Modification of the pressure-probe technique permits controlled

intracellular microinjection of fluorescent probes

K. J. OPARKA1*, R. MURPHY2, P. M. DERRICK1, D. A. M. PRIOR1 and J. A. C. SMITH2

1Department of Cellular and Environmental Physiology, Scottish Crop Research Institute, Inuergowrie, Dundee DD2 5DA, UK2Department of Plant Sciences, University of Oxford, South Parks Road, Oxford 0X1 3RB, UK

* Author for correspondence

Summary

The pressure probe has been widely used to study thewater relations of plant cells. Here we describe asimple modification of the pressure-probe techniquethat permits the controlled microinjection of fluor-escent probes into plant cells while simultaneouslymeasuring cell turgor pressure. Using the pressureprobe, less than lnl of the membrane-impenneantfluorescent dye Lucifer Yellow CH was introducedinto micropipettes and subsequently injected intoleaf trichome cells of Nicotians clevelandli. Disrup-tion of cell contents could be minimized by raisingthe hydrostatic pressure in the probe prior toimpalement to a value approaching the anticipatedcell turgor pressure. Injections to the cytosol resultedin intercellular symplastic transport of the dye in

both acropetal and basipetal directions. In contrast,no symplastic transport was observed following aninjection of dye into the vacuole. As measured withthe pressure probe, cell turgor pressures were in therange 0.18 to 0.36 MPa; the half-time for waterexchange across the cell boundary was approxi-mately 10 s. The potential of this technique for thestudy of turgor-pressure-dependent intercellulartransport and the hydraulic conductivities of thetonoplast, plasmalemma and plasmodesmata is dis-cussed.

Key words: cell water relations, Lucifer Yellow CH,microinjection, pressure probe, symplastic transport.

Introduction

The pressure probe has been used extensively as aninstrument for studying the water relations of individualplant cells. Following its introduction by Zimmermann etal. (1969) for use on giant algal cells, the pressure probewas subsequently miniaturized by Hilsken et al. (1978) foruse on the much smaller cells of higher plants. Theinstrument consists of an oil-filled microcapillary that ishydraulically connected to a pressure chamber or syringe,also filled with oil. A small pressure transducer trans-forms the pressure generated within the system into avoltage output. When a cell is impaled, cell sap is forcedinto the shank of the micropipette as the turgor pressure isreleased. The resulting sap/oil meniscus can be observedunder an optical microscope and its position controlled byadjustments of a syringe plunger coupled to a micrometer.To obtain an estimate of cell turgor pressure, the meniscusis returned to its position just prior to impalement, andthen held in this position until a stable pressure reading isobtained (typically less than lmin). In addition to thedirect measurement of cell turgor pressure, the pressureprobe can also be used to determine the cell volumetricelastic modulus and the hydraulic conductivity of the cellboundary (Husken et al. 1978; Zimmermann and Steudle,1978, 1980; Steudle et al. 1980).

Developments in the use of the pressure probe have beenparalleled by a growing interest in microinjection pro-cedures for introducing fluorescent probes into the plantsymplast. For example, studies of this sort have providedJournal of Cell Science 98, 539-544 (1991)Printed in Great Britain ©The Company of Biologists Limited 1991

valuable information on the molecular size-exclusion limitof plasmodesmata and on factors influencing the inter-cellular transport of fluorescent probes (Robards andLucas, 1990). Two types of microinjection procedure arecommonly used: iontophoretic injection and pressureinjection. In the former, an electrical current is used todrive the dye out of the micropipette tip into the cell. Thismethod has an advantage in that transmembrane electri-cal potentials can be measured simultaneously (van derSchoot and van Bel, 1989). However, the relatively highcurrents required to eject dye from the micropipette tipmay irreversibly disrupt cytoplasmic streaming (Goodwinet al. 1990) and render the tonoplast leaky (van der Schootand van Bel, 1989). In the latter method, pressure injectionrequires the expulsion of dye into the cell by raising thehydrostatic pressure in the microcapillary tip above thatof the opposing turgor pressure of the cell. Both the abovemethods are prone to tip blockages during impalementwhen cell sap is driven into the micropipette shank, aphenomenon referred to by van der Schoot and van Bel(1989) as 'backfiring'.

