High-voltage electron microscopy of inorganic particles in intact blood cells

Preliminary results obtained by examining intact fixed human red and white blood cells containing inorganic particles under the high-voltage transmission electron microscope are described. Iron filings and ferritin ingested in vitro by granulocytes were observed as were inorganic particles in red cells from a case of lead poisoning. The nature of the particles in red cells is discussed. A faint intracellular network was seen in normal red cells and in cells from a case of lead poisoning. It was found possible to focus the electron microscope at different planes within the cell. The optimum accelerating voltage for red cells appeared to be around 750 kV, whereas a clear image of granulocytes was obtained at voltages between 750 and 1000 kV. The first results indicate that it is possible to examine intact blood cells under the high-voltage electron microscope. Further work is in progress to determine if more information can be acquired by this technique.     

Ultrastructural and cytochemical characteristics of megakaryoblastic leukemic cells

Transmission electron microscopy, scanning electron microscopy, and ultrastructural cytochemistry were utilized to study megakaryoblastic cells from four patients suffering from megakaryoblastic leukemia. The results show that megakaryoblastic leukemic cells have a unique ultrastructural appearance, surface architecture, and cytochemical activity. The cells are positive for platelet peroxidase cytochemical reaction, which is localized in the perinuclear space and endoplastic reticulum, but not in the Golgi apparatus and cytoplasmic granules. They have a rather smooth surface and display blebs or tuberculi which are different from those in other types of leukemic cells as seen under the scanning electron microscope. The megakaryoblastic leukemic cells also show a special appearance under the transmission electron microscope, such as a cytoplasm which contains numerous small mitochondria, mostly concentrated in one pole of the cell. These ultrastructural and cytochemical characteristics of the megakaryoblastic leukemic cells revealed by the combined techniques are very useful in the diagnosis of megakaryoblastic leukemia.     

Early detection of cytotoxic events between hepatic natural killer cells and colon carcinoma cells as probed with the atomic force microscope.

The atomic force microscope (AFM) is a powerful tool to investigate surface and submembranous structures of living cells under physiological conditions at high resolution. These properties enabled us to study the interaction between live hepatic natural killer (NK) cells, also called pit cells, and colon carcinoma cells in vitro by AFM. In addition, the staining for filamentous actin and DNA was performed and served as a reference, because actin and nuclear observations at the light microscopic level during the cytotoxic interaction between these two cell types have been presented earlier. In this study, we collected evidence that conjugation of hepatic NK cells with CC531s colon carcinoma cells results in a decreased binding of CC531s cells to the substratum as probed with the AFM in contact mode as early as 10 min after cell contact (n = 11). To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was performed on hepatic NK/CC531s cell conjugates, resulting in identical observations (n = 3). In contrast, the first cytotoxic signs, as determined with the nuclear staining dye Hoechst 33342, could be observed 3 h after the start of the co-culture. This study illustrates that the AFM can be used to probe early cytotoxic effects of effector to target cell contact in nearby physiological conditions. Other routine cytotoxicity tests detect the first cytotoxic effects after 1.5-3 h co-incubation at the earliest.     

Transmission X-ray microscopy of intact hydrated PtK2 cells during the cell cycle

Transmission X-ray microscopy makes it possible to investigate biological specimens, i.e. cells and cell organelles, in their natural wet environment. The main processes determining the contrast in X-ray microscopy are photoelectric absorption and phase shift. X-ray microscopic experiments can therefore be carried out in both amplitude and phase contrast. The Göttingen X-ray microscope at the BESSY storage ring in Berlin is described. PtK2 cells were examined during different stages of the cell cycle. All major constituents of the mitotic apparatus, e.g. chromosomes, centromeres, microtubules and centrosomes, could be visualized, as well as the main structural compartments and organelles of the interphase cell, e.g. nuclear membrane, interphase chromatin, nucleolus and cytoplasmic mitochondria, as well as parts of the cytoskeletal apparatus. In this way new information can be obtained with regard to the ultrastructure of the constituents of intact and unstained cells at a resolution which bridges the gap between light microscopy and electron microscopy. The prospects for the future application of transmission X-ray microscopy in biomedical research are discussed.     

