Carboxypeptidase M, a glycosylphosphatidylinositol-anchored protein, is localized on both the apical and basolateral domains of polarized Madin-Darby canine kidney cells.

Carboxypeptidase M, a glycosylphosphatidylinositol-anchored membrane glycoprotein, is highly expressed in Madin-Darby canine kidney (MDCK) cells, where it was previously shown that the glycosylphosphatidylinositol anchor and N-linked carbohydrate are apical targeting signals. Here, we show that carboxypeptidase M has an unusual, non-polarized distribution, with up to 44% on the basolateral domain of polarized MDCK cells grown on semipermeable inserts. Alkaline phosphatase, as well as five other glycosylphosphatidylinositol-anchored proteins, and transmembrane gamma-glutamyl transpeptidase exhibited the expected apical localization. Basolateral carboxypeptidase M was readily released by exogenous phosphatidylinositol-specific phospholipase C, showing it is glycosylphosphatidylinositol-anchored, whereas apical carboxypeptidase M was more resistant to release. In contrast, the spontaneous release of carboxypeptidase M into the medium was much higher on the apical than the basolateral domain. In pulse-chase studies, newly synthesized carboxypeptidase M arrived in equal amounts within 30 min on both domains, indicating direct sorting. After 4-8 h of chase, the steady-state distribution was attained, possibly due to transcytosis from the basolateral to the apical domain. These data suggest the presence of a unique basolateral targeting signal in carboxypeptidase M that competes with its apical targeting signals, resulting in a non-polarized distribution in MDCK cells.

Emperipolesis of erythroblasts within Kupffer cells during hepatic hemopoiesis in human fetus.

The state in which cells can inhabit other cells without damage is known as emperipolesis. Emperipolesis has been found in various physiological and pathological conditions. We performed a study of emperipolesis of erythroblasts within Kupffer cells in the human fetal liver. We found that Kupffer cells, identified by CD68 immunolabeling, contained 4-8 erythroblasts in a hypertrophic cytoplasm on light microscopy. Emperipoletic erythroblasts were present in various maturation stages from proerythroblast to reticulocyte. By electron microscopy, we found that erythroblasts occupied membrane-bound vacuoles that were separated from each other by thin partitions of Kupffer cell cytoplasm. Neither emperipoletic erythroblasts nor their Kupffer cell hosts showed evidence of damage. Emperipoletic cells in mitosis were found, which suggests the capacity for the proliferation of erythroblasts within Kupffer cells. Some Kupffer cells were seen to contain both emperipoletic cells and phagosomes, without evidence of interaction. Erythroblasts and other hemopoietic cells were also found to be closely associated with the sinusoidal surface of Kupffer cells. However, intercellular junctions, if present, were inconspicuous. On occasion, Kupffer cells engorged with erythroblasts nearly occluded the sinusoidal lumen. Our results demonstrate that emperipolesis of erythroblasts within Kupffer cells occurs in human fetal hepatic hemopoiesis. We suggest that emperipolesis may be one of the mechanisms that support the maturation of erythroblasts in the fetal liver.

Detection of neutral endopeptidase 24.11 (neprilysin) in human hepatocellular carcinomas by immunocytochemistry.

BACKGROUND: We have reported previously that neutral endopeptidase 24.11 (neprilysin; NEP; CALLA, CD10) activity was very high in rat hepatomas and a cultured human hepatocarcinoma cell line (SK-HEP1). MATERIALS AND METHODS: While continuing these studies, we detected the presence of NEP in SK-HEP 1 cells by immunocytochemistry and in paraffin-embedded human hepatocellular carcinomas as well. IgG purified from polyclonal antisera to human NEP was employed as a source of antibody. RESULTS: SK-HEP 1 cells gave a strong positive reaction to the IgG fraction of the antisera. In control studies, where IgG was preabsorbed with recombinant NEP, the results were negative. Of the 18 hepatocellular carcinomas tested, NEP was expressed in 14 (78%) malignant tumors, while adjacent liver tissue did not show the presence of NEP. CONCLUSIONS: It is suggested that, because none of the known hepatocellular carcinoma markers are highly specific, the detection of NEP in these malignant cells can be an additional useful diagnostic tool.

