The structure and dynamics of patch-clamped membranes: a study using differential interference contrast light microscopy

We have developed techniques for micromanipulation under high power video microscopy. We have used these to study the structure and motion of patch-clamped membranes when driven by pressure steps. Patch-clamped membranes do not consist of just a membrane, but rather a plug of membrane-covered cytoplasm. There are organelles and vesicles within the cytoplasm in the pipette tip of both cell-attached and excised patches. The cytoplasm is capable of active contraction normal to the plane of the membrane. With suction applied before seal formation, vesicles may be swept from the cell surface by shear stress generated from the flow of saline over the cell surface. In this case, patch recordings are made from membrane that was not originally present under the tip. The vesicles may break, or fuse and break, to form the gigasealed patch. Patch membranes adhere strongly to the wall of the pipette so that at zero transmural pressure the membranes tend to be normal to the wall. With transmural pressure gradients, the membranes generally become spherical; the radius of curvature decreasing with increasing pressure. Some patches have nonuniform curvature demonstrating that forces normal to the membrane may be significant. Membranes often do not respond quickly to changes in pipette pressure, probably because viscoelastic cytoplasm reduces the rate of flow through the tip of the pipette. Inside-out patches may be peeled from the walls of the pipette, and even everted (with positive pressure), without losing the seal. This suggests that the gigaseal is a distributed property of the membrane-glass interface.     

A look at membrane patches with a scanning force microscope.

We combined scanning force microscopy with patch-clamp techniques in the same experimental setup and obtained images of excised membrane patches spanning the tip of a glass pipette. These images indicate that cytoskeleton structures are still present in such membrane patches and form a strong connection between the membrane and the glass wall. This gives the membrane patch the appearance of a tent, stabilized by a scaffold of ropes. The lateral resolution of the images depends strongly on the observed structures and can reach values as low as 10 nm on the cytoskeleton elements of a (inside-out) patch. The observations suggest that measurements of membrane elasticity can be made, opening the way for further studies on mechanical properties of cell membranes.     

Ion fluxes in giant excised cardiac membrane patches detected and quantified with ion-selective microelectrodes

We have used ion-selective electrodes (ISEs) to quantify ion fluxes across giant membrane patches by measuring and simulating ion gradients on both membrane sides. Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE. For detection on the extracellular (pipette) side, ISEs are fabricated from flexible quartz capillary tubing (tip diameters, 2-3 microns), and an ISE is positioned carefully within the patch pipette with the tip at a controlled distance from the mouth of the patch pipette. Transport activity is then manipulated by solution changes on the cytoplasmic side. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. The sensitivity and utility of the methods are demonstrated with cardiac membrane patches by measuring (a) potassium fluxes via ion channels, valinomycin, and Na/K pumps; (b) calcium fluxes mediated by Na/Ca exchangers; (c) sodium fluxes mediated by gramicidin and Na/K pumps; and (d) proton fluxes mediated by an unknown electrogenic mechanism. The potassium flux-to-current ratio for the Na/K pump is approximately twice that determined for potassium channels and valinomycin, as expected for a 3Na/2K pump stoichiometery (i.e., 2K/charge moved). For valinomycin-mediated potassium currents and gramicidin-mediated sodium currents, the ion fluxes calculated from diffusion models are typically 10-15% smaller than expected from the membrane currents. As presently implemented, the ISE methods allow reliable detection of calcium and proton fluxes equivalent to monovalent cation currents <1 pA in magnitude, and they allow detection of sodium and potassium fluxes equivalent to <5 pA currents. The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion-coupled transporters in giant patches.     

Cell membrane patches are supported by proteoglycans

A cell membrane patch isolated on a patch clamp pipette incorporates in addition to the phospholipid bilayer, an extracellular matrix and cytoskeletal components. The significance of the extracellular matrix for the patch formation was studied in aortic smooth muscle and cerebellar granule cells grow in the presence of an inhibitor of proteoglycan synthesis, -d -xyloside. The xyloside improved the seal success rate, and after patch excision membrane vesicles were formed instead of inside-out patches. When amphotericin B was included in the pipette solution, perforated outside-out vesicles were formed in 96% of cells. The findings suggest, that membrane patches are supported by the extracellular matrix or by structures that relate to this matrix.     

