Difficulties in Differentiating Coronaviruses from Subcellular Structures in Human Tissues by Electron Microscopy.

Efforts to combat the coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have placed a renewed focus on the use of transmission electron microscopy for identifying coronavirus in tissues. In attempts to attribute pathology of COVID-19 patients directly to tissue damage caused by SARS-CoV-2, investigators have inaccurately reported subcellular structures, including coated vesicles, multivesicular bodies, and vesiculating rough endoplasmic reticulum, as coronavirus particles. We describe morphologic features of coronavirus that distinguish it from subcellular structures, including particle size range (60-140 nm), intracellular particle location within membrane-bound vacuoles, and a nucleocapsid appearing in cross section as dense dots (6-12 nm) within the particles. In addition, although the characteristic spikes of coronaviruses may be visible on the virus surface, especially on extracellular particles, they are less evident in thin sections than in negative stain preparations.

Label-free prediction of three-dimensional fluorescence images from transmitted-light microscopy.

Understanding cells as integrated systems is central to modern biology. Although fluorescence microscopy can resolve subcellular structure in living cells, it is expensive, is slow, and can damage cells. We present a label-free method for predicting three-dimensional fluorescence directly from transmitted-light images and demonstrate that it can be used to generate multi-structure, integrated images. The method can also predict immunofluorescence (IF) from electron micrograph (EM) inputs, extending the potential applications.

Quantitative analysis of F-actin redistribution in astrocytoma cells treated with candidate pharmaceuticals.

Actin fibers (F-actin) control the shape and internal organization of cells, and generate force. It has been long appreciated that these functions are tightly coupled, and in some cases drive cell behavior and cell fate. The distribution and dynamics of F-actin is different in cancer versus normal cells and in response to small molecules, including actin-targeting natural products and anticancer drugs. Therefore, quantifying actin structural changes from high resolution fluorescence micrographs is necessary for further understanding actin cytoskeleton dynamics and phenotypic consequences of drug interactions on cells. We applied an artificial neural network algorithm, which used image intensity and anisotropy measurements, to quantitatively classify F-actin subcellular features into actin along the edges of cells, actin at the protrusions of cells, internal fibers and punctate signals. The algorithm measured significant increase in F-actin at cell edges with concomitant decrease in internal punctate actin in astrocytoma cells lacking functional neurofibromin and p53 when treated with three structurally-distinct anticancer small molecules: OSW1, Schweinfurthin A (SA) and a synthetic marine compound 23'-dehydroxycephalostatin 1. Distinctly different changes were measured in cells treated with the actin inhibitor cytochalasin B. These measurements support published reports that SA acts on F-actin in NF1(-/-) neurofibromin deficient cancer cells through changes in Rho signaling. Quantitative pattern analysis of cells has wide applications for understanding mechanisms of small molecules, because many anti-cancer drugs directly or indirectly target cytoskeletal proteins. Furthermore, quantitative information about the actin cytoskeleton may make it possible to further understand cell fate decisions using mathematically testable models.

Visualizing cells and humans in 3D: biomedical image analysis at nanometer and meter scales.

Researchers analyzed and presented volume data from the Visible Human Project (VHP) and data from high-resolution 3D ion-abrasion scanning electron microscopy (IA-SEM). They acquired the VHP data using cryosectioning, a destructive approach to 3D human anatomical imaging resulting in whole-body images with a field of view approaching 2 meters and a minimum resolvable feature size of 300 microns. IA-SEM is a type of block-face imaging microscopy, a destructive approach to microscopic 3D imaging of cells. The field of view of IA-SEM data is on the order of 10 microns (whole cell) with a minimum resolvable feature size of 15 nanometers (single-slice thickness). Despite the difference in subject and scale, the analysis and modeling methods were remarkably similar. They are derived from image processing, computer vision, and computer graphics techniques. Moreover, together we are employing medical illustration, visualization, and rapid prototyping to inform and inspire biomedical science. By combining graphics and biology, we are imaging across nine orders of magnitude of space to better promote public health through research.

Effects of a combretastatin A4 analogous chalcone and its Pt-complex on cancer cells: A comparative study of uptake, cell cycle and damage to cellular compartments.