Potentiation of the actions of bradykinin by angiotensin I-converting enzyme inhibitors. The role of expressed human bradykinin B2 receptors and angiotensin I-converting enzyme in CHO cells.

Part of the beneficial effects of angiotensin I-converting enzyme (ACE) inhibitors are due to augmenting the actions of bradykinin (BK). We studied this effect of enalaprilat on the binding of [3H]BK to Chinese hamster ovary (CHO) cells stably transfected to express the human BK B2 receptor alone (CHO-3B) or in combination with ACE (CHO-15AB). In CHO-15AB cells, enalaprilat (1 mumol/L) increased the total number of low-affinity [3H]BK binding sites on the cells at 37 degrees C, but not at 4 degrees C, from 18.4 +/- 4.3 to 40.3 +/- 11.9 fmol/10(6) cells (P < .05; Kd, 2.3 +/- 0.8 and 5.9 +/- 1.3 nmol/L; n = 4). Enalaprilat preserved a portion of the receptors in high-affinity conformation (Kd, 0.17 +/- 0.08 nmol/L; 8.1 +/- 0.9 fmol/10(6) cells). Enalaprilat decreased the IC50 of [Hyp3-Tyr(Me)8]BK, the BK analogue more resistant to ACE, from 3.2 +/- 0.8 to 0.41 +/- 0.16 nmol/L (P < .05, n = 3). The biphasic displacement curve of the binding of [3H]BK also suggested the presence of high-affinity BK binding sites. Enalaprilat (5 nmol to 1 mumol/L) potentiated the release of [3H]arachidonic acid and the liberation of inositol 1,4,5-trisphosphate (IP3) induced by BK and [Hyp3-Tyr(Me)8]BK. Moreover, enalaprilat (1 mumol/L) completely and immediately restored the response of the B2 receptor, desensitized by the agonist (1 mumol/L [Hyp3-Tyr(Me)8]BK); this effect was blocked by the antagonist, HOE 140. Finally, enalaprilat, but not the prodrug enalapril, decreased internalization of the receptor from 70 +/- 9% to 45 +/- 9% (P < .05, n = 7). In CHO-3B cells, enalaprilat was ineffective. ACE inhibitors in the presence of both the B2 receptor and ACE enhance BK binding, protect high-affinity receptors, block receptor desensitization, and decrease internalization, thereby potentiating BK beyond blocking its hydrolysis.

Expression of rat kallikrein and epithelial polarity in transfected Madin-Darby canine kidney cells.

Many properties of urinary kallikrein are well characterized, but the intracellular processing of prokallikrein and release by kidney cells have yet to be clarified. We report here on the synthesis of prokallikrein in Madin-Darby canine kidney (MDCK) cells transfected with rat submaxillary gland kallikrein cDNA and on its activation by MDCK cells and by an enriched liver Golgi membrane preparation. Transfected MDCK cells secreted only prokallikrein at both the apical and basolateral sides in about a 4:1 ratio, but cells transfected with kallikrein cDNA in reverse orientation or untreated cells released only traces of the enzyme. Prokallikrein, in culture medium or in homogenized MDCK cells, was fully activated by trypsin but activated only to 44% by thermolysin. Prokallikrein was synthesized and released into the medium at a high rate: the enzyme secreted by 5 x 10(6) cells in 24 hours cleaved 46 nmol/min D-Val-Leu-Arg-7-amino-4-methylcoumarin and liberated 63 ng/min bradykinin after activation. Immunocytology indicated the association of prokallikrein with the Golgi apparatus in the transfected cells. Antiserum to rat urinary kallikrein detected a single band in a Western blot of conditioned medium and also immunoprecipitated the enzyme. Aprotinin inhibited activated prokallikrein. Although MDCK cells released prokallikrein, their homogenates activated prokallikrein at both pH 5.5 and 7.5. Prokallikrein was also activated by a highly enriched liver Golgi membrane fraction and by an endoplasmic reticulum preparation, but the Golgi preparation was 38-fold more active. The activation was blocked significantly by inhibitors of serine proteases and less by cysteine protease inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Carboxypeptidase M activity is increased in bronchoalveolar lavage in human lung disease.