Calculation of time constants for intracellular diffusion in whole cell patch clamp configuration.

We present a simplified model to identify and analyze the important variables governing the diffusion of substances from pipettes into canine cardiac Purkinje cells in the whole cell patch clamp configuration. We show that diffusion of substances through the pipette is the major barrier for equilibration of the pipette and cellular contents. We solve numerically the one-dimensional diffusion equation for different pipette geometries, and we derive a simple analytic equation which allows one to estimate the time necessary to reach the steady state of intracellular concentration. The time constant of the transient to steady state is given by a pipette geometric factor times the cell volume divided by the diffusion coefficient of the substance of interest. The geometric factor is shown to be given by the ratio of pipette resistance to the resistivity of the filling solution. Additionally from our modeling, we concluded that pipette perfusion at distances greater than 20 microns from the pipette tip would not substantially reduce the time necessary to achieve the steady state.     

USING ATOMIC FORCE MICROSCOPY TO INVESTIGATE PATCH-CLAMPED NUCLEAR MEMBRANE

Nuclear patch clamp is an emerging research field that aims to disclose the electrical phenomena underlying macromolecular transport across the nuclear envelope (NE), its properties as an ion barrier and its function as an intracellular calcium store. The authors combined the patch clamp technique with atomic force microscopy (AFM) to investigate the structure—function relationship of NE. In principle, patch clamp currents, recorded from the NE can indicate the activity of the nuclear pore complexes (NPCs) and/or of ion channels in the two biomembranes that compose the NE. However, the role of the NPCs is still unclear because the observed NE current in patch clamp experiments is lower than expected from the known density of the NPCs. Therefore, AFM was applied to link patch clamp currents to structure. The membrane patch was excised from the nuclear envelope and, after electrical evaluation, transferred from the patch pipette to a substrate. We could identify the native nuclear membrane patches with AFM at a lateral and a vertical resolution of 3nm and 0.1nm, respectively. It was shown that complete NE together with NPCs can be excised from the nucleus after their functional identification in patch clamp experiments. However, we also show that membranes of the endoplasmic reticulum can contaminate the tip of the patch pipette during nuclear patch clamp experiments. This possibility must be considered carefully in nuclear patch clamp experiments.     

Currents recorded through small areas of squid axon membrane with an internal virtual ground voltage clamp.

A new voltage-clamp apparatus for the squid axon has been implemented to enable recording of currents through small areas of axon membrane. The performance of this clamp was tested by recording total sodium currents from perfused axons (I total) and sodium currents from small membrane patches (I patch), which were recorded from inside the axon with an L-shaped pipette. The I patch records, although four orders of magnitude smaller than I total, were stable and showed normal kinetics and voltage dependence, and appeared to reflect the activation of a small population of normal sodium channels. The size of the current recorded from the patch was mainly a function of the tip diameter of the L-shaped pipette and of the shunt resistance between inside the pipette and the axoplasm.     

Exocytosis of single chromaffin granules in cell-free inside-out membrane patches

In chromaffin cells, exocytosis of single granules and properties of the fusion pore--the first connection between vesicular lumen and extracellular space --can be studied by cell-attached patch amperometry, which couples patch-clamp capacitance measurements with simultaneous amperometric recordings of transmitter release. Here we have studied exocytosis of single chromaffin granules and endocytosis of single vesicles in cell-free inside-out membrane patches by patch capacitance measurements and patch amperometry. We excised patches from chromaffin cells by using methods developed for studying properties of single ion channels. With low calcium concentrations in the pipette and bath, the patches showed no spontaneous exocytosis, but exocytosis could be induced in some patches by applying calcium to the cytoplasmic side of the patch. Exocytosis was also stimulated by calcium entry through the patch membrane. Initial conductances of the fusion pore were undistinguishable in cell-attached and excised patch recordings, but the subsequent pore expansion was slower in excised patches. The properties of exocytotic fusion pores in chromaffin cells are very similar to those observed in mast cells and granulocytes. Excised patches provide a tool with which to study the mechanisms of fusion pore formation and endocytosis in vitro.     