The combretastatin A4 analogous chalcone (2E)-3-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one 1 and its dichloridoplatinum(II) (6-aminomethylnicotinate) complex 2 were previously found to be highly active against a variety of cancer cell lines while differing in their apoptosis induction and long-term regrowth retardation (Schobert et al. [1]). Further differences were identified now. The cellular uptake of complex 2, like that of oxaliplatin, occurred mainly via organic cation transporters (OCT-1/2; ∼32%) and copper transporter related proteins (Ctr1; ∼24%), whereas that of chalcone 1 was dependent on endocytosis (∼80%). Complex 2 was more tumour-specific than 1 concerning neural cells. This was apparent from the ratios of IC(50)(48h) values against primary astrocytes versus human glioma cells U87 (>7000 for complex 2; 55 for compound 1). In tubulin-rich neurons and 518A2 melanoma cells complex 2 disrupted microtubules and actin filaments. Cancer cells treated with 2 could repair the cytoskeletal damage but ceased to proliferate and perished. Complex 2 was particularly cytotoxic against P-gp-rich cells. It acted as a substrate for ABC-transporters of types BCRP, MRP3, and MRP1 and so was less active against the corresponding cancer cell lines. Complex 2 arrested the cell cycle of the melanoma cells in G(1) and G(2)/M phases. A fragmentation of their Golgi apparatus was observed by TEM for incubation with complex 2 but not with 1. In conclusion, unlike chalcone 1, its platinum complex 2 is highly cell line specific, is taken up via cell-controlled transporters and induces apoptosis by triggering multiple targets.

Synchrotron radiation FTIR imaging in minutes: a first step towards real-time cell imaging.

FTIR microscopy with a focal plane array (FPA) of detectors enables routine chemical imaging on individual cells in only a few minutes. The brilliance of synchrotron radiation (SR) IR sources may enhance the signal obtained from such small biosamples containing small amounts of organic matter. We investigated individual cells obtained from a cell culture specifically developed for transmission FTIR imaging using either a Globar or an SR source coupled to the same instrumentation. SR-IR source focussing was optimized to control the energy distribution on the FPA of detectors. Here we show that accessing the IR absorption distribution from all the organic contents of cells at 1 x 1 microm pixel resolution was possible only with high circulating current (> or = 1.2 A) illuminating a limited number of the FPA's detectors to increase the signal-to-noise ratio of IR images. Finally, a high-current SR ring is mandatory for collecting FTIR images of biosamples with a high contrast in minutes.

Preparation of cells for assessing ultrastructural localization of nanoparticles with transmission electron microscopy.

We describe the use of transmission electron microscopy (TEM) for cellular ultrastructural examination of nanoparticle (NP)-exposed biomaterials. Preparation and imaging of electron-transparent thin cell sections with TEM provides excellent spatial resolution (approximately 1 nm), which is required to track these elusive materials. This protocol provides a step-by-step method for the mass-basis dosing of cultured cells with NPs, and the process of fixing, dehydrating, staining, resin embedding, ultramicrotome sectioning and subsequently visualizing NP uptake and translocation to specific intracellular locations with TEM. In order to avoid potential artifacts, some technical challenges are addressed. Based on our results, this procedure can be used to elucidate the intracellular fate of NPs, facilitating the development of biosensors and therapeutics, and provide a critical component for understanding NP toxicity. This protocol takes approximately 1 week.

Trait variability of cancer cells quantified by high-content automated microscopy of single cells.

Mapping quantitative cell traits (QCT) to underlying molecular defects is a central challenge in cancer research because heterogeneity at all biological scales, from genes to cells to populations, is recognized as the main driver of cancer progression and treatment resistance. A major roadblock to a multiscale framework linking cell to signaling to genetic cancer heterogeneity is the dearth of large-scale, single-cell data on QCT-such as proliferation, death sensitivity, motility, metabolism, and other hallmarks of cancer. High-volume single-cell data can be used to represent cell-to-cell genetic and nongenetic QCT variability in cancer cell populations as averages, distributions, and statistical subpopulations. By matching the abundance of available data on cancer genetic and molecular variability, QCT data should enable quantitative mapping of phenotype to genotype in cancer. This challenge is being met by high-content automated microscopy (HCAM), based on the convergence of several technologies including computerized microscopy, image processing, computation, and heterogeneity science. In this chapter, we describe an HCAM workflow that can be set up in a medium size interdisciplinary laboratory, and its application to produce high-throughput QCT data for cancer cell motility and proliferation. This type of data is ideally suited to populate cell-scale computational and mathematical models of cancer progression for quantitatively and predictively evaluating cancer drug discovery and treatment.