In the same way that iontophoretic injection permits themeasurement of membrane potential, so pressure injectionsystems have the potential to be modified to permit theconcurrent measurement of cell turgor pressure. However,such a combined pressure-probe/pressure-injection sys-tem has not been developed. Here we describe a simpleprocedure for recording cell turgor pressure with apressure probe, whilst simultaneously microinjectingfluorescent probes into either the cytosol or the vacuole of

539

plant cells. This approach affords a means of studyingdirectly the hydraulic conductivities of the tonoplast,plasmalemma and plasmodesmata, as well as the influ-ence of turgor pressure on intercellular transport. Anothernotable feature of the technique is that 'backfire' can beminimized by raising the hydrostatic pressure in thepressure probe prior to impalement. This significantlyreduces cellular disruption and the likelihood of tipblockage.

Materials and methods

Plant materialLeaves of glasshouse-grown Nicotiana cleuelandu (Gray) wereexcised from approximately six-week-old rosette plants. The endof the cut petiole was inserted into a bench centrifuge tubecontaining distilled water and the tube sealed with Nescofilm(Nippow Shoji Kaisha Ltd, Osaka, Japan). This operation tookonly several seconds to complete and prevented wilting of the leafon the microscope stage (see Derrick et al. 1990).

Pressure probeMicroinjections and turgor pressure measurements were per-formed with a manually operated pressure probe similar to thatdescribed by Itoh et al. (1987). In our version of the probe (Murphyand Smith, 1989), the micropipette is separated from themicrometer by a length of flexible HPLC tubing (Teflon or PVC).This mechanical isolation facilitates micromanipulation andmanual operation of the micrometer. In the present study, themicropipette was inserted into the hydraulic arm of a Narishigemicromanipulator (model MN-3), and directed with a three-axisjoystick (MO-202; Narishige) hydraulically coupled to the stagemicromanipulator. The micromanipulator arm was connected tothe HPLC tubing via an HPLC coupling (Anachem (UK) Ltd).Pressures were monitored with a PDCR 200 pressure transducer(Druck Ltd, UK) and recorded on a flat-bed chart recorder.

Introduction of Lucifer Yellow CH into micropipettesBorosilicate glass capillaries (GC 120-10; Clark Electromedicalinstruments, Pangbourne, Berks, UK) were pulled on a Narishigemicropipette puller (model PB-7), adjusted so as to producemicrocapillaries with a slowly tapering shank. This afforded moreaccurate control of meniscus position with the pressure probe. Themicropipettes were backfilled with low-viscosity silicone oil (DowCorning 200/20cs; BDH Ltd, UK) and inserted into the arm of themicroinjector. The tip of the oil-filled micropipettes was thensubmerged in a droplet of 30molm~3 aqueous Lucifer Yellow CH(LYCH; Sigma, UK) mounted on a glass slide. Oil, and any airbubbles trapped in the micropipette tip, were expelled into theLYCH solution by positive pressure generated with the mi-crometer of the pressure probe. The expelled oil droplets wereimmiscible with the LYCH and, when the micropipettes were freeof any air, could be removed from the micropipette tip by gentlemovements of the micromanipulator. The tip of the micropipettewas then filled with LYCH solution by reducing the pressurebelow ambient for several seconds. Uptake of dye (typically<10pi) was halted by raising the micropipette out of the LYCHsolution.

Microinjection of LYCH and the measurement of cellturgor pressureIndividual leaves of N. clevelandii were mechanically supportedon a glass slide. Trichomes were clearly visible at the leaf marginsand were held in place against a blunted micropipette, orientatedat right angles to the cell wall of an individual cell of thetrichome. The retaining micropipette was moved with a secondmicromanipulator (type MN-3; Narishige) also attached to themicroscope stage. When viewed under a combination of bright-field and fluorescence optics, the oil/LYCH interface was clearlyvisible in the micropipette tip (e.g. Fig. 1A). The oil/dye interface

appeared slightly out of focus when the objective was focused onthe micropipette tip, but could be brought into sharp focusfollowing the successful impalement of the cell (Fig. 1H).

