Ultrastructural immunocytochemistry of the adenohypophysis in the rat: A review

Immunocytochemistry has made great strides in the morphology of endocrine glands, especially the adenohypophysis, because the localization of hormones can be clearly demonstrated by this method in the microscopic preparations both for light and electron microscopy. In the adenohypophysis, electron microscopic immunocytochemistry is useful for identifying the producer cell of each hormone. The second contribution is its application to the cell biology of secretion mechanisms. The pituitary hormones, their precursors, derivatives, and fragments were artificially synthesized and their antibodies were produced. Using these antibodies the intracellular sites of synthesis, condensation, processing, and sorting were studied under the electron microscope. The ultrastructure of each cell organelle and its alteration due to the changing function was studied. It was proved that the intracisternal granules in the thyroidectomy cells contain thyroid-stimulating hormone (TSH). The trans-Golgi network or GERL contains a peculiar supporting structure, intracisternal skeleton. Transport of secretory granules may be performed in relation to the microtubules, actin, and some related substances. The most frequently observed mode of hormone release in the adenohypophysis is exocytosis. Sometimes multigranular exocytosis occurs. Vesiculation of membrane around the secretory granules often occur inward or outward. The inward vesiculation forms pinocytotic vesicles, through which the membrane material may be retrieved. The outward vesiculation forms vesicle-like fragments of cytoplasm being discarded to the extracellular space. By these mechanisms the surface area of the cell is maintained constantly.     

Effect of drugs on pituitary ultrastructure.

Various drugs and hormones influence the light microscopic and especially the electron microscopic structure of the anterior pituitary and its tumors. Many structural effects are known only from animal experiments since specimens from human pituitaries are mostly not available. The structure of growth hormone (GH) cells is relatively stable. A massive GH cell hyperplasia is known only in rare cases with growth hormone releasing factor (GRF) excess from tumors. Prolactin cells can be stimulated by drugs, neurotransmitters, and hormones which decrease the dopamine inhibition. Adrenocorticotropic hormone (ACTH) cells are stimulated by stress, some hormones, loss of adrenals, and drugs which activate the alpha 1- and beta-receptors or inhibit the alpha 2-receptors. They are suppressed and changed into Crooke's cells by treatment with glucocorticoids. Thyroid-stimulating hormone (TSH) cells increase in number and size in states for overstimulation especially by thyrotropin releasing hormone (TRH). A decrease results from hyperthyroidism and possibly from somatostatin, L-dopa, and dopamine. Gonadotroph cells transform into castration cells in strongly hyperactive states (gonadectomy, antiandrogens, gonadotropin releasing hormone [Gn-RH]agonists, aminoglutethimide). Special types of pituitary adenomas can be treated with drugs which suppress hormone production and proliferation. Dopamine agonists and somatostatin reduce the tumor size of varying proportions of GH secreting adenomas in acromegaly. Ultrastructurally, a decrease of cytoplasmic and nuclear volume and an increase of lysosomes are found. Bromocriptine and other dopamine agonists are established in the treatment of prolactin secreting adenomas. They induce a shrinkage in many cases. Ultrastructurally, a reduction of cellular and nuclear size, an increase in number of secretory granules and of lysosomes, and a reduction of rough endoplasmic reticulum can be demonstrated.     

Hypothalamic regulatory peptides and their receptors: Cytochemical studies of their role in regulation at the adenohypophyseal level