Endoplasmic reticulum stress reduces the export from the ER and alters the architecture of post-ER compartments.

In eukaryotic cells several physiologic and pathologic conditions generate the accumulation of unfolded proteins in the endoplasmic reticulum (ER), leading to ER stress. To restore normal function, some ER transmembrane proteins sense the ER stress and activate coordinated signalling pathways collectively called the Unfolded Protein Response (UPR). Little is known on how the UPR relates to post-ER compartments and to the export from the ER of newly synthesized proteins. Here, we report that the ER stress response induced by either thapsigargin or nitric oxide modifies the dynamics of the intracellular distribution of ERGIC-53 and GM130, two markers of the ER Golgi Intermediate Compartment and of the cis-Golgi, respectively. In addition, induction of ER stress alters the morphology of the ERGIC and the Golgi complex and interferes with the reformation of both compartments. Moreover, ER stress rapidly reduces the transport to the Golgi complex of the temperature sensitive mutant of the Vesicular Stomatitis Virus G Glycoprotein (VSV-G) fused with the Green Fluorescent Protein (ts045G), without apparently decreasing the amount of the protein competent for export. Interestingly, a parallel rapid reduction of the number of Sec31 labelled fluorescent puncta on the ER membranes does occur, thus suggesting that the ER stress alters the ER export and the dynamic of post-ER compartments by rapidly targeting the formation of COPII-coated transport intermediates.

Subcellular immunocytochemical analysis detects the highest concentrations of glutathione in mitochondria and not in plastids.

The tripeptide glutathione is a major antioxidant and redox buffer with multiple roles in plant metabolism. Glutathione biosynthesis is restricted to the cytosol and the plastids and the product is distributed to the various organelles by unknown mechanisms. In the present study immunogold cytochemistry based on anti-glutathione antisera and transmission electron microscopy was used to determine the relative concentration of glutathione in different organelles of Arabidopsis thaliana leaf and root cells. Glutathione-specific labelling was detected in all cellular compartments except the apoplast and the vacuole. The highest glutathione content was surprisingly not found in plastids, which have been described before as a major site of glutathione accumulation, but in mitochondria which lack the capacity for glutathione biosynthesis. Mitochondria of both leaf and root cells contained 7-fold and 4-fold, respectively, higher glutathione levels than plastids while the density of glutathione labelling in the cytosol, nuclei, and peroxisomes was intermediate. The accuracy of the glutathione labelling is supported by two observations. First, pre-adsorption of the anti-glutathione antisera with glutathione reduced the density of the gold particles in all organelles to background levels. Second, the overall glutathione-labelling density was reduced by about 90% in leaves of the glutathione-deficient Arabidopsis mutant pad2-1 and increased in transgenic plants with enhanced glutathione accumulation. Hence, there was a strong correlation between immunocytochemical and biochemical data of glutathione accumulation. Interestingly, the glutathione labelling of mitochondria in pad2-1 remained very similar to wild-type plants thus suggesting that the high mitochondrial glutathione content is maintained in a situation of permanent glutathione-deficiency at the expense of other glutathione pools. High and constant levels of glutathione in mitochondria appear to be particularly important in cell survival strategies and it is predicted that mitochondria must have highly competitive mitochondrial glutathione uptake systems. The present results underline the suggestion that subcellular glutathione concentrations are not controlled by a global mechanism but are controlled on an individual basis and it is therefore not possible to conclude from global biochemical glutathione analysis on the status of the various organellar pools.

Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids.

The merozoite stage of the malaria parasite that infects erythrocytes and causes the symptoms of the disease is initially formed inside host hepatocytes. However, the mechanism by which hepatic merozoites reach blood vessels (sinusoids) in the liver and escape the host immune system before invading erythrocytes remains unknown. Here, we show that parasites induce the death and the detachment of their host hepatocytes, followed by the budding of parasite-filled vesicles (merosomes) into the sinusoid lumen. Parasites simultaneously inhibit the exposure of phosphatidylserine on the outer leaflet of host plasma membranes, which act as "eat me" signals to phagocytes. Thus, the hepatocyte-derived merosomes appear to ensure both the migration of parasites into the bloodstream and their protection from host immunity.