To measure cell turgor pressure, the micropipette was advancedtowards a trichome cell, while simultaneously tracking theoil/dye meniscus (see below). On successfully impaling the cell,sap was observed to backfire rapidly into the shank of themicropipette. Using the micrometer, the pressure was then raisedso as to return the oil/dye meniscus to its original position (i.e.that just prior to impalement). The meniscus was held in thisposition until a stable pressure reading was obtained. During theinitial backfire, the dye moved as a pulse with the bulk-flow of sap(Fig. IB and C). However, on reversing the flow and returning themeniscus towards its original position, the dye diffused rapidlyinto the cell (Fig. 1D-G).

MicroscopyMicroinjections and the intercellular spread of LYCH wereobserved under a Nikon Optiphot microscope with fluorescenceattachment. A X10 long-working-distance objective providedadequate clearance to permit microinjection. A blue combinationfilter (Nikon B-2E, dichroic mirror 610 nm, excitation filter450-490 nm, barrier filter 520-660 nm) was used for fluorescencemicroscopy. Microinjection sequences were recorded using a SonyDXC-750P 3-chip CCD high-resolution video camera attached tothe camera port of the microscope. Video sequences were recordedin colour on a JVC Panasonic NV-180 portable video recorderusing Kodak Eastman Professional Video Cassette E-180 XHGfilm. Stills from the video sequences were made using a Sony UP-5000P colour video printer. The video system also incorporated anRMC-D3 image analyser (Reece Scientific, UK) that generated a'mouse'-controlled cursor on the monitor. This greatly facilitatedtracking of the oil/dye meniscus during impalement. Onsuccessfully impaling a cell, the cursor was 'frozen' so as toprovide an unambiguous reference point to which the meniscuswas returned.

Results

Two typical impalements of leaf trichome cells of Nico-tiana clevelandii, one to the cytoplasm and the other to thevacuole, are shown in Figs 1 and 2, respectively. InFig. 1A, the appearance of the LYCH/oil interface isshown under combined bright field/fluorescence optics.The same field is shown under fluorescence alone inFig. IB. Fig. 1C shows the release of turgor pressure fromthe cell, which resulted in the movement of cytoplasm intothe tip of the micropipette. Fig. 1D-G demonstrates themovement of the LYCH/oil interface back to its originalposition, whilst simultaneously introducing LYCH intothe cytoplasm. Note that the nucleus became intenselyfluorescent following a cytoplasmic injection. In Fig. 1Gthe objective is focused on the specimen, rather than thedye/oil interface, whereas Fig. 1H shows the appearanceof the interface in sharp focus. Subsequent acropetal andbasipetal spread of LYCH through cells of the trichomefollowing withdrawal of the micropipette is shown at alower magnification in Fig. II.

The sudden release of turgor pressure on impaling thecell caused relatively large amounts of cytoplasm to enterthe micropipette tip. In such cases cytoplasmic streamingwas often irreversibly halted following the microinjection.The extent of this "backfire' could be reduced considerablyby raising the pressure in the dye-filled micropipette tipprior to impalement to a value slightly lower than theanticipated turgor pressure of the cell (the latter beingdetermined from previous impalements using differenttrichomes). As a result, little cytoplasmic disruptionoccurred during impalement and very small quantities ofdye could be introduced into the cell in a controlled

540 K. J. Oparka et al.

Fig. 1. Microinjection of LYCH into the cytosol of a trichome cell using the pressure probe. (A) Combined bright-field/fluorescenceimage showing a micropipette with LYCH in the tip and an adjacent trichome cell supported by a holding pipette.(B,C) Epifluorescence images showing impalement and the resulting 'backfire' of cell sap into the micropipette. (D-G) Return of theoil/dye meniscus to its pre-impalement position (see Fig. 3). (H) Oil/dye meniscus in sharp focus. (I) Acropetal and basipetal spreadof LYCH through the file of trichome cells; the arrow indicates the injected cell, n, nucleus. Bars: 50/an (A-H); 100/nn (I).

manner. Furthermore, cytoplasmic streaming resumedrapidly following such less-traumatic injections.