Hypothalamic regulatory peptides bind to specific receptors on target cells in the pituitary and control secretion. They in turn can be regulated at the pituitary level by steroid and peptide modulators. Affinity cytochemical techniques are important tools for the identification of specific target binding sites for these regulatory peptides. This presentation reviews the work in which potent, biotinylated ligands of gonadotropin releasing hormone (bio-GnRH), corticotropin releasing hormone (bio-CRH), and arginine vasopressin (bio-AVP) were applied to study the target cell responses. Bio-GnRH, bio-CRH, and bio-AVP bind to membrane receptors on specific anterior pituitary cells. Dual labeling for either gonadotropin or adrenocorticotropin (ACTH) antigens further identified the target cells. After 1–3 minutes, the label was in patches or capped on the surface. After 3 minutes, it was internalized in small vesicles and sent to receptosomes and vacuoles in the Golgi complex. Eventually the biotinylated peptides, or a metabolite, was found in the lysosomes (multivesicular bodies) and a subpopulation of secretory granules. The route and rate of uptake was similar to that described for the classical receptor-mediated endocytosis process. In contrast, intermediate lobe corticotropes internalized the bio-CRH in less than 1 minute. The route through the Golgi complex appeared to be bypassed. Instead the labeled peptide was in vesicles, on the membranes of scattered vacuoles, and in multivesicular bodies. Modulation of ligand binding by steroids showed that changes in receptor numbers correlated with changes in the number of cells that bound the ligand. In male rats, dihydrotestosterone reduced the percentage of GnRH-bound cells by 50%. Most of the reduction appeared in cells that stored luteinizing hormone (LH) antigens. In diestrous female rats, estradiol increased the percentage of bio-GnRH-bound cells. However, the steroid decreased the percentage of GnRH-bound cells in cells from proestrous rats. Glucocorticoids decreased the percentage of CRH-bound corticotropes in as little as 10 minutes. Potentiation of secretion by these ligands was correlated with increases in the percentage of ligand-bound cells. AVP pretreatment of corticotropes increased the percentage of cells that bound bio-CRH. It also increased the rate of receptor-mediated endocytosis of CRH and changed the route so that the Golgi complex was bypassed. This effect could be mimicked by activation of its second messengers (calcium and protein kinase C). Similarly, CRH pretreatment increased the percentage of corticotropes that bound AVP. Thyrotropin releasing hormone (TRH) pretreatment also increased the percentage of thyrotropes that bound AVP. Finally, calcium or sodium channel blockers altered CRH binding so that fewer cells were labeled. This binding by CRH was not dependent on extracellular calcium and tests with a calcium channel agonist showed that it was related to activation of calcium channels. To summarize, these affinity cytochemical studies have identified specific target cells in the pituitary for GnRH, CRH, and AVP. They have also identified heterogeneity in the population. They have demonstrated new information about the direct modulatory effects of steroids, ion channels, and neuropeptides on neuropeptide binding by subpopulations of these target cells.     

The defensive secretion of Carabus lefebvrei Dejean 1826 pupa (Coleoptera,Carabidae): Gland ultrastructure and chemical identification

This study documents the defensive function of flavored humor secreted by the abdominal glands of Carabus lefebvrei pupae. The morphology and the ultrastructure of these glands were described and the volatile compounds of glands secretion were identified by gas chromatography/mass spectrometry. The ultrastructure analysis shows an acinose complex formed by about 50 clusters. Each cluster has 20 glandular units and the unit—composed of one secretory and one canal cell lying along a duct—belongs to the class 3 cell type of Quennedey (1998). In the cytoplasm, the secretory cell contains abundant rough endoplasmatic reticula, glycogen granules, numerous mitochondria, and many well-developed Golgi complexes producing electron-dense secretory granules. Mitochondria are large, elongated, and often adjoining electronlucent vesicles. The kind and the origin of secretory granules varying in size and density were discussed. The chemical analysis of the gland secretion revealed the presence of a mixture of low molecular weight terpenes, ketones, aldehydes, alcohols, esters, and carboxylic acids. Monoterpenes, especially linalool, were the major products. We supposed that ketones, aldehydes, alcohols, esters, and carboxylic acids have a deterrent function against the predators and monoterpenes provide a prophylaxis function against pathogens. Microsc. Res. Tech., 2009. © 2008 Wiley-Liss, Inc.     

Cytochemistry of sialoglycoconjugates,lysozyme, and β‐defensin in eccrine glands of porcine snout skin as studied by electron microscopy

In most mammals except for humanoid primates, eccrine glands are confined to the skin of a series of specific body regions. Sialic acids and antimicrobial substances exhibit various functional properties and serve as a component of nonspecific defense against micro‐organisms, respectively. In this study, the distribution of these moieties was studied by electron microscopic histochemical methods. The eccrine glandular acini consisted of two types of dark cells as well as clear cells. The secretory granules and Golgi apparatus of both types of dark cells contained sialic acid residues linked to α2‐6Gal/GalNAc. On the other hand, sialoglycoconjugates with Siα2‐3Galβ1‐4GlcNAc sequence were confined to those of the Type II dark cells. In addition, lysozyme and β‐defensin were mainly detected in the secretory granules of the Type II dark cells. These secretory products may create a defensive barrier against microbial invasion and play an essential role in preservation of the integrity of porcine snout skin as a sensory organ. Microsc. Res. Tech. 2013. © 2012 Wiley Periodicals, Inc.     