Metabolic state dependent preservation of cells by fixatives for electron microscopy

The preservation of mitochondria, cytoplasmic vacuoles and cytoplasm by various fixatives after various pretreatments of ethothelial heart cells from Xenopus laevis tadpoles in tissue culture was investigated. The study was based on phase contrast cinemicrographic recordings and on qualitative and quantitative observations with the electron microscope. Three fixatives were used: 3% glutaraldehyde in phosphate buffer, followed by 1% osmium tetroxide postfixation, fixation only with 1% osmium tetroxide in phosphate buffer and the fixing medium according to Dalton. Cells were either not treated or pretreated for 20 min: 10 microM FCCP (Carbonylcyanide-p-trifluoromethoxy-phenylhydrazone) or 4 mM KCN. The superiority of glutaraldehyde was exemplified by its very rapid action, good preservation of cytoplasm, vacuoles, and mitochondria. It was the only medium which maintained an electron density of the mithochondria matrix. In both of the other fixatives swelling of mitochondria and coagulated appearance of cytoplasm (in phase contrast) was more pronounced in cells pretreated with metabolic inhibitors than in controls. Observations with the light microscope have been confirmed by morphometry of electron micrographs of mitochondria. The relation of matrix space to intracristal space is changed in opposite directions after glutaraldehyde and after the Dalton-type fixation. The results indicate a higher sensitivity against fixation artifacts in cells under pathological conditions than normal cells.     

SLIM: a new method for molecular imaging

We used spectrally resolved fluorescence lifetime imaging (SLIM) to investigate the mitochondria staining dye rhodamine 123 and binding of DAPI to RNA and DNA in cells. Moreover, different components of the photosensitizer Photofrin were resolved in cell cultures by SLIM. To record lifetime images (tau-mapping) with spectral resolution we used a laser scanning microscope equipped with a spectrograph, a 16 channel multianode PMT, and multidimensional time-correlated single photon counting. A Ti:Saphir laser was used for excitation or alternatively a ps diode laser. With this system the time- and spectral-resolved fluorescence characteristics of different fluorophores were investigated in cell cultures. As an example, the mitochondria staining dye rhodamine I23 could be easily distinguished from DAPI, which binds to nucleic acids. Also different binding sites of DAPI could be discriminated. This was proved by the appearance of different lifetime components within different spectral channels. Moreover, we were able to detect monomeric and aggregated forms of Photofrin in cells. Different lifetimes could be attributed to the various compounds. In addition, a detailed analysis of the autofluorescence by SLIM could explain changes of mitochondrial metabolism during Photofrin-PDT.     

Quantitative subcellular imaging of boron compounds in individual mitotic and interphase human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS)

Boron measurements at subcellular scale are essential in boron neutron capture therapy (BNCT) of cancer as the nuclear localization of boron‐10 atoms can enhance the effectiveness of killing individual tumour cells. Since tumours contain a heterogeneous population of cells in interphase as well as in the M phase (mitotic division) of the cell cycle, it is important to evaluate the subcellular distribution of boron in both phases. In this work, the secondary ion mass spectrometry (SIMS) based imaging technique of ion microscopy was used to quantitatively image boron from two BNCT agents, clinically used p‐boronophenylalanine (BPA) and 3‐[4‐(o‐carboran‐1‐yl)butyl]thymidine (N4), in mitotic metaphase and interphase human glioblastoma T98G cells. N4 belongs to a class of experimental BNCT agents, designated 3‐carboranyl thymidine analogues (3CTAs), which presumably accumulate selectively in cancer cells due to a process referred to as kinase‐mediated trapping (KMT). The cells were exposed to BPA for 1 h and N4 for 2 h. A CAMECA IMS‐3f SIMS ion microscope instrument capable of producing isotopic images with 500 nm spatial resolution was used in the study. Observations were made in cryogenically prepared fast frozen, and freeze‐fractured, freeze‐dried cells. Three discernible subcellular regions were studied: the nucleus, a characteristic mitochondria‐rich perinuclear cytoplasmic region, and the remaining cytoplasm in interphase T98G cells. In metaphase cells, the chromosomes and the cytoplasm were studied for boron localization. Intracellular concentrations of potassium and sodium also were measured in each cell in which the subcellular boron concentrations were imaged. Since the healthy cells maintain a K/Na ratio of approximately 10 due to the presence of Na‐K‐ATPase in the plasma membrane of mammalian cells, these measurements provided validation for cryogenic sample preparation and indicated the analysis healthy, well preserved cells. The BPA‐treated interphase cells revealed significantly lower concentrations of boron in the perinuclear mitochondria‐rich cytoplasmic region as compared to the remaining cytoplasm and the nucleus, which were not significantly different from each other. In contrast, the BPA‐treated metaphase cells revealed significantly lower concentration of boron in their chromosomes than cytoplasm. In addition, the cytoplasm of metaphase cells contained significantly less boron than the cytoplasm of interphase cells. These observations provide valuable information on the reduced uptake of boron from BPA in mitotic cells for BPA‐mediated BNCT. SIMS observations on N4 revealed that boron was distributed throughout the interphase and mitotic cells, including the chromosomes. The presence of boron in chromosomes of metaphase cells treated with N4 is indicative of a possible incorporation of this thymidine analogue into DNA. The 3‐D SIMS imaging approach for the analysis of mitotic cells shown in this work should be equally feasible to the evaluation of other BNCT agents.     