Injections into the vacuole resulted in no intercellulardye movement (see also Derrick et al. 1990). In Fig. 2A-I,illustrating a vacuolar injection, the pressure in themicropipette tip was raised prior to impalement asdescribed above. Consequently, there was relatively littlebackfire as turgor pressure was released from the cell(arrow, Fig. 2C). On returning the meniscus to its pre-impalement position, the dye spread rapidly around thecentrally located nucleus to fill the remainder of thevacuole (cf. the appearance of the nucleus during acytoplasmic injection in Fig. 1G).

Pressure recordings for a cytoplasmic and a vacuolarimpalement are shown in Fig. 3A and B, respectively.Measured turgor pressures were in the range 0.18 to0.36 MPa, with a mean±s.D. of 0.29±0.06MPa (8 measure-ments). In three cases pressure-relaxation experimentswere performed (Hiisken et al. 1978; Zimmermann andSteudle, 1978, 1980; Steudle et al. 1980; see also Dainty,1963, 1976). In such experiments, a small volume ofsolution is withdrawn or injected, and the oil/dyemeniscus maintained in its new position by adjustments ofthe pressure-probe micrometer. This is illustrated inFig. 2G-I, where the objective is focused on the oil/dyeinterface rather than the specimen. Note that the

interface remains clearly visible as the turgor pressure israised (Fig. 2G,H) or lowered (Fig. 2H,I). The resultingpressure record is then an indication of the water fluxacross the cell boundary. Fig. 4A and B show suchexamples for cytoplasmic and vacuolar impalements,respectively. The estimated half-times for water exchangewere 8 s for a cytoplasmic impalement and 9 s and 10 s fortwo vacuolar impalements.

Discussion

The introduction of fluorescent probes into the cytoplasmor vacuole of plant cells with the pressure probe extendsthe existing uses of the probe to studies of intercellulartransport processes. A common problem encountered insuch studies is the severe backfiring encountered as turgorpressure is suddenly released from the impaled cell. In thecase of sieve elements in the phloem, which haveextremely high turgor pressures, van der Schoot and vanBel (1989) overcame this problem by lowering the cellturgor by defoliation. Using the pressure probe it ispossible to raise the pressure in the tip of the micropipette,without loss of the dye, to a value closer to that of the cellturgor pressure. This approach may prove to be of valuewhere minimal cellular disturbance is required during

Plant cell water relations 541

Fig. 2. Similar sequence to Fig. 1, but for a vacuolar injection. Note that there is litle backfire (see arrow in C) because thehydrostatic pressure in the pressure probe was raised prior to impalement. (D,E) Return of the meniscus to its pre-impalementposition. (G-I) Control of meniscus position during pressure-relaxation experiments (see Fig. 4). Note that there is no intercellularmovement of the dye. n, nucleus. Bar: 50/an.

£ 0.3

0.020 40 60

Time (s)

microinjection, for example during the introduction of pH-or Ca2+-sensitive probes into the cytosol.

The retention of dye in the micropipette tip may reflectsurface tension effects. In theory, a concave air/watermeniscus at the probe tip could sustain a pressure (P)relative to ambient approaching 2y/r, where y is thesurface tension in the meniscus (73 mN m~1 at 20 °C) and ris the tip radius (Sears et al. 1978). Hence a 1-jnn diameterpipette tip could sustain a pressure of nearly 0.29 MPa.Measurements with distilled water in the micropipette tipgave values for P of 0.1 to 0.3 MPa for tip diameters of1-5 /an. A tip diameter of 1 fan probably represents theuseful lower limit for pressure probe work, since thehydraulic resistance of the micropipette tip must remainsmall compared to that of the cell boundary (Zimmermannand Steudle, 1978). However, the ability to achieve suchpressures in the probe prior to injection will be useful forreducing backfire even when the turgor pressures of theimpaled cells are very high (1 MPa or more), since the cellvolumetric elastic modulus (e) generally increases with

Fig. 3. Measurement of cell turgor pressures with the pressureprobe while simultaneously introducing LYCH into either thecytosol (A) or vacuole (B). Following the initial backfire onimpalement (see Fig. 1 and 2), the pressure in the pressureprobe is raised so as to return the oil/dye meniscus to itsoriginal position. At this point the recorded pressure is takenas equal to the cell turgor pressure.