Carboxypeptidase M (CPM) cleaves the C-terminal arginine and lysine of peptides; it is expressed in the lung, especially on the plasma membrane of alveolar type I cells. Here, we report on CPM in human bronchoalveolar lavage (BAL) collected from 69 patients and analyzed for activity, cell number and type, and protein level. Seventy-six percent of CPM activity, measured at pH 7.5 with 5-dimethylamino-naphthalene-1-sulfonyl-alanyl-arginine (Dansyl-Ala-Arg) substrate, was immunoprecipitated with polyclonal antibody to purified human enzyme. In patients without active lung disease, CPM activity in BAL was 7.69 (+/- 2.12) nmol/h/mg protein, but in patients with acute pneumonia, it was 29.25 (+/- 4.06) (p < 0.01). In patients with Pneumocystis carinii pneumonia, CPM activity was elevated to 26.00 (+/- 4.85) (p < 0.01) and in patients with lung cancer, to 30.95 (+/- 4.12) (p < 0.01). The activity was not associated with the cellular elements of BAL. The highest specific activity was in the large aggregate fraction of surfactant, which also contained the highest concentration of phosphorus. Transmission electron microscopy of this fraction revealed the presence of typical lamellar bodies and tubular myelin structures. The high CPM activity may stem from its induction and release in acute lung disease. In addition, CPM may be a marker of infection with certain pathogens and an indicator of type I cell injury in parenchymal lung diseases.

Plasma membrane-bound and lysosomal peptidases in human alveolar macrophages.

Alveolar macrophages protect the lungs against noxious agents. Proteases and peptidases are essential for this defense and many metabolic activities. Human alveolar macrophages were evaluated for the presence of six important peptidases. Deamidase, a serine peptidase identical with the lysosomal protective protein and possibly with cathepsin A, had high specific activity in alveolar macrophages and is also present in cultured mouse J774A.1 and human U937 cells, used for the sake of comparison. In fractionated J774A cells, most of the deamidase activity was in the lysosomal fraction and in the final supernatant. Deamidase in human alveolar macrophages, obtained by bronchoalveolar lavage from 23 patients, cleaved dansyl-Phe-Leu-Arg at a rate of 2.26 mumol/h/mg protein and hydrolyzed the chemotactic peptide N-f-Met-Leu-Phe even faster, at a rate of 53.1 mumol/h/mg protein, the highest activity for this enzyme with any of the cells we tested. Rabbit antiserum, elicited with the recombinant partial sequence of the enzyme, immunoprecipitated 77-88% of the macrophage deamidase. In immunocytochemistry, this antiserum localized deamidase within the human macrophages. The enzyme was inhibited by diisopropylfluorophosphate (DFP; 1 mM) and by ebelactone B (10 microM), noncompetitively. The mRNA of deamidase was detected in mouse macrophages by Northern blot; the two protein chains of deamidase were shown in human macrophages by Western blot. In addition, two other serine peptidases were also highly active in macrophages: dipeptidyl peptidase IV (1.38 mumol/h/mg protein) and prolylcarboxypeptidase (0.72 mumol/h/mg protein). The activity of plasma membrane zinc metallopeptidases, neutral endopeptidase 24.11 and carboxypeptidase M, in contrast, was low or absent (angiotensin I converting enzyme; kininase II).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of bradykinin B2 receptors in adult myocardium and neonatal rat cardiomyocytes.