In-vivo observation of cells with a combined high-resolution multiphoton-acoustic scanning microscope

We present a combined multiphoton-acoustic microscope giving collocated access to the local morphological as well as mechanical properties of living cells. Both methods relay on intrinsic contrast mechanisms and dispense with the need of staining. In the acoustic part of the microscope, a gigahertz ultrasound wave is generated by an acoustic lens and the reflected sound energy is detected by the identical lens in a confocal setup. The achieved lateral resolution is in the range of 1 mum. Contrast in the images arises mainly from the local absorption of sound in the cells related to viscose damping. Additionally, acoustic microscopy can access the sound speed as well as the acoustic impedance of the cell membrane and the cell shape, as it is an intrinsic volume scanning technique. The multiphoton image formation bases on the detection of autofluorescence due to endogenous fluorophores. The nonlinearity of two-photon absorption provides submicron lateral and axial resolution without the need of confocal optical detection. In addition, in the near-IR cell damages are drastically reduced in comparison with direct excitation in the visible or UV. The presented setup was aligned with a dedicated procedure to ensure identical image areas. Combined multiphoton/acoustic images of living myoblast cells are discussed focusing on the reliability of the method.     

Morphometric,quantitative, and three‐dimensional analysis of the heart muscle fibers of old rats: Transmission electron microscopy and high‐resolution scanning electron microscopy methods

This research investigated the morphological, morphometric, and ultrastructural cardiomyocyte characteristics of male Wistar rats at 18 months of age. The animals were euthanized using an overdose of anesthesia (ketamine and xylazine, 150/10 mg/kg) and perfused transcardially, after which samples were collected for light microscopy, transmission electron microscopy, and high‐resolution scanning electron microscopy. The results showed that cardiomyocyte arrangement was disposed parallel between the mitochondria and the A‐, I‐, and H‐bands and their M‐ and Z‐lines from the sarcomere. The sarcomere junction areas had intercalated disks, a specific structure of heart muscle. The ultrastructural analysis revealed several mitochondria of various sizes and shapes intermingled between the blood capillaries and their endothelial cells; some red cells inside vessels are noted. The muscle cell sarcolemma could be observed associated with the described structures. The cardiomyocytes of old rats presented an average sarcomere length of 2.071 ± 0.09 μm, a mitochondrial volume density (Vv) of 0.3383, a mitochondrial average area of 0.537 ± 0.278 μm², a mitochondrial average length of 1.024 ± 0.352 μm, an average mitochondrial cristae thickness of 0.038 ± 0.09 μm and a ratio of mitochondrial greater length/lesser length of 1.929 ± 0.965. Of the observed mitochondrial shapes, 23.4% were rounded, 45.3% were elongated, and 31.1% had irregular profiles. In this study, we analyzed the morphology and morphometry of cardiomyocytes in old rats, focusing on mitochondria. These data are important for researchers who focus the changes in cardiac tissue, especially changes owing to pathologies and drug administration that may or may not be correlated with aging. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Quantification of red blood cells using atomic force microscopy

