Cd Caldum (1990) 11, 291-297

Q Longman Grasp UK Ltd 1880

Confocal imaging of ionised calcium in living plant cells

D.A. WILLIAMS’, S.H. CODY’, C.A. GEHRING2, R.W. PARISH2 and P.J. HARRIS’

’ Department of Physiology, The University of Melbourne, Parkvile, Victoria, Australia 250tany Department, La Trobe University, Bundoora, Victoria, Australia

Abstract - Laser-scanning confocal microscopy has been used in conjunction with Fluo-3, a highly fluorescent visible wavelength probe for Ca2+, to visualise Ca2+-dynamics in the function of living plant cells. This combination has overcome many of the problems that have limited the use of fluorescence imaging techniques in the study of the role of cations (Ca2+ and H+) in plant cell physiology and enables these processes to be studied in single cells within intact plant tissue preparations. Maize coleoptiles respond to application of ionophores and plant growth hormones with elevations in cytosolic Ca2+ that can be resolved with a high degree of spatial resolution and can be interpreted quantitatively.

It has been possible to localise ionised calcium within living animal cells with a high degree of spatial and temporal resolution. This information has enhanced our understanding of the role of calcium in essential cellular processes such as contraction [l, 21, celI motility 131, signal transduction [4], and mitosis [5]. Such investigations have generally involved use of isolated cell systems, highly fluorescent specific calcium probes (Fura-2, Indo-1) and digital video imaging techniques [see Special Issue on Imaging of Cell Calcium [Cell Calcium 1990, 11, 55-24911. In contrast, plant cells have proven to be resistant to the application of many of these elegant imaging techniques. Single cells cannot readily be isolated mechanically or enzymatically from plant tissues. Plant cells are difficult to load with fluorescent calcium indicators such as Quin 2, Fura- and Indo-1, possibly due to extracellular ester hydrolysis [6], or incomplete internal dye hydrolysis [7]. The few studies that have been carried out have resorted to introduction

of dyes through microinjection, iontophoresis or acid loading [8].

Problems of a different kind arise with both intact plant and animal tissues, involving uncertainty in analysing signals emanating from individual cells within a given image plane because of contaminating out-of-focus information [9-l 11. With the recent realisation of the importance of calcium ions in mediation of plant cellular responses (for review see [12]), there is impetus to improve existing technologies to enable monitoring of intracellular calcium dynamics especially in response to growth hormones and other physiological stimuli such as gravity and light. To circumvent some of the technical difficulties evident with plant cells we have devised methods to emplo

21 a new selective fluorescent indicator of Ca ’ (l?lu+3) and a laser scan confocal microscope (Lasersharp MRC-500, BioRad) that allow investigation of cation distributions and changes accompanying physiological stimuli.

291

Fig. 1 Schematic diagram of the experimental apparatus illustrating the coupling between the laser-scanning confocal (MRC-500) and

standard widefield epifiuorescence microscope (for details see text)

Materials and Methods

Preparation and dye loading

Maize (Zea mays, L. cv Iochief) seeds were soaked in tap water (10 h) and then grown in vermiculite for 5 days at 26°C in the dark Coleoptiles were cut l-2 cm below the apex, the primary leaf removed, and the tip region (0.5 cm) or small slices (1 mm) were loaded with fluorescent probes by incubation with esterified forms of the Ca2+-indicator Fluo-3 (25-50 p.M in DMSO) for 1 h at room temperature (20-24°C) in the dark. Although the small coleoptile slices successfully internal&d Fluo-3, initial attempts to load coleoptile tips with Fluo-3/AM resulted in mixed success as little Ca2+-sensitive fluorescence was recorded within individual plant cells in this period. Scoring or removal of the outer waxy cuticle with adhesive tape from large tissue samples such as tips, enhanced the loading process and was adopted as an essential step of the loading protocol. Dye access was terminated by rinsing in fresh tap water (3 times), and loaded tissue was then bathed in tap water during the imaging process. The free [Ca*‘] of the tap water, as determined by

potentiometric titration [13], was 40 pM. Intemalised dye, isolated by mechanical homogenisation of plant tissue, was found to be fully Ca2+ sensitive [14].

Coleoptile slices were also incubated with the membrane permeant, fluorescent, vital stain for DNA and RNA, Ethidium Bromide. This probe provided an effective means for efficiently distinguishing between nuclei and other potential intracellular sites of fluorescence heterogeneity.