Membrane-pipette interactions underlie delayed voltage activation of mechanosensitive channels in Xenopus oocytes.

To investigate the mechanism for the delayed activation by voltage of the predominant mechanosensitive (MS) channel in Xenopus oocytes, currents were recorded from on-cell and excised patches of membrane with the patch clamp technique and from intact oocytes with the two-electrode voltage clamp technique. MS channels could be activated by stretch in inside-out, on-cell, and outside-out patch configurations, using pipettes formed of either borosilicate or soft glass. In inside-out patches formed with borosilicate glass pipettes, depolarizing voltage steps activated MS channels in a cooperative manner after delays of seconds. This voltage-dependent activation was not observed for outside-out patches. Voltage-dependent activation was also not observed when the borosilicate pipettes were either replaced with soft glass pipettes or coated with soft glass. When depolarizing voltage steps were applied to the whole oocyte with a two-electrode voltage clamp, currents that could be attributed to MS channels were not observed. Yet the same depolarizing steps activated MS channels in on-cell patches formed with borosilicate pipettes on the same oocyte. These observations suggest that the delayed cooperative activation of MS channels by depolarization is not an intrinsic property of the channels, but requires interaction between the membrane and patch pipette.     

The use of quartz patch pipettes for low noise single channel recording.

Quartz has a dissipation factor of approximately 10(-4), which is an order of magnitude less than that of the best glasses previously used to fabricate patch pipettes; it's dielectric constant of 3.8 is also lower than that of other glasses. On the basis of these electrical characteristics it is expected that patch pipettes pulled from quartz tubing will produce significantly less noise than pipettes made from other glasses. Our work confirms these expectations and we describe theoretical and practical aspects of the use of quartz pipettes for single channel patch voltage clamp measurements. Methods for pulling quartz pipettes with a laser-based puller and coating them with low-loss elastomers are discussed, as are precautions that are necessary to achieve low noise recordings. We have shown that quartz pipettes can be pulled from tubing with outer diameter to inner diameter ratios as large as 3 and a method of applying heavy elastomer coatings all the way to the tip of pipettes is presented. Noise sources arising from the pipette and its holder are described theoretically, and it is shown that measured noise is in good agreement with such predictions. With low noise capacitive feedback electronics, small geometry holders, and thick-walled quartz pipettes coated with low-loss elastomers we have been routinely able to achieve noise of 100 fA rms or less in a 5-kHz bandwidth with real cell patches and a pipette immersion depth of approximately 2 mm. On occasion we have achieved noise as low as 60 fA rms in this bandwidth.     

A pressure-polishing set-up to fabricate patch pipettes that seal on virtually any membrane,yielding low access resistance and efficient intracellular perfusion

When performing whole-cell configuration recordings, it is important to minimize series resistance to reduce the time constant of charging the cell membrane capacitance and to reduce error in membrane potential control. To this end, an existing method was improved by widening the patch pipette shank through the calibrated combination of heat and air pressure. The heat was produced by passing current through a filament that was shaped appropriately to ensure a homogeneous heating of the pipette shank. Pressurized air was applied to the lumen of a pipette, pulled from a borosilicate glass microcap, via the pressure port of a modified commercial holder. The pipette reshaping was viewed on an LCD monitor connected to a contrast-intensified CCD camera and coupled to a modified bright-field stereomicroscope. By appropriately regulating the timing of air pressure and the application of heating, the pipette shank and, independently, the tip opening diameter were widened as desired. The methods illustrated here to fabricate and use the patch pipettes, using just one glass type, allowed the sealing of a wide variety of cell types isolated from different amphibian, reptilian, fish, and mammalian tissues as well as a variety of artificial membranes made with many different lipid mixtures. The access resistance yielded by pressure-polished pipettes was approximately one-fourth the size of the one attained with conventional pipettes; besides improving the electrical recordings, this minimized intracellular ion accumulation or depletion as well. Enlarged shank geometry allowed for fast intracellular perfusion as shown by fluorescence imaging, also via pulled quartz or plastic tubes, which could be inserted very close to the pipette tip.     

