Mapping cholesteryl ester analogue uptake and intracellular flow in Paramecium by confocal fluorescence microscopy

In Paramecium primaurelia the uptake and intracellular flow of cholesteryl ester was studied by fluorescence confocal laser scanning optical microscopy and by the fluorescent analogue cholesteryl‐BODIPY^® FL C₁₂ (BODIPY‐CE). The BODIPY FL fluorophore has the characteristic of emitting green fluorescence, which is red‐shifted as the probe concentrates. In cells incubated with 25 µm BODIPY‐CE for 30 s, fluorescence is found in vesicles located around the cytopharynx in the posterior half of the cell. Successively, the lipid is internalized by food vacuoles, the fluorescent vesicles are distributed throughout the cell and the intracellular membranes are labelled. The food vacuole number is maximum after 10–15 min of continuous labelling, then it decreases until no food vacuoles are found in 30‐min fed cells. BODIPY‐CE accumulates in red‐labelled cytoplasmic droplets located in the anterior half of the cell. When food vacuole formation is inhibited by trifluoperazine, fluorescence is found on cellular membranes and in small green‐labelled vesicles at the apical pole. The inhibition of clathrin‐mediated endocytosis does not interfere in P. primaurelia with BODIPY‐CE intracellular flow: intracellular membranes and storage droplets in the cell anterior part are dyed. Conversely, the use of sterol‐binding drugs prevents the lipid accumulation in droplets, stopping the lipid within the cytoplasmic membranes. Furthermore, the cells treated with monensin and cytochalasin B show a labelling of the cellular membranes and lipid droplets, whereas NH₄Cl reduces the lipid storage. Low temperature (4 °C) does not prevent the internalization of BODIPY‐CE that, however, is localized at the cytoplasmic membrane level and does not accumulate in storage droplets. In addition, BODIPY‐CE inhibits phagocytosis, as evidenced by comparing the kinetics of food vacuole formation of control cells, only fed with latex particles, with that of cells fed with latex particles and BODIPY‐CE. In conclusion, this study points out that in P. primaurelia the cholesteryl ester enters the cell via food vacuoles and through the plasma membrane and, inside the cell, it alters cell functions.     

Receptor autoradiography and 2-deoxyglucose techniques in neuroscience

The use of the receptor autoradiography and 2-deoxyglucose (2-DG) techniques in neuroscience are reviewed. Receptors and other binding sites can be visualized autoradiographically in microtome tissue sections after labelling with radioligand in vivo or in vitro. Autoradiograms are generated by apposition of the labelled tissue to photographic emulsions. Combined with computerized image analysis, this technique can be used to analyse and quantify the microscopic distribution of receptors and receptor alterations associated with lesions or disease in human and animal tissues. The 2-DG technique permits microscopic analysis of modifications in brain glucose utilization induced by physiological and pharmacological manipulations. Limitations of these techniques and attempts to optimize their resolution are also discussed.     

Autoradiographic studies of DNA synthesis in lymphoid tissues

Using long exposures of stripping film autoradiographs before processing, mixtures of weakly and strongly labelled nuclei were seen in different areas of the mouse spleen. Previous results (Harris et al., 1973) led to the conclusion that many cells, not in division cycle, were labelling with (³H) thymidine and that this process was important for the development of specific antibody-producing cells following stimulation with an antigen such as sheep red cells (SRC). The present data are an analysis of the (³H) thymidine labelling kinetics in the spleens of mice reared in conventional or germ-free conditions. The labelling seen in the 24 h following an injection of (³H) thymidine could best be interpreted on the basis of synthesis of unstable DNA. The changes in the pattern, and distribution of labelled nuclei as well as the intensity of their labelling was not compatible with cell division only, but was also the result of movement of labelled material between the lymphoid cells of the organ. Germ-free mice were followed for 24 days following a single injection of (³H) thymidine. The rate of uptake of label into the spleen was much slower than has been found previously in mice reared in conventional conditions. When SRC were injected 2 h after giving (³H) thymidine the labelling of lymphoid cells in the spleen and blood was quite different to controls given (³H) thymidine alone. Detailed analysis indicated that turnover of labelled material, presumably DNA, as well as cells was involved. This turnover of DNA could be considered to be metabolic in the sense that renewal, increase in amount, loss, and transfer to other cells were involved. These, and other studies, in vivo (Harris & Olsen, 1973) and in vitro (Harris et al. 1975) indicate that such processes, involving DNA, are highly relevant to the development of antibody-producing capacity by cells responding to antigenic challenge.     