Confocal microscopy

Apical coleoptiles or coleoptile slices loaded with Fluo-3 were placed on their bases on a glass coverslip (thickness 0.017 mm), bathed in tap water, and viewed on an Olympus IMT-2 inverted microscope. The experimental system is schematically represented in Figure 1. Intracellular fluorescence was excited usiug the 488 nm baud (selected with conventional 10 nm band pass filter) of an Argon-ion laser scanned through the computer-controlled galvanometric mirrors of a laser scan confocal microscope (Lasersharp MRC-500, BioRad). Emitted fluorescence passed through a

CONFOCAL IMAGING OF IONISED CALCIUM IN LIVING PLANT CELLS 293

Fig.2 Standard widefield microscopic (A) and confocal images (B) of a single full coleoptile slice loaded with Flw-3.

A. low magnification (x4) image, captured with a SIT-66 csmera

and the hardware system illustrated in Figure 1

long pass filter (515 nm cut-on), was collected by a photomultiplier tube (PMT-1) enclosed in the same housing and displayed as a 768 x 512 pixel resolution image through the frame store of the host computer (standard PC-AT). Optical section thickness was controlled by varying the size of an adjustable pin hole aperture (PH) in the detector light-path. For images of complete coleoptile cross-sections a low-power Nikon CF-Fluor objective (x10, n.a. 0.5) was used and provided optical sections which ranged to a miuimum of 1.6 pm. A Nikon CF UV-F glycerine immersion objective (x40, n.a. 1.30) provided higher magnification images and allowed for optical sections, theoretically as small as 0.26 pm to be viewed. As whole cell responses were the primary interest in this study optical sections greater than 10 nM were generally viewed. To reduce photo- bleaching of cellular Flue-3 coleoptiles were only scanned during the acquisition of images, and laser intensity was attenuated by incorporation of a variable neutral density filter (ND 2, 3% transmission) into the illumination ath. All images

5+ were corrected for leakage of Ca -sensitive dye (2-10% of total dye per hour) from the coleoptile slices during the experimental protocols.

Wide-field microscopy

Coleoptile slices loaded with fluorescent calcium

B. Confocal image of the same slice captured immediately after

that shown in A. Confocal section thickness is approximately 28

pm. Both images are the average of 8 axsecutive frames. Scale

bar 250 pm for both images

indicators were also viewed on the Olympus MT-2 during illumination with a 100 W mercury source. Excitation wavelengths were selected with narrow baud pass filters (bandwidth 12 nm) housed in a filter cube which was not resident in the light path in confocal mode. Images were captured with a silicon-intensified-target (SIT-66) camera (Dage MTI), and digitised, frame-averaged and stored with the frame grabber and other hardware of the Lasersharp confocal microscope system (see Fig. 1).

REWlt.9

Widefeldfluorescence of coleoptiles

Conventional widefleld microscopic images of Fluo-3 loaded coleoptiles illustrate many of the optical problems which make quantitative evaluation of cellular fluorescence with plant cells diffkult. A low magnification (Fig. *, x10) image of a coleoptile slice as captured by a SIT-66 camera shows the presence of diffuse fluorescence with little indication of cellular detail. The autofluorescent areas representing the two major vascular bundles are the only discernible features. The contaminating fluorescence emanates from focal planes above and below the image plane of interest and is a problem in all conventional widefield imaging systems.

294 CELL CALCIUM

Fig. 3 A. High magnifiiation confocal image (8 frames

averaged) of a small segment of a Fluo-3 loaded coleoptile slice

as captured with a glycerine immersion, high mune-rical apetIure

(1.3) Nikon CF UV-F objective. The estimated confocal section

depth was 2 m. The in&a- and extracellular heterogeneity of

fluorescence intensity is readily apparent. Scale bar 25 pm

Confocal microscopy of coleoptiles

The same coleoptile slice, imaged with the confocal microscope employing the same objectives and image acquisition system (see Fig. l), is shown in Figure 2B. There is a marked improvement in resolution of individual cells within the coleoptile slice. An epidermaI layer can be clearly distinguished surrounding the cortical cell mass. Spatial heterogeneity of the fluorescence intensity of individual cells is also apparent

The basis for cellular heterogeneity becomes apparent when higher magnification images of cells are viewed in a 1 to 2 pm focal plane at the cut surface of each fluorescently labelled coleoptile slice (see Fig. 3A). Individual epidemral cells (35 x 20 p) with distinct basal nuclei (16 unr) cover a layer of polygonal cortical cells (diameter 30 jun) with large central vacuoles within a thin layer of cytoplasm and a peripheral nucleus. The location of cell nuclei could clearly be discerned within coleoptile slices stained with Ethidium Bromide (Fig. 3B). Where optical sections included vacuoles, little fluorescence was seen suggesting that the majority of intemalised Fluo-3 was cleaved and retained by the small volume of cytosol surrounding the vacuoles, After 60 min there was increased

Fig. 3 B. Coleoptile slice stained with Ethidium Bromide (same

objective) to identify cell structures and particularly illustrate the

location of cell nuclei in cortical cells. Scale bar 50 w

vacuolar fluorescence, providing evidence of leakage or transport of fluorescent indicators from the cytosol into the vacuoles.