Demonstration of acid phosphatase-containing vacuoles in hyphal tip cells of Sclerotium rolfsii.

A lysosomal system was demonstrated in hyphal tip cells of Sclerotium rolfsii by light and electron microscopy observations of the sites of acid phosphatase activity visualized by a modified Gomori lead nitrate method. The cytochemical reaction product was found to be present in numerous vacuoles, each aout 0.5 mum in diameter, which were seen as chains of spheres when viewed with the light microscope. They usually did not occur in the first 30 to 40 mum of the hyphal tip cell, but were concentrated in a zone extending from 30 to 200 mum from the hyphal apex. As shown by the electron microscope, the vacuoles were sometimes interconnected by narrow channels. Acid phosphatase reaction product was also occasionally localized in vacuoles of the older hyphal cells, but never in apical vesicles, lipid bodies, or microbodies. It is proposed that this vacuolar system may orginate from the endoplasmic reticulum.     

Preparation and some properties of giant liposomes and proteoliposomes

Optimal conditions for formation of giant liposomes and proteoliposomes were investigated. A suspension of small unilamellar vesicles made of various phospholipids in a buffer of 0-3 M KCl, 0.1 mM EDTA, and 20 mM MOPS (pH 7.0) was subjected to a freeze-thaw treatment. Giant multilamellar liposomes of diameter ranging from 10 to 60 microns were found to form from phospholipid mixtures containing phosphatidylethanolamine as a major component and phosphatidylserine as a minor component. The concentration of KCl optimal for the giant vesicle formation was 30-500 mM. By applying a patch-pipette to a giant liposome, suitable conditions for obtaining a high-resistance (giga-ohm) seal were sought. It was found that use of a patch-pipette of relatively small tip diameter (less than 1 micron), the presence of divalent metal cations in the suspension medium and inflation of vesicles in a hypotonic solution facilitated giga-seal formation. In a suspension of asolectin (soybean phospholipid) vesicles which had been subjected to the freeze-thaw treatment, giant unilamellar vesicles were found. They could be held on the tip of a suction pipette and impaled with a microelectrode filled with an EGTA solution. Small unilamellar proteoliposomes were prepared by the cholate-dialysis method from asolectin and sarcoplasmic reticulum vesicles, and were subjected to a freeze-thaw cycle. When the ratio of exogenous phospholipid to protein was larger than 10, giant multilamellar vesicles were formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphology of the macula neglecta in sharks of the genus Carcharhinus

Ears from several species of carcharhinid sharks were studied by gross dissection, light microscopy, transmission electron microscopy, and scanning electron microscopy. Structures along a possible sound transmission path to the ear are described, but main consideration is given to the structure of the macula neglecta. The macula neglecta is composed of two patches of sensory epithelium which line part of the posterior canal duct. In an adult shark the larger of these contains 224,000 sensory hair cells oriented so as to detect forces directed posteroventrolaterally in the duct. The smaller patch contains 43,000 hair cells oriented so as to detect oppositely directed forces. These receptor cells project through numerous small terminals to a total for both patches of 4,700 myelinated nerve fibers. Cytostructural variations throughout the hair cell population are also reported. Estimated acoustic properties of the tissues in this complex and the processing potential of the neural elements are interpreted as suggestive of auditory function. A mechanism based on the geometry of the receptor arrays is proposed to explain behaviorally observed instantaneous sound localization from the farfield. Evolution of the macula neglecta is reviewed, and evidence for homology of the macula neglecta and amphibian papilla is presented.     

Closure of potassium M-channels by muscarinic acetylcholine-receptor stimulants requires a diffusible messenger.

