Three-dimensional cytomorphology in fine needle aspiration biopsy of subacute thyroiditis

OBJECTIVE: To elucidate 3-dimensional (3-D) cytomorphology in fine needle aspiration biopsy (FNAB) of subacute thyroiditis. STUDY DESIGN: Ultrasound-guided FNAB was performed on the inflamed area of the thyroid from 4 patients with subacute thyroiditis. The aspirates were stained and observed under a light microscope (LM). The aspirates were also fixed, dehydrated, critical point dried, spattered with gold ions and observed with a scanning electron microscope (SEM). For transmission electron microscopy (TEM), the specimen was fixed, dehydrated, embedded in an Epon mixture, cut with an ultramicrotome, mounted on copper grids, electron doubly stained with uranium acetate and lead citrate, and observed with TEM. Findings with SEM were correlated with those with LM and TEM. RESULTS: Under SEM, 3-D cytomorphology of subacute thyroiditis displayed loss of a uniform, honeycomb cellular arrangement; variation in size; and decrease or shortening of microvilli in follicular cells, which corresponded to varying degrees of cellular degeneration under TEM. Giant cells that were round or ovoid were also noted with SEM. CONCLUSION: Loss of a uniform, honeycomb cellular arrangement; variation in size and decrease or shortening of microvilli in follicular cells; and appearance of round or ovoid giant cells were characteristic 3-D cytomorphology findings in FNAB of subacute thyroiditis.     

Cytologic characteristics and origin of naked nuclei in breast aspirate smears

To elucidate the origin of "naked nuclei" in breast aspiration smears, 17 cases of fibroadenoma were studied by light and electron microscopy. The ATPase reaction was also studied at both levels. The aspirates contained two types of naked nuclei: denuded degenerated nuclei and oval to spindle-shaped nuclei with very scanty cytoplasm. The cytoplasm of the latter was rich in free ribosomes and rough-surfaced endoplasmic reticulum but was devoid of cytoplasmic filaments and dense bodies. These cells were negative for ATPase activity. Stromal cells, not myoepithelial cells, characteristically demonstrated such cellular features in the aspirates and tissue sections studied. We conclude that most naked nuclei are derived from stromal cells.     

Cytologic, ultrastructural and immunologic features of intracytoplasmic hyaline bodies in fine needle aspirates of hepatocellular carcinoma

Intracytoplasmic hyaline bodies in malignant cells from an aspirate of a liver mass are suggestive of hepatocellular carcinoma. Such inclusions were studied by light and electron microscopy and by immunocytochemistry in fine needle aspirates from five cases of hepatocellular carcinoma. Seen by light microscopy, the inclusions were round or ovoid and were surrounded by a prominent halo. By both light and electron microscopic immunocytochemistry, the hyaline bodies showed negative staining for alpha-fetoprotein, alpha-1-antitrypsin and cytokeratin. Ultrastructurally, they were not membrane bound and were composed of filamentous, finely granular material, resembling the early stages of Mallory bodies.     

Three-dimensional cytomorphology in fine needle aspiration biopsy of parathyroid lesions

OBJECTIVE: To elucidate three-dimensional (3-D) cytomorphology in fine needle aspiration biopsy (FNAB) of parathyroid lesions. STUDY DESIGN: Ultrasound-guided FNAB was performed on parathyroid lesions from 10 patients with hyperparathyroidism. The aspirates were stained and observed under a light microscope (LM). The aspirates were also fixed, dehydrated, critical point dried, spattered with gold ions and observed with a scanning electron microscope (SEM). Findings under SEM were correlated with the appearances under LM as well as with serum parathyroid hormone (PTH) concentrations. RESULTS: Under LM, nine cases displayed isokaryosis and one case, anisokaryosis. These appearances corresponded to isocytosis or anisocytosis under SEM. Under SEM, 3-D cytomorphology of parathyroid lesions displayed isocytotic, scattered cells in five cases, uniform cellular arrangements in four cases and anisocytotic, scattered cells in one case. The cell surface was rather smooth in five cases. The other five cases had significant granules on the cell surfaces; these all had serum PTH concentrations > or = 268 pg/mL. CONCLUSION: 3-D cytomorphology in FNAB of parathyroid lesions was a rather smooth cell surface in cases with low serum PTH and a granular cell surface in cases with significantly increased serum PTH. These characteristics and the absence of microvilli might be helpful in the differential diagnosis between parathyroid and follicular thyroid lesions.     