Ca” responsiveness of internalised dye

Cytoplasmic Fluo-3 accurately signals changes in free calcium ion concentrations as shown by the response of coleoptile slices to the addition of the ionophore A23187. Figum 4A shows an image of the longitudinal aspect of the epidermis of a coleoptile tip (laid on its side) loaded with Fluo-3 after gently scoring the waxy cuticle with a blade. Large areas of cytoplasmic fluorescence were apparent, as were a number of individual cells which do not exhibit discernible fluorescence. These cells were in areas of the coleoptile tip still covered by the cuticle and underlined the need to remove this barrier for effective dye intemalisation in large samples of plant tissue. Following the addition of A23187 to the tissue there was a rapid increase (within 1 min) in cytosolic fluorescence to a stable level maintained over the period of observation (Fig. 4B; after 30 min). Addition of Ca” (1-2 mM) to the bathing medium caused no further change in cytosolic fluorescence level. The fluorescence intensity of non-loaded cells did not change following ionophore addition confirming the absence of Fluo-3 from these compartments.

In the presence of the growth hormone Indole-acetic-acid (LU, l-10 p.lvI), Fluo-3 loaded

CONFOCAL IMAGING OF IONISED CALCIUM IN LIVING PLANT CELLS 295

Fig. 4 A. Confocal image of Fluo-3 loaded coleoptile slice

viewed from a longitudinal aspect after placing the coleoptile on

its side rather than its base. In this orientation huge areas of

cytoplasm or vacuoles can be analysed separately from other cell

structures. Cells that did not exhibit obvious fluorescence were

those cells still masked by a covering of waxy cuticle which

prevented entry of Fluo-3/AM

B. The same coleoptile slice 30 min after addition of the cation

ionophore A23187 (10 @I). Increases in fluorescence are

detected only within cytosolic areas and not within vacuoles

coleoptile slices exhibited marked elevations in cytosolic Ca2+ levels as shown in Figure 5. This illustrates that these procedures are well suited for investigations of the hormonal responses of plant tissue and in particular enable direct studies of the second messenger signalling systems (paper in preparation; see also [HI).

Photobleaching

Photobleaching of intracellular Fluo-3 was investigated by laser scanning a small subsection of the available 2-dimensional field-of-view of the coleoptile. The scanning protocol (number and timing of scans, degree of high voltage and neutral density attenuation of the laser) was identical to that used in acquiring the timecourse of growth hormone effect on cellular calcium (see Fig. 5). The kinetics of dye photobleaching were determined by comparing average cellular fluorescence intensities of scanned and the adjacent unscanned areas at each time-point. Intracellular intensities decreased mono-exponentially with total intensity reduced by 27% during the period of the experiment (12 mitt).

Calibration of resting calcium levels

Ratiometric fluorescence measurements provide the most accurate and direct estimates of intracellular levels in living cells. Unlike the related fluorescent Ca2+-sensitive dyes such as Fura-2, Fluo-3 does not

Fig. 5 Cmfocal microscopic images of Fhto-3 loaded coleoptile

slice before (A), and 12 min. at&r (B), exposure to growth

hormone (Indole acetic acid - IAA)

allow this type of calibrated signal since no spectral shift occurs upon blndin the cation. Instead, the

2f absolute level of [Ca I, and the changes in concentration within coleoptile cells were estimated from cellular fluorescence intensities with similar paradigms to those recently described for cardiac &Is [16]. Ionophore addition was used to determine fluorescence limits as has commonly been done in isolated mammalian celI systems with other Ca2+-sensitive dyes [17]. This method indicated that cytosolic Ca2’ levels in epidermal cells (see Fig. 4) averaged 264 nM. The changes in [Ca2+] in response to a physiological stimulus (e.g. IAA, see Fig. 5) were then calculated from the following information: (i) the cytosolic resting Ca2+ level; assumed to be

the level determined using ionophores (264 + 6 nM, n = 9, mean + SEM, see above),

(ii) the absolute fluorescence enhancement of cellular Fluo-3 with a known degree of Ca2+-binding (approx. 36-fold increase in fluorescence per molecule; from cuvette studies), and

(iii) the in-vitro dissociation constant Kd, which was determined in a solution of standard intracellular ionic content, and with this optical system to be 437 nM.