542 K. J. Oparka et al.

0.50

0.0010 20 30

Time (s)40

Fig. 4. With the pressure probe impaled in a cell, severalpicolitres of solution were ejected from the micropipette tip intothe cytosol (A) or vacuole (B), so raising the cell turgorpressure. When the oil/dye meniscus is held in its newposition, the resulting relaxation of pressure can be interpretedin terms of an efflux of water across the cell boundary. Half-times (£j) for water equilibration are indicated.

turgor pressure (Zimmermann and Steudle, 1978; Tyreeand Jarvis, 1982). As the volume of sap ejected into themicropipette tip will be inversely proportional to e, most ofthe volume backfire will occur as the cell approaches zeroturgor. However, this method is only applicable when thetarget cell is in air.

With the pressure probe, turgor pressure can not only bemeasured but also can be 'clamped' at any desired value(Wendler and Zimmermann, 1982; Cosgrove and Dur-achko, 1986; Murphy and Smith, 1989). Hence, the presenttechnique offers the potential to study, directly at thecellular level, the dependence of symplastic transport oncell turgor pressure. In this regard, Fensom and hiscolleagues have obtained evidence that plasmodesmatamay function as pressure-sensitive valves, gating inresponse to pressure differentials between cells (Barclayand Fensom, 1984; Zawadski and Fensom, 1986; Cote etal.1987; Trebacz and Fensom, 1989). Such pressure differen-tials may exert their effect on the neck constrictions ofplasmodesmata, the dimensions of which are crucial forthe regulation of both diffusive and convective transport(Gunning, 1976; Gunning and Robards, 1976; Blake,1978). Sphincter-like structures in the neck regions ofplasmodesmata have been described by Willison (1976),Evert et al. (1977) and Olesen (1979). Extension of thepresent technique to phloem tissues may offer a valuabletool with which to study simultaneously the turgorpressures and symplastic continuities of the cells involvedin phloem loading and unloading.

A further advantage of the present technique is that theimpaled intracellular compartment (cytosol or vacuole)can be identified according to the distribution andintercellular transport of the injected dye. In fact, if thedye can be chemically fixed (as in the case of LYCH), boththe cell type and the injected compartment could beidentified even when the impaled cell lies several celllayers below the surface, and so is obscured duringmeasurements. This raises the possibility of determining

the individual hydraulic conductivities of the tonoplast,plasmalemma and plasmodesmata by pressure-clamp andpressure-relaxation experiments. In our opinion, theclarity of the oil/dye interface under the fluorescencemicroscope is superior to that observed with conventionalpressure-probe techniques employing optical microscopyalone. This may be useful in automated versions of theprobe (Cosgrove and Durachko, 1986; Buchner et al. 1987),where the meniscus must be tracked by a feedback controlsystem.

This brief report describes a simple extension of thepressure-probe technique that permits the controlledintroduction of fluorescent probes into plant cells. Theprincipal advantages are: (1) simultaneous microinjectionand measurement of turgor pressure; (2) minimal cellulardisruption during microinjection; and (3) identification ofthe impaled intracellular compartment. Future studieswill be aimed at a more mechanistic understanding of therole of turgor pressure in influencing intercellular trans-port processes.

K. J. Oparka, P. M. Derrick and D. A. M. Prior acknowledge thefinancial support of the Department of Agriculture and Fisheriesfor Scotland. R. Murphy and J. A. C. Smith are grateful to theAgriculture and Food Research Council (UK) for financialsupport.

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(Received 22 November 1990 - Accepted 4 January 1991)

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