Specific [125I-Tyr8]bradykinin (BK) binding was observed on myocardial membranes from adult guinea pigs, dogs, rats, and rabbits that was displaced by unlabeled BK with an IC50 between 0.1 and 30 nmol/L. In the adult guinea pig ventricular myocardium, which displays both high- and low-affinity binding, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 100 mumol/L) eliminated high-affinity binding and reduced total specific [2,3-prolyl-3,4-3H(N)]BK ([3H]BK) binding by > 60%. Agonist competition binding to rat myocardial membranes was characterized as being of one affinity for BK in the nanomolar range, and it was not altered by GTP gamma S. Saturation binding studies with [125I-Tyr8]BK and [3H]BK, performed on cultured neonatal rat cardiac myocytes, revealed a single class of BK binding sites with a Kd and Bmax of 0.24 +/- 0.04 nmol/L and 18.4 +/- 1.1 fmol/mg protein, respectively (approximately 1500 receptors per cell). In competitive binding assays, unlabeled BK, Hoe 140 (a specific BK B2 receptor antagonist), and des-Arg9,[Leu8]BK (a BK B1 receptor antagonist) displaced [125I-Tyr8]BK with an IC50 of 4.3, 0.041, and 307 nmol/L, respectively. In the presence of 100 mumol/L GTP gamma S, [3H]BK binding to myocyte membranes was reduced by 40%, but the IC50 did not change. Cardiac fibroblasts, evaluated in parallel to the myocytes, contain a single class of [3H]BK binding sites (248 +/- 72 fmol/mg) with a 130-fold lower relative affinity (32.4 +/- 11.3 nmol/L) than that determined in rat cardiomyocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

High concentration of carboxypeptidase M in lungs: presence of the enzyme in alveolar type I cells.

The presence of high concentrations of membrane-bound carboxypeptidase M in human, baboon, dog, and rat lung was established by employing a variety of techniques. The activity of the enzyme in the membrane-enriched fractions of human, baboon, dog, and rat lung, measured with fluorescent dansyl substrate (DNS-Ala-Arg), was 198, 261, 484, and 153 nmol/h/mg protein, respectively. This activity in the lung was much higher than that found in the heart, liver, or kidney. The enzyme, optimally active around neutral pH, was completely inhibited by 10 microM 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid and was activated by 1 mM CoCl2 to 170%. Antibody to human carboxypeptidase M immunoprecipitated the solubilized carboxypeptidase from human (98%), baboon (81%), and dog (88%) lung membrane fractions. Carboxypeptidase M is attached to lung membranes by a phosphatidylinositol glycan anchor; thus, it is released with bacterial phospholipase C. Membrane fractions from cultured human pulmonary arterial endothelial cells also contained high carboxypeptidase M activity (254 nmol/h/mg protein). A Northern blot of poly(A)+ RNA from various human tissues showed the presence of a high level of carboxypeptidase M mRNA in human lung and placenta. Finally, immunohistochemistry, employing purified antibody to the enzyme, revealed in fluorescent light microscopy that carboxypeptidase M is present in alveolar type I pneumocytes and in macrophages in apparently lower concentration. In contrast, type II alveolar epithelial cells gave negative results. Because carboxypeptidase M cleaves a variety of active peptides (e.g., bradykinin, anaphylatoxins), it may protect the alveolar surface from the effects of these peptides. In addition, carboxypeptidase M could be a marker enzyme for type I cells.

Carboxypeptidase M in brain and peripheral nerves.