Cryoultramicrotomy versus plastic embedding: comparative immunocytochemistry of rat anterior pituitary cells

The anterior pituitary of the rat is used as a model for the study of the effects of freezing or plastic embedding on the maintenance of antigenicity. Rat anterior pituitaries are fixed in 2.5% glutaraldehyde in 0.1 m phosphate buffer pH 7.4. Some of the blocks are post-fixed before being divided into two lots. One batch is frozen, while the other is dehydrated and embedded. The indirect antibody enzyme method is applied to ultrathin sections obtained by cryoultramicrotomy after freezing or by sectioning after embedding. All six pituitary hormones are detected by both methods. Comparison shows that the morphological characteristics are identical for both techniques, though ultrastructural preservation is better after embedding. Immunoreactivity is found in secretory granules and sometimes in the endoplasmic reticulum. Osmium postfixation may reduce or even abolish antigenicity in plastic-embedded tissue. After cryoultramicrotomy, however, even after osmium fixation, antibody may be used 1000 times more diluted than after plastic embedding. Embedding preserves ultrastructure and limited antigenicity while the use of cryoultramicrotomy is a far more sensitive technique.     

Confocal laser scanning microscopic analysis of ectopic sublingual gland‐like tissue inside the hamster submandibular gland

Based on its histochemical properties, the secretory portion of the hamster submandibular gland has been classified as seromucous cells. The presence of endogenous peroxidase (PO) reaction was shown in the nuclear envelope, cisternae of endoplasmic reticulum and Golgi apparatus. The 3,3′‐diaminobenzidene, tetrahydrochloride (DAB) method revealed bipartite secretory granules containing a PO‐positive dense core surrounded by a less dense halo in these cells. In the present investigation, serous and mucous‐like cells were found in resin‐embedded semi‐thin sections of the DAB‐reacted hamster submandibular gland. These sections were already on glass slides for routine light microscopic observations, therefore electron microscopic analysis could be unrealizable. We then used reflectance‐mode confocal laser scanning microscopy to visualize additional sites of PO activity as detected in these sections. Using this approach, we found mucous cells with PO activity‐negative secretory granules and seromucous cells with PO activity‐positive spot‐like secretory granules of the regular sublingual gland most frequently adjacent to the serous cells with typical electron‐dense secretory granules. These cells clearly differ from the seromucous cells with bipartite secretory granules and the granular duct cells with typical electron‐dense secretory granules of the hamster submandibular gland. Additionally, secretory endpieces of the ectopic sublingual gland‐like tissue empty into the duct of the hamster submandibular gland lobule. Thus, our findings suggest that a mass of sublingual gland tissue extends into the hamster submandibular gland during its development, and PO may be synthesized and secreted into the same duct. Microsc. Res. Tech. 76:1284–1291, 2013. © 2013 Wiley Periodicals, Inc.     

Application of ion etching to immunoscanning electron microscopy

A method for achieving both the light and electron microscopic observations of the same immunolabeled semithin section is described. Mild ion etching (IE) was performed on the semithin LR white resin sections of rat pancreas to evaluate conditions for scanning electron microscopic secondary electron image observations. Before immunocytochemical staining, very mild, rapid etching was conducted as follows: ionization voltage 300 V, operating vacuum 35 Pa, and etching time 1 min, employing an ion coater above sections on glass slides. The sections were immunohistochemically stained with anti-insulin and immunogold in association with silver enhancement techniques for light microscopic observation, in which B cells in pancreatic islets were positively stained brown. Subsequently, essential mild IE was performed over the stained section as follows: 350 V, 38 Pa, 29 min. The samples were coated with platinum for scanning electron microscopic secondary electron images, in which the cores of secretory granules of the B cells were positively labeled with gold-silver particles. The present method is suitable for detection of substances involving immunogold labeling. It enables us to obtain high-resolution images at low magnification that can be correlated with light microscopic observations. Middle to high magnifications are applicable for detailed observations with secondary electron imaging scanning electron microscopy.     

Fine structure of the male accessory glands of Triatoma rubrofasciata (De Geer, 1773) (Hemiptera, Triatominae)

Male of Triatoma rubrofasciata has four elongated sac-like reproductive mesodermic accessory glands, lined by an inner single layer of secretory cells, with basal plasma membrane infolds and short apical microvilli, and externally enveloped by a thin visceral muscle layer. The secretory cells have a well-developed rough endoplasmic reticulum, Golgi complex, mitochondria, and secretory granules. In one day old adult the gland cells are poorly developed, presenting small, electron-transparent secretory granules scattered among the rough endoplasmatic reticulum, whereas in three days old adult these cells have the cisternae of the rough endoplasmatic reticulum varing size degree, filled with granular electrondense content. In five days old males the secretory granules increase in diameter, being released to the gland lumen. Therefore, there is an increase of the secretory activity according to male maturation.     