Computerized microscopic analysis of prostatic fine needle aspirates. Comparison with breast aspirates

Computerized image analysis was employed to analyze fine needle aspiration smears of the prostate and breast using both high-resolution images of individual cells and medium-resolution images of scenes and clusters (contextual analysis). A linear discriminant analysis was used to demonstrate the computer's ability to discriminate between benign and malignant categories for both types of tissue. Correct classification as benign or malignant using contextual analysis was achieved in 22 of 26 prostatic aspirates and in 15 of 18 breast aspirates, as determined by comparison with histology. The addition of high-resolution single-cell analysis resulted in correct classification of 24 of 26 prostatic aspirates and all breast aspirates. For virtually all features, the distinction between benign and malignant was more subtle for prostatic than for breast tissue. The data indicate that contextual analysis may be less effective as an adjunct to high-resolution single-cell microscopy of prostatic specimens than it is for breast specimens.     

Differential diagnosis of lymph node aspirates by intermediate filament typing of tumor cells

Twenty-one lymph node aspirates for which a differential diagnosis was difficult or not possible by light microscopy alone were selected for further study by intermediate filament typing. The use of four monoclonal antibodies specific for different keratin subsets or for vimentin resulted in a definitive diagnosis in all instances. The results show that use of these antibodies can improve the accuracy of cytologic diagnosis and provide further evidence that intermediate filament typing can help differentiate (1) malignant melanoma from adenocarcinoma, (2) malignant lymphoma from small-cell anaplastic carcinoma and (3) squamous-cell carcinoma from adenocarcinoma. Intermediate filament typing can also be used to identify very small numbers of carcinoma cells in specimens that are apparently negative for tumor cells by light microscopy. The method is quick, relatively simple, reliable and unambiguous, provided that appropriate questions are asked and antibodies with well-defined specificities are used.     

Transmission and scanning electron microscopic study of the same cytologic material

The same cytologic material was successively examined by light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). After the SEM examination, the specimens were rehydrated for a long period of time to allow the penetration of Epon 812 into the cells. The TEM examination showed the cell organelles to be comparatively well preserved. These consecutively performed LM-SEM-TEM examinations provided useful information on cytologic subjects, especially concerning the origin of the cells.     

Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis

A portfolio is presented documenting economic, high-resolution correlative focused ion beam scanning electron microscopy (FIB/SEM) in routine, comprising: (i) the use of custom-labeled slides and coverslips, (ii) embedding of cells in thin, or ultra-thin resin layers for correlative light and electron microscopy (CLEM) and (iii) the claim to reach the highest resolution possible with FIB/SEM in xyz. Regions of interest (ROIs) defined in light microscope (LM), can be relocated quickly and precisely in SEM. As proof of principle, HeLa cells were investigated in 3D context at all stages of the cell cycle, documenting ultrastructural changes during mitosis: nuclear envelope breakdown and reassembly, Golgi degradation and reconstitution and the formation of the midzone and midbody.     

Molecular genetic analysis in the diagnosis of lymphoma in fine needle aspiration biopsies. II. Lymphomas versus nonlymphoid malignant tumors

The configurations of immunoglobulin genes and T-cell receptor beta chain genes were analyzed by Southern blotting in DNA derived from nonlymphoid malignant tumors and lymphomas. Gene rearrangements were not detected in any of the 35 cases of nonlymphoid malignant tumors. On the contrary, they were shown in all 14 cases of non-Hodgkin's lymphomas, 2 of 3 cases of Hodgkin's disease and 2 cases diagnosed as non-Hodgkin's lymphoma or angioimmunoblastic lymphadenopathy. The differentiation by light microscopy between lymphoma and nonlymphoid malignant tumors was a diagnostic problem in five cases; the molecular genetic analysis of DNA was contributory in all five diagnostically difficult aspirates. By gene rearrangement studies, the diagnosis of lymphoma was confirmed in two cases and nonlymphoid malignant tumors were accurately indicated in aspirates diagnosed finally as rhabdomyosarcoma (one case) and carcinoma (two cases).     