Carboxypeptidase M (CPM), a plasma membrane-bound enzyme, cleaves C-terminal basic amino acids with a neutral pH optimum. We studied its distribution in human, baboon, and dog brain and in dog peripheral nerves. Areas were dissected, homogenized, centrifuged, and assayed for activity with dansyl-Ala-Arg. The corpus callosum and the pyramidal and optic tract were especially rich in CPM, whereas basal ganglia and cortex had low activity. The identity of the basic carboxypeptidase activity with CPM was shown by similarities in subcellular localization, membrane attachment, substrate hydrolysis, inhibition by a specific basic carboxypeptidase inhibitor, and cross-reaction with anti-human CPM antiserum. This antiserum immunoprecipitated an average of 85% of the activity in human and baboon brain and approximately 66% in dog brain. CPM co-purified with myelin extracted from the brain. Consistent with results obtained in placenta and cultured kidney cells, CPM in the brain appears to be membrane-bound via a phosphatidylinositol glycan anchor. In the peripheral nerves, the specific activity in dog sciatic nerve and in vagus was high (98 and 149 nmol/h/mg of protein, respectively). In immunohistochemical studies, glia in the brain, which appear to be oligodendrocytes or astrocytes, and the outer aspects of myelin sheaths and Schwann cells in sciatic and vagus nerves were stained. We conclude that in some areas of the CNS and the PNS, CPM is closely associated with myelin and myelin-forming cells. Northern blot analysis revealed the presence of mRNA coding for CPM in the brain, showing that the enzyme is indeed synthesized there.

Carboxypeptidase M in Madin-Darby canine kidney cells. Evidence that carboxypeptidase M has a phosphatidylinositol glycan anchor.

Carboxypeptidase M, a plasma membrane-bound enzyme, is present in many human organs and differs from other carboxypeptidase that cleave basic COOH-terminal amino acids. Cultured Madin-Darby canine kidney (MDCK) distal tubular cells contain a kininase I-type enzyme that inactivates bradykinin by releasing Arg9. We found the properties of this kininase to be identical with carboxypeptidase M. In fractionated cells, carboxypeptidase activity sediments with membranes; and detergents, trypsin, and phosphatidylinositol-specific phospholipase C solubilize it, similar to results with human placental carboxypeptidase M. Ten microM 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid and 1 mM o-phenanthroline inhibit, whereas 1.0 mM CoCl2 activates the enzyme. It has a neutral pH optimum and cleaves COOH-terminal Arg or Lys in bradykinin and in shorter peptides. The relative hydrolysis rates of peptides in the presence or absence of 1 mM CoCl2 were similar to those obtained with human carboxypeptidase M. The carboxypeptidase in MDCK cells (54 kDa) cross-reacts with antibodies to human carboxypeptidase M in Western blotting, but not with antibodies to plasma carboxypeptidase N. The enzyme is a glycoprotein; chemical deglycosylation reduced the size to 48 kDa. The presence of the enzyme on the cell membrane of MDCK cells was also shown with transmission electron microscopy using immunogold, which indicated that the enzyme is on the apical side. In addition, MDCK cells contain neutral endopeptidase 24.11 (enkephalinase) and prolylcarboxypeptidase (angiotensinase C) activities. Partitioning of solubilized carboxypeptidase M into Triton X-114 and water indicates that trypsin and phospholipase C remove a hydrophobic tail, while detergent solubilization leaves the hydrophobic moiety intact. Labeling of MDCK cells with [3H]ethanolamine resulted in the synthesis of radiolabeled carboxypeptidase M as determined by immunoprecipitation and fluorography. Thus, MDCK cells contain membrane-bound carboxypeptidase M, which is anchored to the plasma membrane via phosphatidylinositol-glycan. As a major kininase of the distal tubules, it may regulate salt and water excretion.

Endocytosis of serum albumin-gold conjugates by microvascular endothelial cells in rat adrenal gland: regional differences between cortex and medulla.