This information allowed the measured change in cellular fluorescence intensity (corrected for background intensity and photobleaching; see

296 CELL cALc1uh4

above) to be converted to absolute changes in d e saturation (i.e. CaFluo--3/Flu~3) by S+ Ca (described elsewhere, [16]). Free calcium concentrations could then be directly derived from the equilibrium equation:

[Ca2+l = Kd l [CaFluo-3]/[nu~3]

Using this procedure the fluorescence response following IAA addition (Fig. 5) represented a 3-fold increase in [Ca2+] from 264 to 756 nM. Changes of this order of magnitude were also recorded in preliminary experiments with Fura- loaded coleoptiles viewed with wide-field microscopy.

Discussion

The study of intracellular function in animal tissues has benefited enormously as a result of imaging procedures that allow localisation of intracellmar calcium and can follow dynamic responses to external stimuli such as hormones. However, there are very few reports of successful imaging of calcium distributions and changes with cell function in plant tissue. Most of this limitation probably results from either unsuccessful cleavage of fluorescent Ca2’ indicators by plant cells [ 101, or the contamination of the signal emission of individual cells by other cells within the plant tissue [9, 111. It is clear from this report that these restrictions can be effectively eliminated by i) removing the waxy cuticle that presents the major barrier for dye intemakation in plant tissues and ii) employing confocal microscopy to reduce the 3dimensional spread of contaminating out-of-focus information in image planes. A remarkable increase in spatial detail was evident in confocal images when compared to conventional wide-field microscopy of the same coleoptile slice (Fig. 2). Increased spatial resolution with confocal microscopy has also recently been reported in cardiac muscle cells [18, 191, and could also be expected by employing the elegant image restoration algorithms recently described for conventional widefield images of smooth muscle cells 19, 201. However, the application of these techniques is beyond the computing resources of most research groups.

In plants, confocal imaging has obvious advantages over the use of other techniques for the measurement of both cytosolic Ca2’ and pH in individual cells. Ion-selective microelectrodes have to date been the major tool for investigation of cation activities related to physiological function in plants. However, within a vacuolated cell the cytosol surrounding the vacuole represents a small target for microelectrode insertion, a point which is clearly reflected by the low success rate of such measurements in previous studies [21]. To improve the success rate, plant preparations which have unusually large areas of cytoplasm, such as mot hair cells or giant algal cells may be employed [lo, 221. Clearly this restricts the range of cell and tissue types that can be used and the physiological processes that can be investigated. Integration of total cellular fluorescence intensities, as obtained in studies using widefield imaging systems, would result in the dilution of fluorescence changes because of the presence of large volumes which do not contribute to the absolute fluorescence intensity (i.e. vacuoles). However, within confocal fluorescence images, the cytoplasm of coleoptile cells is a compartment that can be clearly distinguished from other cellular areas such as nuclei or vacuoles (e.g. see Fig. 3), allowing for discrete analysis of cytosolic [Ca2’l during physiological function free from out-of-focus contaminating fluorescence.

The absence of fluorescence within vacuoles suggests that intemalised Fluo-3 is cleaved efficiently by cytosolic esterases before it is able to invade the vacuolar compartment. The slow leakage, or transport of dye using anion transport mechanisms [23], into vacuoles ensures that changes in fluorescence recorded within 60 min of loading exclusively reflect changes in cytosolic [Ca2’l.

The confocal microscopic views of Ca2+-distribution in coleoptile cells offer detail that exceeds that in light microscopic images, and approaches that seen in electron micrographs, as reported for example with gravistimulated calcium redistribution in antimonate-fixed plant tissue [24]. The ma’or advantages of the present techniques are

rl+ that Ca measurements can be made dynamically in living, unfiied cells, and that only the physiologically active form of calcium, free ion&d

CONFOCAL IMAGING OF IONJSED CALCIUM JN LMNG PLANT CELLS 297

calcium, is recorded. Using the techniques and paradigms described here we have now been able to visualise changes in [Ca2’] iu epiderml and cortical cells as a result of physiological stimuli such as gravitropism or phototropism [15].

Acknowledgements 15.

We are grateful for the scientific support of Professor T.O.

Morgan, and acknowledge the expert photographic assistance of David Llewellyn Thomas.

16.

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Please send reprint requests to: Dr D.A. Williams, Department of Physiology, University of Melbourne. Parkville, Victoria, Australia, 3052.

Received : 16 October 1989 Revised : 28 November 1989 Accepted : 1 December 1989