The M-current (IK(M)) is a slow voltage-gated K+ current which can be inhibited by muscarinic acetylcholine-receptor (mAChR) agonists. In the present experiments we have tested whether this inhibition results from a local (membrane-delimited) interaction between the receptor and adjacent channels, or whether channel closure is mediated by a diffusible messenger. To do this, single KM(+)-channel currents were recorded from membrane patches in dissociated rat superior cervical sympathetic neurons by using cell-attached patch electrodes. Channel activity was inhibited when muscarine was applied to the cell membrane outside the patch but persisted when channels were exposed to muscarine added to the pipette solution. We conclude that a diffusible molecule (or molecules) is (are) required to induce intrapatch channel closure following activation of extra-patch receptors.     

Comparison of monoclonal luteinizing hormone (LH) receptor antibody and LH binding sites in vibratome sections of rat ovary by immunohistochemistry

To identify luteinizing hormone (LH) receptors, a monoclonal antibody (MAb) was produced by immunization of Balb/c mice with rat luteal cell membranes. Hybridomas, produced by a method for proteins of low antigenicity, were selected by competition with [125I]-hCG (LH) for luteal membrane binding. Conditions for analysis of LH receptor antibody (IgG2b isotype) binding by immunohistochemistry with an avidin-biotin-peroxidase complex were examined and results compared to localization of bound hCG, to detect receptors. By light microscopy, both bound hCG and the LH receptor antibody were located on luteal cell surfaces. In addition, the LH receptor antibody was associated with luteal cell cytoplasm. Cell surface membrane binding, but not cytoplasmic staining, was reduced in ovaries from rats injected with hCG. By electron microscopy, LH receptor antibody was observed in patches on luteal cell surface membranes and was associated with polysomes, small vesicles, and occasionally with discrete areas of endoplasmic reticulum. Therefore, detection of LH receptors with bound hCG may be limited to receptors found on cell surfaces, while additional LH receptors are revealed by use of a receptor antibody. The cytoplasmic LH receptor may represent stages in the processing of receptor protein. Furthermore, the methodology used in this study should be generally useful for immunohistochemistry with other MAb to receptors.     

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as α4 L9′A mice ¹ and α6 L9′S mice ², allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.     

Distribution and movement of membrane-associated platelet glycoproteins: use of colloidal gold with correlative video-enhanced light microscopy, low-voltage high-resolution scanning electron microscopy, and high-voltage transmission electron microscopy

Scanning electron microscopy (SEM), especially low-voltage (1 KeV) high-resolution SEM, can be used in conjunction with stereo pair high-voltage (1 MeV) transmission electron microscopy (HVEM) of whole spread cells or thick sections effectively to correlate surface structure with internal structure. Surface features such as microvilli, pits, pseudopodia, ruffles, attached virus, and other surface-related morphologic characteristics can be identified using SEM, while underlying cytoskeletal structure and organelle organization can be viewed by HVEM of the same preparation. However, the need to "prepare" cells for electron microscopy precludes observation in the living state. The use of several types of video-enhanced light microscopy (VLM) permits observation of living cells such that certain surface and internal features can be observed at a relatively high level of resolution or detection. Thus, changes in living cells can be followed, and at appropriate times the cells may be chemically fixed or rapidly frozen and prepared for ultrastructural examination by electron microscopy. We have utilized VLM in conjunction with SEM and HVEM to correlate changes in shape and surface structure with changes in the internal structure of platelets. In addition, we have found it advantageous to use colloidal gold-labeling procedures, because these markers are detectable by all three forms of microscopy. Using this approach we have labeled platelet membrane GPIIb/IIIa, a receptor for RGD-containing adhesive proteins, with gold-fibrinogen or gold-anti-IIb/IIIa. The initial binding and subsequent movement of gold-fibrinogen-IIb/IIIa complexes in living platelets was followed by VLM. The movement of individual labels could be mapped. Subsequent observation by low-voltage (1 KeV) high-resolution SEM and HVEM permits visualization of the same individual receptors tracked by LM. The final position on the membrane or the position-in-transit when fixative was added was determined relative to surface ultrastructure (SEM) and internal, particularly cytoskeletal, ultrastructure (HVEM).     