We investigated the distribution of serum albumin (bovine) modified by adsorption to 15 nm diameter colloidal gold (Au15BSA) in rat adrenal vasculature after perfusion of Au15BSA in situ for 6-20 min. A striking difference in permeability to and uptake of Au15BSA by adrenal cortical and medullary endothelial cells was found. The fenestrated capillary endothelial cells in the cortex were permeable to Au15BSA particles, but showed no significant endocytosis of these conjugates. Endothelial cells of the medullary capillaries and venous tributaries of the central vein were markedly less fenestrated than cortical capillaries, and resembled morphologically the sinusoidal endothelial cells of bone marrow. These endothelial cells, unlike those in the cortex, were not permeable to the perfused Au15BSA, and showed endocytosis of the gold-conjugated protein by means of coated pits and vesicles. No evidence of transcytosis of Au15BSA by means of non-coated, plasmalemmal vesicles was found. Uptake of Au15BSA by medullary endothelia was not appreciably diminished by the presence of one hundred-fold excess, unmodified, monomeric BSA. One hundred-fold excess of serum albumin which was modified by treatment with formaldehyde (FmBSA), on the other hand, did effectively compete for uptake of Au15BSA. These results indicate that adrenal medullary endothelial cells, in contrast to those of the cortex, selectively take up serum albumin modified by adsorption to colloidal gold beads, or modified by treatment with formaldehyde. The endocytosis appears to involve selective adsorption of the Au-modified albumin to sites, perhaps receptors, at coated regions of the luminal endothelial surface.

Silver staining of Pneumocystis carinii in the rat's lung.

The backscattered electron imaging mode of the scanning electron microscope was used to study the ontogenetic acquisition of argyrophilia in Pneumocystis carinii in rats. Silver staining continually increased from the late trophozoite to the mature cyst stage. The silver uptake began with a fine outline at the surface of the bodies of the late trophozoites; their cellular extensions, however, did not stain. The oblate precyst forms acquired the silver in heterogeneous patches. On spherical cysts the silver staining became more uniform and intense with at least one dense spot. The spherical and collapsed cysts also had short silver staining projections that may represent microvilli. Collapsed forms were paler than spherical ones and appear to be cysts that have undergone partial or complete release of sporozoites. These cell surface observations confirm and amplify previous transmission electron microscopical and histochemical studies indicating that silver staining correlates with the acquisition of the cell pellicle.

Endocytosis by endothelial phagocytes: uptake of bovine serum albumin-gold conjugates in bone marrow.

The endocytic function of the endothelial cells of the venous sinuses ("sinusoids") in rat bone marrow was investigated. Marrow sinusoidal endothelial cells (MSEC) were exposed to pulse injections of bovine serum albumin-gold (BSA-Au) conjugates in vivo and examined after timed intervals by electron microscopy. BSA-Au particles were rapidly taken up by MSEC. Within 1 min, BSA-Au was internalized by means of coated pits and vesicles (exclusively) and processed through pleomorphic endosomes to dense bodies known to be secondary lysosomes. In this processing, no involvement of the Golgi apparatus was observed. The continuous association of BSA with gold throughout endocytic processing to lysosomes was verified by immunolabeling with anti-BSA antibody. Although the nature of the affinity mediating endocytosis of BSA-Au by MSEC, in particular the possibility of specific adsorption of BSA and receptor involvement, remains to be established, it is suggested that marrow sinusoidal endothelial cells internalize the albumin-coated particles and process them through to lysosomal structures for purposes of degrading the albumin component.

Preservation of ultrastructure in bone marrow with a glutaraldehyde-artificial blood perfusate.

A method of perfusion-fixation of bone marrow using a commercially available blood substitute as a vehicle for glutaraldehyde has been developed. Low magnification observations of sections by transmission electron microscopy revealed a high degree of integrity throughout the hematopoietic tissue. The continuity and fine structure of the sinusoidal endothelial cells were well preserved as evidenced by exclusion of ruthenium red/OsO4, and occasionally, stages of blood cell egress (diapedesis) across the sinusoidal lining were observed. The quality of fixation of cellular organelles was judged to be equal or superior to that obtained with the use of conventional buffer-based fixatives.