Benign squamous cells with pleomorphic microvilli in urine or bladder washings

Exfoliated cells in catheterized urine or bladder washings from 40 patients were observed by light microscopy (LM) and by scanning electron microscopy (SEM). The specimens from seven of these patients (six postmenopausal females and one 85-year-old male) contained squamous cells with pleomorphic microvilli (PMV) on their surfaces. Four of these cases had no bladder lesions by cystoscopic examination. Three patients had recurrent papillary transitional-cell carcinoma of the bladder, and the cytologic specimens from two of them contained transitional cells with PMV. The distinction between squamous and transitional cell is readily made by SEM, based primarily on cell shape and thickness. The presence of PMV on otherwise-benign-appearing squamous cells in urine or bladder washing specimens may be a source of confusion in the interpretation of SEM findings. The presence of PMV on exfoliated squamous cells in cytologic material from the human urinary tract does not seem to have the same diagnostic and prognostic significance as the presence of PMV on transitional cells.     

Surface configuration of mesothelial cells in effusions. A comparative light microscopic and scanning electron microscopic study.

Surface configuration of mesothelial cells identified by light microscopy (LM) has been studied by scanning electron microscopy (SEM). It has been shown that mesothelial cells may have a variable SEM appearance. The surfaces of a small proportion of mesothelial cells are covered by regular microvilli (MV) and show openings of the pinocytotic vesicles. The surfaces of the majority of these cells are covered by vesicles or blebs. An intermediate population of mesothelial cells, i.e., cells displaying side-by-side blebs and MV, has also been observed. The latter cells no longer display pinocytotic vesicles. Occasional mesothelial cells have smooth surfaces. It has been shown by LM and transmission electron microscopy that cells with blebs are viable and capable of mitotic activity. It is concluded that mesothelial cells, detached from their epithelial setting, lose microvilli and pinocytotic vesicles and acquire surface blebs. The possible relationship between mesothelial cells and macrophages based on surface features has been discussed.     

Scanning electron microscope analysis of structural changes and aberrations in human chromosomes associated with the inhibition and reversal of inhibition of ultraviolet light induced DNA repair

Metaphase chromosomes appear decondensed in preparations from mitotic cells that have been irradiated with ultraviolet light (UV) and incubated with inhibitors of DNA synthesis; under these conditions DNA repair is inhibited and both single and double strand DNA breaks accumulate. After reversal of the inhibition chromosomes are condensed, but are often damaged. In this paper we show by scanning electron microscopy (SEM) that decondensed HeLa chromosomes are composed of fibre clusters similar to those previously described for the large chromosomes of the Indian muntjac. This suggests that the clusters may be a universal higher order packing unit in mammalian metaphase chromosomes. We also examine by SEM the nature of the aberrations that appear after the reversal of periods of inhibited repair; these include gaps, breaks, deletions and telomeric packing abnormalities. SEM analysis allows an extension and reconsideration of conclusions about chromatid continuity based on the study of conventional light microscope (LM) preparations.     

Cell surface characteristics and DNA content of macrophages in murine bone marrow cultures. A study using simultaneous scanning electron microscopy and fluorescence microscopy

An instrument combining scanning electron microscopy (SEM) and light microscopy (LM) was used to study the cell surface characteristics and DNA content of macrophages in murine bone marrow cultures. After a quantitative Feulgen DNA staining, the DNA content of the individual macrophages was measured and their cell surface morphology was studied immediately thereafter with the SEM part of the instrument. The cells were divided into six groups according to the number of microvilli and/or microridges present on their surface. A proportion of macrophages showed a DNA content more than occurs in diploid cells, which could indicate a future division. No special surface morphology could be detected in this cell type.     

Cell surface characteristics and DNA content of macrophages in murine bone marrow cultures

Summary An instrument combining scanning electron microscopy (SEM) and light microscopy (LM) was used to study the cell surface characteristics and DNA content of macrophages in murine bone marrow cultures. After a quantitative Feulgen DNA staining, the DNA content of the individual macrophages was measured and their cell surface morphology was studied immediately thereafter with the SEM part of the instrument. The cells were divided into six groups according to the number of microvilli and/or microridges present on their surface. A proportion of macrophages showed a DNA content more than occurs in diploid cells, which could indicate a future division. No special surface morphology could be detected in this cell type.     

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).     