Progress in applying the high-voltage electron microscope to biomedical research

This review attempts a physical definition of the technical problems and achievements in applying the high-voltage electron microscope (HVEM) to biological and medical research. It is hoped that the review will summarize for biologists, funding agencies, and institutions the achievements of the HVEM, its future prospects, and the main problem areas that still need to be explored. At present it is not known whether future HVEMs will favor the fixed beam or the scanning transmission electron microscopy (STEM) mode. The STEM mode offers reduced radiation damage as a result of more efficient electron detection and ease of manipulation of the collected signals by separating the elastic and inelastic signals. Energy filtration to remove the inelastic signal provides a means to enhance the contrast and improve the resolution for thick specimens. Several prototype STEM-mode HVEMs are now under development and it is expected that, in a few years, comparisons of fixed beam and STEM modes will be possible. The review discusses several HVEM instrument features that remain poorly developed. In the area of image recording a photographic emulsion has been designed to give optimized performance at an acceleration voltage of 1 MV. However, this remains unavailable commercially. Conversion of the HVEM electron image to a usable light image by phosphors etc., involves some difficulties, making it difficult to obtain good performance from TV systems. Since the HVEM is particularly useful for three-dimensional imaging, the further development of improved goniometers for stereo viewing and image reconstruction is important. The large volume available in the objective specimen volume and the increased penetration at high acceleration voltages make the HVEM particularly suitable for the application of environmental chambers in the microscopy and electron diffraction of thick wet specimens. An improved signal-to-noise ratio improves the prospects for elemental analysis at high acceleration voltages. When carefully carried out, improved resolution can be obtained in dark-field over that obtainable at 100 kV. Dark-field provides the easiest way to obtain high contrast on weakly stained or unstained objects. Its further improvement requires the use of specially thick and shaped beam stops and apertures that are not penetrated by the 1 MV beam. Recent HVEM studies of whole cells and microorganisms are reviewed. These studies already show that the former thin-section approach led to some incorrect ideas about the shape of some organelles and their three-dimensional relationships. This new information is proving important in helping to establish the function of fibrillar and membranous components of the cell. The most important limitation in examining thick sections is the large depth of field that causes excessive overlap of in-focus structures in stereo views of thick sections. In a few cases special specific heavy metal stains have been developed to overcome this problem, but an optical solution would be more generally applicable. Attempts are now being made to unscramble overlapped detail by applying the image reconstruction techniques of tomography and holography. It is concluded that even with existing techniques, the HVEM examination of thick sections provides a very useful improvement in sampling statistics and in three-dimensional imaging of cell structures over that obtainable by examining thin sections at a lower acceleration voltage (100 kV). Randomized author sequence.     

Combined immunofluorescence and field emission scanning electron microscope study of plasma membrane-associated organelles in highly vacuolated suspensor cells of white spruce somatic embryos

Highly vacuolated suspensor cells of spruce somatic embryos were examined by immunofluorescence light microscopy using butyl-methyl-methacrylate (BMM) and polyethylene glycol (PEG) embedded sections, transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM). The use of PEG embedded embryos provided a rapid method for light microscope detection of antigens before committing to FESEM analysis. BMM embedded specimens provided well preserved suspensor cells for immunofluorescence. FESEM permitted high resolution observation of large areas of the inner surface of the plasma membrane and associated cell organelles. Suspensor cells contained mostly transversely oriented cortical microtubules linked to the plasma membrane and adjacent microtubules by cross- bridges. Light and electron microscopy revealed numerous clathrin coated structures on the plasma membrane. These included flat patches of clathrin, coated pits and coated vesicles. Many coated vesicles were associated with microtubules. Both tubular and lamellar endoplasmic reticulum were observed on the plasma membrane by FESEM.