Statistical analysis of labelling patterns of mammary carcinoma cell nuclei on histological sections

It is of central interest for tumour biology to explore the mechanisms of tumour cell proliferation. In this study, methods of spatial statistics were used to study the spatial distribution of proliferating cells within tumour tissue quantitatively and objectively. Mammary cancer tissue was studied as an example. It was attempted to clarify whether cell division occurs entirely at random (random labelling), i.e. the process of division occurs at random, independently from the state of the neighbouring nuclei, or whether the spatial distribution of proliferation is more complex, e.g. in the form of actively proliferating clusters alternating with relatively silent zones. In the case of random labelling, the reduced second moment functions K(r) of the labelled and the unlabelled nuclei would be identical. The same would hold for the pair correlation functions g(r) . The alternative hypothesis is that the second‐order properties of the processes of the labelled and the unlabelled nuclei are different. Twenty cases of invasive ductal mammary carcinomas were studied. The nuclei of proliferating cells were stained immunohistochemically with the monoclonal antibody MIB‐1, which detects specifically the proliferation‐associated nuclear antigen Ki 67. The planar coordinates of the tumor cell nucleus profiles from two rectangular visual fields per case were recorded. For each visual field, the following investigations were performed: estimation of the explorative summary characteristics K(r) and g(r) , fitting of the parameters of a stationary Strauss hard‐core model to the observed point patterns, estimation of two distance‐dependent Simpson indices and Monte Carlo tests of all individual patterns on the null hypothesis of random labelling. Significant differences between the mean K‐functions and the mean g‐functions of the labelled and the unlabelled nuclei were found. Moreover, the mean interaction parameter γ of the stationary Strauss hard‐core model was significantly higher for the labelled nuclei than for the unlabelled nuclei. The estimates of the two distance‐dependent Simpson indices showed a tendency of points with the same label towards a positive spatial correlation. In the Monte Carlo tests, the null hypothesis of random labelling was rejected for the majority of the visual fields. These four lines of investigation led to the concordant conclusion that the labelling of mammary carcinoma nuclei by MIB‐1 is not simply random. The data suggest that the second‐order properties of the point process of the labelled nuclei are significantly different from those of the unlabelled nuclei. In particular, the process of the labelled nuclei shows a higher degree of clustering (increased strength of interaction) than the process of the unlabelled points.     

Statistical methods for in situ hybridization: identification of autoradiographically labelled cells and structures

In situ hybridization experiments frequently use autoradiography to identify labelled structures. Ideally, labelled cells will be overlain with a dense accumulation of particles, allowing one to discriminate them from unlabelled cells easily. However, if noise is high or the density of labelling is low, it can be difficult to distinguish bona fide labelling ‘by eye’. In such situations, labelled cells could be overlooked. This paper evaluates two statistical solutions to this problem: (1) a parametric method proposed by Hashimoto and co‐workers and (2) Wang & Wessendorf's non‐parametric method using contingency testing (i.e. the chi‐square or Fisher's exact tests). The Hashimoto method determines the mean and standard deviation of the density of background labelling, using sense‐strand controls as the source of background levels. Cells labelled at densities greater than two standard deviations above the mean (P < 0.0455) are defined as significantly labelled. Contingency testing determines whether the grain density over a cell is significantly higher than that over the remainder of the image. When compared, the two methods gave similar results. The Hashimoto method may be more sensitive if most cells are labelled but contingency testing requires no assumptions about the uniformity of non‐specific labelling.     