Review: Studies on the reproductive and developmental biology of <I>Cichlasoma dimerus</I> (Percifomes,Cichlidae)

Many characteristics of the South American teleost fish Cichlasoma dimerus (body size, easy breeding, undemanding maintenance) make it amenable to laboratory studies. In the last years, many of the fundamental aspects of its reproductive and developmental biology have been addressed in our laboratory. Rather recently, the immunohistochemical localization of pituitary hormones involved in reproduction and in background color adaptation has been described in both adult and developing individuals, and the role of FSH in ovarian differentiation has been established. These findings have been correlated with mapping of some of their brain-derived controlling hormones. The latter include brain-derived gonadotropins which were shown to be active in vitro in the control of pituitary hormone secretions. The emerging picture shows C. dimerus as an interesting species in which many of their basic features have already been investigated and which conform a solid platform for comparative studies correlating neurohormones, pituitary hormones and behavior, from the molecular to the organismic level.     

Utilization of electron microscopic techniques in the in vitro study of adenohypophysial function and regulation.

Morphologic studies of human adenohypophysial cells using immunocytochemistry and electron microscopy have characterized the hormone-producing cell types of the normal gland and pituitary adenomas. The classifications which have emerged allow more accurate clinicopathologic correlations than ever before, but have also raised new questions concerning cytogenesis, pathogenesis, and structure-function correlations. We report the results of studies which marry the conventional morphologic techniques of light microscopy, immunohistochemistry, electron microscopy, and ultrastructural immunocytology with functional analyses using tissue culture and radioimmunoassay of hormones released into culture media. The hormone secretory activity of nontumorous and adenomatous pituitary cells is correlated with their structural features; their secretory responses to several adenohypophysiotropic factors are compared with morphologic alterations which are characterized at the light and electron microscopic levels by morphometric analysis. These studies have shown that hypothalamic stimulating hormones increase hormone release by their target cells and alter the ultrastructural appearance of the affected cells by increasing organelles involved in hormone synthesis. Inhibitory drugs and adrenal and gonadal steroids are capable of suppressing hormone release by some tumors and also give rise to morphologic changes which correlate with the functional inhibition. Hormone release by clinically nonfunctioning adenomas has been characterized and the behavior of these tumor cells in vitro sheds some light on the reasons for lack of clinical symptomatology. The plurihormonal nature of several nontumorous and adenomatous pituitary cell types has been characterized in vitro. The results of these studies provide the basis for more accurate structure-function correlations which can be used to study the hormonal milieu in vivo, to predict the role of pathogenetic factors in pituitary tumorigenesis, and to assess the therapeutic value of stimulating or inhibiting hormones and drugs.     

Ultrastructure of differentiating oocytes and vitellogenesis in the giant freshwater prawn, Macrobrachium rosenbergii (de man)

The ultrastructure of oogenesis in Macrobrachium rosenbergii, with reference to vitellogenesis, has not been reported. We used light and electron microscopy, as well as vitellin (Vn) purification and antibody production, to study the temporal and spatial production of Vn in the ovary by immunofluorescence. Histologically, the ovary is subdivided into cone‐shaped ovarian pouches with a central core containing layers of oogonia. These divide to produce oocytes that migrate outwardly and differentiate into mature oocytes. During the course of differentiation, oocytes undergo modifications, including the rearrangement of nuclear chromatin, the accumulation of ribosomes, rough endoplasmic reticulum (RER), and lipid, and the formation of secretory and yolk granules, resulting in four stages. Ultrastructurally, early previtellogenic oocytes (Oc₁) are characterized by the accumulation of new ribosomal aggregates, translocated from the nucleus. Late previtellogenic oocytes (Oc₂) show nuclear heterochromatin with a “clock face” pattern, the presence of RER, and three types of secretory granules. Follicular cells occupy the intercellular spaces and surround the Oc₂. Early vitellogenic oocytes (Oc₃) are larger, with nuclei containing predominantly decondensed euchromatin, and cytoplasm with yolk and secretory granules, and few lipid droplets. Late vitellogenic oocytes (Oc₄) are characterized by completely euchromatic nuclei, an indistinct plasma membrane, yolk platelets and secretory granules, and abundant lipid. Vitellogenin (Vg) in ovaries of M. rosenbergii consist of two main bands at MW 90 and 102 kDa. Our data indicates that Vn is present, and probably synthesized in Oc₃ and Oc₄, but there may be some undetected exogenous Vg production. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Cell biology of the adipokinetic hormone-producing neurosecretory cells in the locust corpus cardiacum.

