Wuming to near baseline levels with the passage of the Ca2’ wave.

The accuracy of this method of estimation was verified using a number of other experimental methods. Firstly, a high time-resolution Fura- fluorescence system [19], was used to make measurements of the peak Ca2’ levels reached in a single central region-of-interest of spontaneously contracting Fura- loaded cells. These spot measurements indicated that Ca2’ oscillated in the viewing area from resting levels of 165-260 nM to peak levels that ranged from 0.5 to 1.6 PM (n = 8) in different cells (results not shown). A recent report

(determined to be 950 nM in cardiac cells), and a much lower fluorescence enhancement with Ca2+-binding to the fluorophore (l&x/l&In 1.50 in cardiac cells) was also used to image calcium fluctuations of spontaneously contracting cells with the BioRad MRCJOO confocal microscope. Changes in intracellular [Ca2’] of the magnitude reported above (238 to 660 nM), would be expected, by the same rationale, to induce changes in the fluorescence intensity of Rhod-2 of less than 10%. Fluorescence changes in spontaneously active cells were all of this magnitude. In some strongly contracting cells a fluorescence change was visually almost undetectable.

The rationale described for Ca2’ estimation from Flu-3 fluorescence intensity data is best applied with accurate knowledge of the initial or resting cytosolic [Ca2T, as can be obtained with ratiometric use of Fura-2. Estimates of the timecourse of Ca2’ transients could also be made by adopting an arbitrarily derived or population average resting [C!a2+J but this will not allow for the large variation in both resting and stimulated [Ca2’] commonly found in individual cells within a population. For example, if the intracellular Ca2’ changes displayed in Figure 4 had been calculated from assumed initial cytosolic levels of 100 nM or 400 r&i, the peak [Ca2’] would be estimated at 247 and 1587 nM, respectively.

The error inherent with single wavelength detection of fluorimetric intensities is reduced by the depth and volume discrimination available in confocal microscopy provided that the optical section (sample volume) is contained entirely within the cell boundaries. As the potential minimum optical section thickness is in the range of 0.25 to 1.0 pm (for high n.a. immersion objectives), this constraint would be met for all cells except the peripheral areas of well-spread cells such as fibroblasts. In these exceptions, the low numbers of photons emanating from extremely thin regions would lead to low si

P -to-noise ratios and high

error in calculated [Ca ‘7 in these areas [lo], in both

CALCIUM IMAGING WITH LASER-SCANNING CONFOCAL MICROSCOPY 597

the Flue-3 confocal images and the individual single wavelength components of a ratiometric Fura- image.

In addition, the depth discrimination may not prevent inaccuracies in Ca2+ determinations under experimental conditions which lead to a large degree of intracellular dye redistribution within the optical section. However, the lack of a detectable change in the single wavelength fluorescence intensity of spontaneously contracting CdlS containing Ca2+-insensitive fluorophores [18], would suggest that this is not a major constraint on the estimation method.

The ability to make measurements from single cells within living tissue samples (e.g. see [ll, 21]), coupled with the depth discrimination and control, outweigh to a large degree the need to make ratiometric measurements to obtain useful quantitative Ca2+ information from some cell and tissue systems. These Ca2+-calibration protocols have proven particularly useful in recent investigations of Ca2’ dynamics involved in hormonal and trophic res~nses of plant cells [ll, 211, and spontaneous Ca + oscillations in isolated cardiac cells [18]. Meanwhile, we await the arrival of Ca2+-sensitive fluorophores, excitable in the visible wavelength range and exhibiting large Ca2+-dependant spectral changes.

10.

11.

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15.

16.

17.

Acknowledgements 18.

I acknowledge the expert technical assistance of Mr S.H. Cody and MS C. MC Phee and am grateful for the loan of an Olympus Xenon light source and power supply from Mr Neville Scott, Selby Anax, Australia.

19.

RePeremes 20.

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2 Pawley JB. (ed) (1990) Han&ook of Biological Confocal Miaoscopy. Revisai Edition. New York, PIenum Press.