For humans the sizes and shapes of their red blood cells are important indicators of well being. In this study, the feasibility of using the atomic force microscope (AFM) to provide the sizes and shapes of red blood cells has been investigated. An immobilisation procedure has been developed that enabled red blood cells to be reliably imaged by contact AFM in air. The shapes of the red blood cells were readily apparent in the AFM images. Various cell quantification parameters were investigated, including thickness, width, surface area and volume. Excellent correlation was found between the AFM-derived immobilised mean cell volume (IMCV) parameter and the mean cell volume (MCV) parameter used in current haematological practice. The correlation between MCV and IMCV values has validated the immobilisation procedure by demonstrating that the significant cell shrinkage that occurs during immobilisation and drying does not introduce quantification artifacts. Reliable IMCV values were obtained by quantifying 100 red blood cells and this typically required 3-5 AFM images of 100 microm x 100 microm area. This work has demonstrated that the AFM can provide in a single test the red blood cell size and shape data needed in the assessment of human health.     

The application of water soluble,mega‐Stokes‐shifted BODIPY fluorophores to cell and tissue imaging

BODIPY (4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene) fluorophores are widely used in bioimaging to label proteins, lipids and nucleotides, but in spite of their attractive optical properties they tend to be prone to self‐quenching because of their notably small Stokes shift. Herein, we compare two BODIPY compounds from a recently developed family of naphthyridine substituted BODIPY derivatives, one a visible emitting derivative (BODIPY‐VIS) and one a near‐infrared emitting fluorophore with a Stokes shift of approximately 165 nm as contrast reagents for live mammalian cells and murine brain tissue. The compounds were rendered water soluble by their conjugation to polyethylene glycol (PEG). Both PEGylated compounds exhibited good cell uptake compared with their parent compounds and confocal fluorescence microscopy revealed all dyes explored to be nuclear excluding, localizing predominantly within the lipophilic organelles; the endoplasmic reticulum and mitochondria. Cytotoxicity studies revealed that these BODIPY derivatives are modestly cytotoxic at concentrations exceeding 10 μM where they induce apoptosis and necrosis. Although the quantum yield of emission of the visible emitting fluorophore was over an order of magnitude greater than the Mega‐Stokes shifted probe, the latter showed considerably reduced tendency to self quench and less interference from autofluorescence. The near‐infrared probe also showed good penetrability and staining in live tissue samples. In the latter case similar tendency to exclude the nucleus and to localize in the mitochondria and endoplasmic reticulum was observed as in live cells. This to our knowledge is the first demonstration of such a Mega‐Stokes BODIPY probe applied to cell and tissue imaging.     

Fine structural study of the red seaweed <i>Gymnogongrus torulosus</i> (Phyllophoraceae,Rhodophyta)

The present study analyzed several characters of the red seaweed Gymnogongrus torulosus, such as cellular structure of the thallus, cuticle, pit plug and cell wall ultrastructure, and morphology of some organelles like plastids, Golgi bodies and mitochondria. Also, anomalous chloroplasts with thylakoid disorganization were found in medullary cells. The significance of this thylakoid disposition is still unclear. This is one of the first studies focused on the fine structure of a red alga recorded in Argentina.     

Application of microscopical techniques in the study of autolysosome dynamics in PC12 neurites

Autophagy is a principal degradation pathway for the turnover of intracellular proteins or cytoplasmic organelles in response to starvation. During autophagic activation, autophagosomes fuse with lysosomes to form autolysosomes where incorporated materials are degraded. However, the dynamics of autolysosomes in neurites of live cells was still poorly known. In this study, various subsets of microscope were applied to analyse the autophagy induction and highly dynamic transport of autolysosomes in rat PC12 neurites. Beading formation was found in degenerating PC12 neurites under phase contrast light microscope after serum deprivation. The monomeric red fluorescence protein‐green fluorescence protein‐light chain 3–labelled autolysosomes accumulated throughout PC12 neurites after 18 h of serum deprivation as revealed by fluorescence microscope analysis. The single‐membrane autolysosomes were also visualized in PC12 cells under transmission electron microscope. Moreover, fluorescence recovery after photobleaching experiment, which was conducted by confocal laser scanning microscope, demonstrated that autolysosomes were motile vesicles and moved along PC12 neurites during starvation. The directional transport of monomeric red fluorescence protein ‐labelled autolysosomes in neurites was further monitored by a motorized video microscope. Both anterograde and retrograde transport of autolysosomes were observed. In addition, the autolysosomes were precisely mapped by using 2D Gaussian fitting and then their highly dynamic movement was robustly tracked by using multidimensional assignment. Collectively, by using different microscopical techniques, our results confirmed the dynamic transport of autolysosomes in starved PC12 neurites and may provide valuable insight into understanding the biophysical characteristics of autolysosomes in neurites under physiological and pathological conditions.     