An unbiased cell morphology-based screen for new, biologically active small molecules.

We have implemented an unbiased cell morphology-based screen to identify small-molecule modulators of cellular processes using the Cytometrix (TM) automated imaging and analysis system. This assay format provides unbiased analysis of morphological effects induced by small molecules by capturing phenotypic readouts of most known classes of pharmacological agents and has the potential to read out pathways for which little is known. Four human-cancer cell lines and one noncancerous primary cell type were treated with 107 small molecules comprising four different protein kinase-inhibitor scaffolds. Cellular phenotypes induced by each compound were quantified by multivariate statistical analysis of the morphology, staining intensity, and spatial attributes of the cellular nuclei, microtubules, and Golgi compartments. Principal component analysis was used to identify inhibitors of cellular components not targeted by known protein kinase inhibitors. Here we focus on a hydroxyl-substituted analog (hydroxy-PP) of the known Src-family kinase inhibitor PP2 because it induced cell-specific morphological features distinct from all known kinase inhibitors in the collection. We used affinity purification to identify a target of hydroxy-PP, carbonyl reductase 1 (CBR1), a short-chain dehydrogenase-reductase. We solved the X-ray crystal structure of the CBR1/hydroxy-PP complex to 1.24 A resolution. Structure-based design of more potent and selective CBR1 inhibitors provided probes for analyzing the biological function of CBR1 in A549 cells. These studies revealed a previously unknown function for CBR1 in serum-withdrawal-induced apoptosis. Further studies indicate CBR1 inhibitors may enhance the effectiveness of anticancer anthracyclines. Morphology-based screening of diverse cancer cell types has provided a method for discovering potent new small-molecule probes for cell biological studies and anticancer drug candidates.

Ultrastructural studies of the Mehlis' gland in Metamicrocotyla macracantha (Monogenea, Microcotylidae) parasite of Mugil liza (Teleostei)

A ultraestrutura da glândula de Mehlis de Metamicrocotyla macracantha, parasita de brânquia coletado de Mugil liza do Rio de Janeiro, Brasil,foi estudado através da microscopia eletrônica de transmissão. A glândula de Mehlis consiste de dois tipos de células secretoras, S1 e S2, cada uma produzindo um corpo secretor diferente. Os corpos S1 são esféricos, em forma de lamelas e observados em diferentes estágios de desenvolvimentos no citoplasma dessas células. Os corpos S2 são esféricos a ovais com conteúdos densos, apresentando uma estrutura cristalina. O citoplasma das células da glândula de Mehlis apresenta também ribossomas livres, retículo endoplasmático granular e complexo de Golgi, organelas características de células secretoras.

A C-terminal region dictates the apical plasma membrane targeting of the human sodium-dependent vitamin C transporter-1 in polarized epithelia.

The human sodium-dependent vitamin C transporter (hSVCT1) mediates sodium-dependent cellular uptake of the essential micronutrient l-ascorbic acid (vitamin C). However, the molecular determinants that control the cell surface expression, subcellular distribution, and dynamics of hSVCT1 remain undefined. To identify molecular determinants involved in hSVCT1 targeting in polarized epithelia, we used live cell imaging approaches to resolve the targeting and trafficking dynamics of hSVCT1 truncation mutants in renal and intestinal cells. Confocal imaging demonstrated that hSVCT1 was expressed at the apical cell surface and video rate measurements revealed hSVCT1 also resided in a heterogeneous population of intracellular organelles with discrete dynamic properties. By progressive truncation of the cytoplasmic C-terminal tail of hSVCT1, we delimited an essential role for an embedded ten amino acid sequence PICPVFKGFS (amino acids 563-572) in defining the physiological targeting of hSVCT1. Intriguingly, this sequence bears significant homology to recently identified apical targeting motifs in two other sodium-dependent transporters, and we suggest this conservation is reflected topologically through the adoption of a beta-turn confirmation in the cytoplasmic C-tail of each transporter. Our results provide the first direct resolution of functional hSVCT1 expression at the apical cell surface of polarized epithelia and define an apical targeting signal of relevance to transporters of diverse substrate specificity.