The adipokinetic cells are neuron-like unipolar cells, the cell bodies and cell processes of which are intermingled within the glandular part of the corpus cardiacum. In Schistocerca gregaria, they produce two adipokinetic hormones, AKH-I and -II, whereas in Locusta migratoria an additional hormone, AKH-III, is present. The three AKHs are produced by the same cells and are co-localized in secretory granules. The biosynthesis and processing of the AKH prohormones to the bioactive hormones, which has been elucidated in detail for AKH-I and -II in S. gregaria, takes less than 75 min and goes on continuously. In older locusts in particular, the adipokinetic cells contain intracisternal granules, widely dilated cisternae of the rough endoplasmic reticulum, which function as stores of prohormones of AKH-I and -II, not of AKH-III. The adipokinetic cells are subjected to regulation by a number of neural and humoral substances, neural influences coming from secretomotor cells in the lateral part of the protocerebrum. Flight activity is the only natural stimulus unequivocally shown to induce the release of AKHs, which in L. migratoria results in parallel secretion of all three AKHs. During secretory stimulation, young secretory granules containing newly synthesized hormones are preferentially released over older granules. Secretory stimulation is not accompanied by a clear increase in the levels of the AKH mRNAs and the AKH prohormones and in the rate of synthesis of the (pro-)AKHs. Apparently, a coupling between release and biosynthesis of the AKHs in the adipokinetic cells is very loose or does not even exist.     

Ultrastructure of Paneth cells in the intestine of various mammals

Paneth cells in the following species were observed under an electron microscope: human, rhesus monkey, hare, guinea pig, rat, nude rat, mouse, golden hamster, and insect feeder bat. Secretory granules containing homogeneous electron-dense materials were observed in the Paneth cells of humans, monkeys, hares, guinea pigs, and bats; mouse Paneth-cell granules were bipartite (central core and peripheral halo), and the Paneth cells in rats and golden hamsters had secretory granules showing various electron densities. In humans, monkeys, and bats, immature granules near the Golgi apparatus sometimes showed bipartite substructure. The number and size of secretory granules were also diverse among various animal species. Some lysosome-like bodies were commonly observed in peri- or supranuclear regions, though the size and shape of the bodies differed from cell to cell. In apical cytoplasm, small clear vesicles (100–200 nm diameter) were more-or-less observed in all species examined, and it was especially note that rat Paneth cells contained many clear vesicles. Small dense-cored vesicles (150–200 nm diameter) were rare. It is unlikely that the various ultrastructural features of Paneth cells correlate with the phylogenetical classification.     

Instructive induction of prostate growth and differentiation by a defined urogenital sinus mesenchyme

Instructive influences of fetal mesenchyme were examined in heterotypic tissue recombinants consisting of urogenital sinus mesenchyme (UGM) from male and female rats and distal ductal tips from adult rat prostate. Tissues were grown under the renal capsule of male hosts for periods up to 28 days. Resultant growths exhibited typical prostate histology. Expression of lobe-specific proteins for the ventral (prostatic steroid binding protein [PSBP]) lateral (seminal vesicle secretion II [SVS II]), and dorsal prostate (secretory transglutaminase [TGase]) were examined by immunocytochemistry. Male or female UGM combined with terminal segments of the ventral or dorsal prostate and immunolabeled with antibodies to lobe-specific proteins demonstrated expression of all three secretory products. The pattern of staining was consistent with a compound inductive response from the UGM. Unique to this study was our ability to use a defined mesenchymal tissue (female ventral mesenchymal pad [VMP]). This tissue is specifically associated with ductal branching morphogenesis and cytodifferentiation of the ventral prostate. Distal ductal tips from the dorsal lobe of the adult male prostate when recombined with female VMP and grown in vivo exhibited transformation of secretory phenotype, and the epithelium expressed mRNAs for PSBP. Immunocytochemistry of serial sections did not demonstrate labeling for TGase in the new epithelial growth. Ultrastructural analysis of the heterotypic recombinants indicated that the epithelium had similar characteristics to those of normal ventral prostate. Early stages of the mesenchymal-epithelial interactions resulted in dedifferentiation of the adult epithelium to solid cords of stratified cells. These findings illustrate the potent instructive capacity of a defined fetal UGM to influence development and cytodifferentiation of adult prostate epithelium. © 1995 Wiley-Liss, Inc.     