Review : Reticulocyte maturation: mitoptosis and exosome release

During the differentiation of erythroid cells, a vast program of maturation takes place, leading to decay or elimination of organelles, including the nucleus, mitochondria, ribosomes, lysosomes, endoplasmic reticulum and Golgi apparatus. During the last step of red cell maturation, remaining organelles, primarily mitochondria and ribosomes but also vestiges of others are finally cleared from the cell. This cleaning session also affects specific proteins that are partially or entirely removed from the cell surface. The interplay of the various events and their causal relationships are approached here.     

Effect of anticoagulation and lavage prior to casting of postmortem material with Mercox® and Batson® 17

In this study we compare the quality of vascular casts, obtained from organs of several animal species from different sources and sacrified under different conditions. Organs from healthy animals were injected with two different polymers such as Mercox and Batson No. 17. When the specimens were observed under a scanning electron microscope structural elements such as endothelial nuclear impressions on vessels and capillaries, endothelial cell borders, venous valves, imprints of smooth muscle cells and intra-arterial cushions were identified. Organs excised post mortem from large animals can be used for microvascular corrosion casting studies with optimal results.     

Autofluorescence of living cells

We have investigated the autofluorescence of viable mammalian cells (DU-145 and V79) with a confocal laser scanning microscope equipped with a UV laser. Our aim was to investigate the autofluorescence dependence on different treatments in mitochondria and lysosomes by using different reagents and to improve the confocal laser scanning microscope image quality by deconvolution. The following conclusions were drawn from the results: (1) not all of the autofluorescence comes from mitochondria; (2) one can significantly affect the signal which comes from the mitochondria; (3) the other organelles involved are probably lysosomes; (4) it is harder to affect the autofluorescence signal from the lysosomes than that from the mitochondria, and (5) deconvoluted autofluorescence images provide better information than undeconvoluted ones.     

Ultrastructural localization of nucleic acids in plant tissues following the use of malachite green or neutral red in the fixative solution

Malachite green and neutral red, when added to glutaraldehyde for fixation of various tissues, yielded high-contrast images of cell ultrastructure. Malachite green, in acid conditions, appeared to increase contrast of heterochromatin material in the nucleus whereas neutral red gave greater clarity to the nucleolus and to cytoplasmic ribosomes. Control tissue fixed under acid conditions showed little damage but there were ‘crystalline’ areas at the periphery of the nucleolus. RNase did not digest cytoplasmic ribosomes from tissue after neutral red glutaraldehyde fixation. These results suggested that neutral red became bound to RNA in the tissues. Fixation with malachite green, at a pH below 6, did not affect the digestion of RNA by RNase but did protect chromatin against the bleaching action of the chelating agent EDTA. The addition of malachite green (pH < 6) or neutral red to glutaraldehyde are useful techniques for the investigation of the ultrastructure of nuclear material and cytoplasmic ribosomes.     

A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g , washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 m m ammonium acetate, 300 m m sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10–11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.     

Automated identification of normoblast cell from human peripheral blood smear images

In this paper, we have presented a new computer‐aided technique for automatic detection of nucleated red blood cells (NRBCs) or normoblast cell from peripheral blood smear image. The proposed methodology initiates with the localization of the nucleated cells by adopting multilevel thresholding approach in smear images. A novel colour space transformation technique has been introduced to differentiate nucleated blood cells [white blood cells (WBCs) and NRBC] from red blood cells (RBCs) by enhancing the contrast between them. Subsequently, special fuzzy c‐means (SFCM) clustering algorithm is applied on enhanced image to segment out the nucleated cell. Finally, nucleated RBC and WBC are discriminated by the random forest tree classifier based on first‐order statistical‐based features. Experimentally, we observed that the proposed technique achieved 99.42% accuracy in automatic detection of NRBC from blood smear images. Further, the technique could be used to assist the clinicians to diagnose a different anaemic condition.