Induction of apoptotic cellular death in lymphatic tissues of cattle experimentally infected with different strains of rinderpest virus.

The presence, type, and extent of cellular death in lymphatic tissues of cattle experimentally infected with rinderpest virus strains of different virulence was investigated morphologically. Cells with DNA strand breaks were identified in histological sections of palatine tonsil, spleen, and mesenteric and mandibular lymph nodes by the TUNEL (terminal desoxynucleotidyl transferase-mediated dUTP nick end labelling) assay. In addition, representative samples of lymphatic tissues were examined by transmission electron microscopy. The results indicated that cellular disassembly in lymphatic tissues was caused by both apoptosis and oncosis. Cells with DNA strand breaks were observed in follicular and parafollicular areas of lymphatic tissues and their numbers were determined. A significant correlation was found between the number of TUNEL-positive cells and viral virulence. These results suggest that, in addition to oncosis, apoptotic cellular death in lymphatic tissues contributes substantially to the pathogenesis of rinderpest.

Serous oligocystic adenoma of the pancreas: a clinicopathological and immunohistochemical study of three cases with ultrastructural findings.

AIMS: Serous oligocystic adenoma of the pancreas is an uncommon benign neoplasm and is a recently described entity. To date, there are 19 adult cases of this tumour. We report three additional cases, two with macrocystic and one with unilocular types. We describe their clinicopathological, immunohistochemical and ultrastructural findings and review the world's literature. METHODS: For a 10-year period, we reviewed all benign cystic lesions of the pancreas with emphasis on serous oligocystic adenoma. We characterised serous oligocystic adenoma as an ill-demarcated or encapsulated mass, composed largely or exclusively of macrocysts (cysts measuring 20mm or more) but few in number (oligolocular). Grossly, it may contain only a single cyst (unilocular) of any size with a few satellite cysts observed on histological examination. Special stains and immunohistochemistry as well as electron microscopy were performed on three and two cases of serous oligocystic adenoma, respectively. RESULTS: Between 1990 and 2000, we collected 26 benign cystic lesions of the pancreas, three of which were serous oligocystic adenomas (two with macrocystic and one with unilocular types). Many of the cells lining the cysts showed PAS positivity. There was negative staining for PAS with diastase digestion, Alcian blue and mucicarmine. All cases showed positive staining for CAM5.2, AE1/AE3, EMA and CK7. The proliferation index marker was low. There was negative staining for CK20, insulin, glucagon, somatostatin, synaptophysin, chromogranin A, CEA and p53. Ultrastructural studies on two cases revealed similar findings. The single row of uniform epithelial cells lining the cysts was composed of simple cuboidal to flat cells which rested on a thin basal lamina. Their nuclei were round to ovoid. Glycogen granules were identified in the cytoplasm. Short microvilli emerged from the epithelial apical surface. Adjacent tumour cells were connected by microfilaments. CONCLUSIONS: Serous oligocystic adenomas of the pancreas are uncommon benign tumours. Prior to this study, 19 adults with these lesions were reported in the world's literature. No correct pre-operative diagnosis was carried out on all 22 cases. The 20 patients with follow-up ranging from 2 months to 5 years did not show tumour recurrence or malignant transformation.

Overexpression of v-myb oncogene or c-myb proto-oncogene in insect cells: characterization of newly induced nucleolus-like structures accumulating Myb protein.

The oncoprotein v-Myb induces myeloid leukemia and its cellular counterpart c-Myb is involved in the regulation of hematopoiesis. Although intensively studied, their precise subcellular localization is not known. In order to expand our knowledge in this respect, we used an artificial system overexpressing these proteins. We investigated the subcellular localization of Myb proteins in cultured non-synchronized insect cells transfected with recombinant baculoviruses overexpressing either v-myb oncogene or c-myb proto-oncogene. The cell expressing Myb proteins underwent extensive nuclear changes and exhibited distinct nuclear structures resembling nucleoli. The bulk of v-Myb and c-Myb proteins accumulated in such nucleolus-like structures which, according to the nucleolar nomenclature, we classified to three types: compact of enlarged size (type I), large ring-shaped (type II) and with nucleolonemas (type III). We investigated these structures for the presence of important nucleolar macromolecules in order to establish whether they were compatible with the function in the production of ribosomes. Strikingly, our results indicated that the different forms of these structures did not represent genuine nucleoli. They rather reflected progressive changes, induced by the virus infection and high expression of v-myb genes, accompanied by the formation of these prominent nucleolus-like structures highly enriched in Myb protein. Gradual changes in number of individual nucleolus-like forms during infection, increasing amount of Myb protein and DNA localized in them together with decreasing amount of RNA and their different interaction with viral particles indicate that the nucleolus-like structure of type I is a precursor of the type II and finally of the type III.