Role of the golgi apparatus in cellular pathology

The Golgi apparatus response to pathological disorders is predominantly as an intermediary component of membrane biogenesis where it is involved in processing, sorting and secretion of materials via secretory granules, and in the formation of lysosomes. A common initial response of the Golgi apparatus to any stress is an alteration or cessation of secretory activity. In the transformed cell, the Golgi apparatus is altered both morphologically and biochemically, suggesting a shift from a secretory to a membrane-generating mode of functioning. However, since fewer or less well-developed Golgi apparatus are frequently found in transformed cells, analytical methods of membrane isolation developed for normal tissues may not always yield equivalent results when applied to tumors. Cell surface alterations characteristic of malignant cells may result from modifications occurring at the level of the Golgi apparatus. Some lysosomal dysfunctions may result from underglycosylation of acid hydrolases by the Golgi apparatus. The use of cell-free systems between endoplasmic reticulum and Golgi apparatus or within Golgi apparatus cisterane is providing a new approach to the elucidation of the role of the Golgi apparatus in normal as well as pathological states.     

Organ culture of the bovine subcommissural organ: evidence for synthesis and release of the secretory material

The subcommissural organ (SCO) is a brain circumventricular organ formed by ependymal and hypendymal secretory cells. It secretes glycoproteins into the cerebrospinal fluid of the third ventricle where they condense into a thread-like structure known as Reissner's fiber (RF). The present study was designed to investigate whether or not the bovine SCO continues to synthesize and release glycoproteins after a long-term culture. Cultured explants of SCO survive for several months. The content of the secretory granules present in the cultured ependymocytes displayed immunoreactive and lectin-binding properties similar to those of the core glycosylated glycoproteins found in the bovine SCO. The explants actively incorporated (35)S-cysteine. In the cultured ependymocytes, the pattern of distribution of the radioactive label and that of the immunoreactive secretory material was similar, thus indicating that this material has been synthesized during culture. At the ultrastructural level, the cultured tissue exhibited a high degree of differentiation comparable to that of the bovine SCO in situ. A striking finding was the observation of similar results when cerebrospinal fluid was used as a culture medium. The addition of antibodies against RF-glycoproteins into the culture medium allowed visualization, by means of different immunocytochemistry protocols, deposits of extracellular immunoreactive secretory material on the free surface of the cultured ependymocytes, indicating that release of secretory glycoproteins into the culture medium does occur. Primary culture of dispersed SCO ependymocytes, obtained either from fresh or organ cultured bovine SCO, showed that these cells release RF-glycoproteins that aggregate in the vicinity of each cell. The present investigation has shown that: (1) two types of secretory ependymocytes become evident in the cultured SCO; (2) under culture conditions, the SCO cells increase their secretory activity; (3) explants of bovine SCO synthesize RF-glycoproteins and release them to the culture medium; (4) after release these proteins aggregate but do not form a RF; (5) a pulse of anti-RF antibodies into the culture medium blocks the secretion of RF-glycoproteins for several days.     

Tracking of secretory vesicles of PC12 cells by total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy is used to detect cellular events near the plasma membrane. Behaviours of secretory vesicles near the cell surface of living PC12 cells, a neuroendocrine cell line, are studied. The secretory vesicles are labelled by over‐expression of enhanced green fluorescent protein‐tagged Rab3A, one of the small G proteins involved in the fusion of secretory vesicles to plasma membrane in PC12 cells. Images acquired by a fast cooled charge‐coupled device camera using conventional fluorescence microscopy and total internal reflection fluorescence microscopy are compared and analysed. Within the small evanescent range (< 200 nm), the movements of the secretory vesicles of PC12 cells before and after stimulation by high K⁺ are examined. The movements of one vesicle relative to another already docked on the membrane are detected. Total internal reflection fluorescence microscopy provides a novel optical method to trace and analyse the exocytotic events and vesicle specifically near a cell membrane without interference of signals from other parts of the cell.