Structural characters of leaf epidermis and their systematic significance in Vitaceae

Leaf epidermis of 37species representing 11 genera of Vitaceae was investigated using light microscopy（LM）and scanning electron microscopy（SEM）.The shapes of leaf epidermal cells are usually irregular or polygonal;the patterns of anticlinal walls are stra     

Neuroendocrine (Merkel-cell) carcinoma of the skin. Cytology, intermediate filament typing and ultrastructure of tumor cells in fine needle aspirates

The light and transmission electron microscopic findings and the intermediate filament typing of tumor cells from fine needle aspirates of primary, recurrent and metastatic neuroendocrine (Merkel-cell) carcinoma of the skin are described. The tumor cells in the smears coexpressed keratins and neurofilaments and were characterized by "intermediate filament buttons," i.e., buttonlike fragments of cytoplasm of tumor cells, which could be observed either by staining with intermediate filament-specific antibodies using immunofluorescence or by their appearance in hematoxylin-and-eosin-stained smears. These features may help in the differential diagnosis of fine needle aspirates of neuroendocrine carcinomas of the skin.     

Gold-FISH: A new approach for the in situ detection of single microbial cells combining fluorescence and scanning electron microscopy

A novel fluorescence in situ hybridisation (FISH) method is presented that allows the combination of epifluorescence and scanning electron microscopy (SEM) to identify single microbial cells. First, the rRNA of whole cells is hybridised with horseradish peroxidase-labelled oligonucleotide probes and this is followed by catalysed reporter deposition (CARD) of biotinylated tyramides. This facilitates an amplification of binding sites for streptavidin conjugates covalently labelled with both fluorophores and nanogold particles. The deposition of Alexa Fluor 488 fluoro-nanogold–streptavidin conjugates was confirmed via epifluorescence microscopy and cells could be quantified in a similar way to standard CARD–FISH approaches. To detect cells by SEM, an autometallographic enhancement of the nanogold particles was essential, and allowed the in situ localisation of the target organisms at resolutions beyond light microscopy. Energy dispersive X-ray spectroscopy (EDS) was used to verify the effects of CARD and autometallography on gold deposition in target cells.     

Fine needle aspiration cytology of primitive neuroectodermal tumors. A report of these cases.

Primitive neuroectodermal tumor (PNET) is a small round cell malignancy arising in soft tissue and bone, predominantly in older children and adolescents. We report the cytomorphologic features and findings of ancillary studies of eight fine needle aspiration (FNA) biopsies from three patients (7-year-old male, 12-year-old female, 9-year-old female). Two of the biopsies suggested the initial diagnosis of PNET of the chest wall, while the remaining six documented recurrent or metastatic disease. In one of these cases the primary diagnosis made by FNA biopsy enabled the pediatric oncologists to give specific therapy for the unresectable tumor and achieve remission. Local recurrences included the chest wall (two cases), pleura (one case) and pericardium (one case), while metastatic disease involved the supraclavicular lymph node and breast. All the cases consisted of small malignant cells with a high nuclear/cytoplasmic ratio and hyperchromatic nuclei without prominent nucleoli. Homer Wright rosettes were seen in only two of the aspirates, and neuropil and ganglion cells were not present. Ancillary studies, including electron microscopy (two cases), immunocytochemistry (four aspirates from two cases) and cytogenetics (11/22 translocation, one case) performed on the aspirated material were aids in making a specific diagnosis and excluded other small round cell tumors of childhood, such as malignant lymphoma, rhabdomyosarcoma and Ewing's sarcoma. The differential diagnosis between PNET and neuroblastoma can be difficult on the basis of an FNA biopsy alone, although light microscopic morphologic differences exist. Clinical features (e.g., age, primary site, metastatic patterns), catecholamine levels, electron microscopy and cytogenetics are necessary in establishing the correct diagnosis.     

Phylloplane fungi in Hong Kong mangroves: evaluation of study methods

Many methods have been used to study phylloplane fungi, most of which have constraints and may result in biased results. This study used light microscopy and scanning electron microscopy (SEM) to investigate fungal abundance on the leaves of the most common mangrove trees in Hong Kong, Kandelia candel and Aegiceras corniculatum. Species richness was investigated using light microscopy and a leaf washing method. Methods to study phylloplane fungi are discussed and the performances of these three investigation methods are evaluated. Seven mitosporic fungal taxa were found by light microscopy, while 30 sporulating taxa and 18 Mycelia sterilia were isolated using the leaf washing method. Fungal abundance in terms of percentage cover investigated with light microscopy was similar using the SEM method, and was significantly higher on Aegiceras corniculatum than on Kandelia candel. Fungal abundance peaked in the summer and was lowest in the winter. This study indicates that light microscopy reveals the most typical phylloplane fungi and is more efficient than SEM, while the leaf washing method reveals many casual species and is not quantitative.