Synaptic vesicles and the nerve-muscle preparation in resinless sections.

Resinless sections have been used to study the fine structural organization of cellular organelles and fibrous components at synapses. Presynaptic elements of the neuromuscular junction, representing the P.N.S., and of cerebral cortical synapses, representing the C.N.S., are examined and compared. Typical ultrastructural features are apparent in presynaptic and postsynaptic components and in the surrounding muscle and neuronal tissue demonstrating the reliability and usefulness of the technique.

Fine structure of synapses of the central nervous system in resinless sections.

The cytoskeleton has been implicated in neuronal function, particularly in axonal transport, excitability at axonal membranes, and movement of synaptic vesicles at preganglionic endings. The present study demonstrates the presence of a pre- and postsynaptic cytoskeleton in resinless sections of CNS tissue by use of the polyethylene glycol (PEG) technique of Wolosewick (1980) viewed by conventional transmission EM, scanning transmission EM, and surface scanning EM. The PEG technique permits visualization of the cytoskeletal network unobscured by the electron scattering properties of epoxy embedment. In the presynaptic process, synaptic vesicles appear to be suspended in a filamentous network that is contiguous with the synaptic vesicle membrane and with the presynaptic plasma membrane and its dense material. In the postsynaptic process, the postsynaptic density (PSD) is seen in intimate contact with the postsynaptic membrane. En face images of the PSD in some synapses appear as a torus. Emanating from the filamentous web of the PSD are filaments which extend to the adjacent plasma membrane. We conclude that membranous synaptic elements are contiguous with a three-dimensional lattice network that is similar to that described in whole unembedded cells (Wolosewick and Porter, 1976). Moreover, the synaptic densities represent a specialized elaboration of the cytoskeleton.

Backscattered electron imaging for the life sciences: introduction and index to applications - 1961 to 1980.

Backscattered electron imaging (BEI) in a scanning electron microscope has been used in biological investigation since 1961. Originally used for imaging specimen surfaces during x-ray microanalysis, applications of this method accelerated rapidly with the recognition of its potential for selective imaging of heavy metal stained biological structures, including those labelled by cytochemical methods. This use of backscattered imaging, to view heavy metal stained structures in cells and tissues, has become the predominate BEI application in the life sciences. This paper presents a brief introduction to BEI as it relates to the use of the scanning microscope for histo - and cytochemistry. Biological applications reported in the years 1961-1980 are cited and indexed by subject and author(s).

Visualization of subsurface structures in cells and tissues by backscattered electron imaging.

A fraction of the beam electrons which interact with a specimen scatter back. The number of backscattered electrons (BE's) increases with the atomic number of the elements encountered. Cell and tissue structures lacking a heavy metal content yield few BE's compared to structures affixed with heavy metals, either vitally or by means of staining methods applied after fixation. The BE imaging mode of a scanning electron microscope (SEM) provides an intensity map of the BE yield from the specimen. BE imaging of selectively stained structures in cells and tissues renders these structures visible in contrast to the unstained surround. Since BE's can emerge from a significant depth within the material, BE imaging can be used to view such heavy metal stained structures beneath intact cell surfaces. The microcontours of the overlying surface can be viewed concurrently by using the surface scanning (i.e., the secondary electron imaging; SEI) mode of the microscope. Methods for selectively contrasting subsurface structures can be adapted from existing light microscope (LM) and transmission electron microscope (TEM) methods. Staining methods have been devised for subsurface viewing of cell organelles, including nuclei, mitochondria, peroxisomes, lysosomes, and phagosomes. A physical model is presented which describes these observations and suggests future possible trends in this subject. Specifically the image contrast and resolution are described in terms of the physical properties of the stain and specimen and of the SEM operating conditions of energy and current. Finally a summary of instrumentation considerations describes present and potential BE detectors, their ancillary electronics, and image processing.