Establishment of a transplantable tumor line (IP) derived from rat pulmonary carcinoma, developing humoral hypercalcemia of malignancy in IP-bearing rats.

A transplantable tumor line (IP) was established in syngeneic rats from a spontaneous pulmonary carcinoma found in a male F344 rat aged 25 months. A tissue fragment of IP grew into a nodule, 2-3 cm in diameter, 2-3 weeks after implant, and IP has been serially passed through 26 generations. Lined by cuboidal or columnar epithelial cells, the primary tumor consisted of completely alveolar architecture. However, IP tumors developed various growth patterns such as glandular, acinar, trabecular, cord, and solid, and consisting of epithelial cells showing cellular atypia. Ultrastructurally, neoplastic cells had microvilli, basement membranes, and desmosomes, and occasional cells possessed dense cytoplasmic granules. Interestingly, it was shown by the immunohistochemistry, the RT-PCR method, and immunoradiometric assay that IP tumor cells produce parathyroid hormone-related protein (PTHrP). During a 3-week observation period after implant, IP-bearing rats showed severe emaciation, hypercalcemia, and hypophosphatemia, as well as an increase in osteoclastic areas and a decrease in shaft thickness of the femurs. These were considered to be due to a marked elevation of plasma PTHrP levels. Furthermore, IP-bearing rats developed calcification in various organs including the kidneys, lungs, and heart. These findings in IP-bearing rats were similar to those of humoral hypercalcemia of malignancy (HHM) reported in human cancer patients. PTHrP plays a central role in the development of HHM, but the mechanisms of HHM remain poorly understood. IP may become a useful model for studying the pathogenesis of HHM and the pathophysiological role of PTHrP.

A novel embedment-free immunoelectron microscopy technique reveals association of apoptosis-regulating proteins with subcellular structures.

By employing two electron microscopy techniques, postembedding double- and triple immunocytochemical gold-labelling combined with embedment-free electron microscopy (EF-EM), we have detected previously unreported nuclear and cytoplasmic complexes between different proapoptatic proteins in a human cancer cell line COLO 205 stimulated to apoptosis by nimesulide, a specific cyclooxygenase-2 inhibitor. Experiments with the use of double- and triple immunolabelling visualized the colocalization of proapoptotic proteins such as Bax with Bid, Bax with Bid and voltage-dependent anion channel protein (VDAC-1), and Bax with Bid and caspase-8, on organellar membranes and within the nucleus. Application of this technique in combination with EF-EM technique augments our knowledge on the precise identification and relationship of subcellular structures containing Bax, Bid, VDAC-1 and caspase-8.

Effects of acetone, methanol, or paraformaldehyde on cellular structure, visualized by reflection contrast microscopy and transmission and scanning electron microscopy.

The authors recently showed variable subcellular immunoreactivity of the Bcl-2 and Bax proteins after fixation of cell monolayers with acetone, methanol, or paraformaldehyde (PF) followed by methanol (PF/methanol). Here, the authors demonstrate by reflection contrast microscopy and transmission electron microscopy that acetone or methanol fixation result in complete loss of integrity of intracellular structures in contrast with PF or glutaraldehyde fixation. Scanning electron microscopy revealed poor preservation of plasma membrane integrity after fixation in acetone or methanol. Fixation with PF before methanol reduced damage to intracellular and plasma membranes. In addition, Western blot analysis demonstrated loss of Bcl-2 and Bax protein during acetone or methanol fixation, whereas PF fixation before methanol permeabilization markedly reduced this loss. For studies on the intracellular localization of soluble or unknown types of antigen, the authors discourage the use of acetone and methanol as single fixatives.