3. Fay FS. CUington W. Fogarty KE (lQ8Q) ThreedimmsionaI moIecuIar distribution in single ceIIs aualysed using the dig&I imaging microscope. J. Microsc, 153,l33-149.

4. Minta A. Rae JPY. Tsien RY. (1989) FIuoreecen t indicators for cytosoIic caIcium based on Rho&mine and FIuorescein chrcinophores. J. Bid. Chem., 264,8171-8178.

5. T.&XI RY. (1989) Pluorescent Indicators of ion concentrations. Methock Cell Biol., 30, 127-156.

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Tsien RY. Pozzm T. (1989) Measurement of cytosolic free Ca” with quin2. Methods Bnaymol., 172,230~262. Grynkiewia G. Poenie hi. Tsien RY. (1985) A new generation of Ca% indicators with greatly improved fluorescence properties. J. Biol. Chem., 260,~~3450. D&ridge I.M. Harris PJ. Morgan To. (1989) Charactaization of single heart ceil contractility by rapid imaging Clin. Bxp Pharmacol. Phyaiol., 16, 179-184. Scardon M. Williams DA Fay FS. (1987) The presence of a Ca%-insensitive form of Fura- associated with pol~orphonucIear leukocytes: Assessment and accurate Ca measurement. J. Biol. Chem., X2,6308-6312. Moore E Becker PL. Fogarty KE Williams DA Fay FS (1990) Ca2+ Imaging in single living cells: Theoretical and practical issues. Cell Calcium, l&157-179. Williams DA Cody SH. Gehring C. Parish R. Harris PJ. (1990) Confocal imaging of ionised Cz?+ in living plant cells. cell Calcium, 11,291~298. Wells KS Sandison DR. Strickier J. Webb WW. (1989) Quantitative fluorescence imaging with laser scanning confocaI microscopy. In: Pawley J. ed. The Handbook of Biological Confocal Micrcecopy. IMR Press, 2335. Williams DA Fogarty KE Tsien RY. Fay FS (1985) Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using Fura-2. Nature, 318, 558-561. WiIIiams DA Fay FS. (1990) IntraceIIuIar calibration of the fluortxent calcium indicator Fura- CeII Calcium, 11, 75-83. Iakowie JR. (1983) Principles of fluorescence spectroscopy. New York, Plenum Press. Niggh E I_e&rer WJ. (1990) Real-time confocal microscopy and calcium measurements in heart muscle cells: towards the development of a fluorescence microscope with high temporal and spatial resolution. CeU Calcium, 11, 121-130. Stem MD. Capogmssi MC. Lakatta EG. (1988) Spxttaneous calcium release from the sarcoplasmic reticulum in myocardial 0eIIsz mechanisms and consequences. Cell Calcium, 9,247-256. Williams DA Delbridge LM. Cody SH. Harris PJ. Morgan TO. (1990) Propagated spontaneous calcium release in isolated cardiac myocytes viewed by confocal microscopy. Am. J. Physiol., Submitted. Williams DA Milhuisen C. Rent M. Hudson I. Durham L. (1988) Versatile multiple wavelength apparatus for cellular fluorescence microscopy. Prcc. Aust. Physiol. Phannacol. Sot., 19, 70P. Bals S. Bechem M. Paffhausen W Pott L (1990) Spontaneous and experimentally evoked [Caa’-@ansients in cardiac myocytcs measured by means of a fast Fun-2 technique. Ceil Calcium, 11,385~3%. Gehring CA WiIIiams DA Cody SH. Parish RW. (1990) Phototropism and geotropism in maize coleoptiks are spatially cone&d with increases in cytosolic free calcium. Nature, 345,528-530.

Please scud reprint requests to : Dr D.A. Williams, Department of Physiology, The University of Melbourne, Parkville, Victoria, Austraha, 3052.

Received : 14 August 1990 Revised : 10 October 1990 Accepted:20October1990