Influence of the microenvironment on gene and protein expression of odontogenic-like and osteogenic-like cells

Progenitor cells play an important biological role in tooth and bone formation, and previous analyses during bone and dentine induction have indicated that they may be a good alternative for tissue engineering. Thus, to clarify the influence of the microenvironment on protein and gene expression, MDPC23 cells (mouse dental papilla cell line) and KUSA/A1 cells (bone marrow stromal cell line) were used, both in vitro cell culture and in intra-abdominal diffusion chambers implanted in 4-week-old male immunodefficient mice (SCID mice). Our results indicate that KUSA/A1 cells differentiated into osteoblast-like cells and induced bone tissue inside the chamber, whereas, MDPC-23 showed odontoblast-like characteristics but with a low ability to induce dentin formation. This study shows that MDPC-23 cells are especial cells, which possess morphological and functional characteristics of odontoblast-like cells expressing dentin sialophosphoprotein in vivo. In contrast, dentin sialophosphoprotein gene and protein expression was not detected in both cell lines in vitro. The intra-abdominal diffusion chamber appears as an interesting experimental model for studying phenotypic expression of dental pulp cells in vivo.     

Three-dimensional volume reconstruction of extracellular matrix proteins in uveal melanoma from fluorescent confocal laser scanning microscope images

The distribution of looping patterns of laminin in uveal melanomas and other tumours has been associated with adverse outcome. Moreover, these patterns are generated by highly invasive tumour cells through the process of vasculogenic mimicry and are not therefore blood vessels. Nevertheless, these extravascular matrix patterns conduct plasma. The three‐dimensional (3D) configuration of these laminin‐rich patterns compared with blood vessels has been the subject of speculation and intensive investigation. We have developed a method for the 3D reconstruction of volume for these extravascular matrix proteins from serial paraffin sections cut at 4 µm thicknesses and stained with a fluorescently labelled antibody to laminin ( Maniotis et al., 2002 ). Each section was examined via confocal laser‐scanning focal microscopy (CLSM) and 13 images were recorded in the Z‐dimension for each slide. The input CLSM imagery is composed of a set of 3D subvolumes (stacks of 2D images) acquired at multiple confocal depths, from a sequence of consecutive slides. Steps for automated reconstruction included (1) unsupervised methods for selecting an image frame from a subvolume based on entropy and contrast criteria, (2) a fully automated registration technique for image alignment and (3) an improved histogram equalization method that compensates for spatially varying image intensities in CLSM imagery due to photo‐bleaching. We compared image alignment accuracy of a fully automated method with registration accuracy achieved by human subjects using a manual method. Automated 3D volume reconstruction was found to provide significant improvement in accuracy, consistency of results and performance time for CLSM images acquired from serial paraffin sections.     

Monitoring of neuronal and glial calcium activity using a novel direct-contact probe

In vivo neuronal and glial calcium activity was monitored using a novel direct‐contact probe that was designed for fluorescence detection deep within biological tissue. A small diameter fibre bundle direct‐contact probe was employed with a laser scanning confocal microscope to detect evoked neuronal and glial activity in the posteromedial barrel subfield of the rat somatosensory cortex in vivo. Resolution of the probe allowed discrimination of single cells, and calcium dynamics spanning milliseconds to several seconds were observed. Initial results indicate that the probe has useful practical applications in the imaging of individual cells and monitoring rapid calcium fluctuations within their cell body and large processes.     

Dual-mode reflectance and fluorescence near-video-rate confocal microscope for architectural, morphological and molecular imaging of tissue

We have developed a near‐video‐rate dual‐mode reflectance and fluorescence confocal microscope for the purpose of imaging ex vivo human specimens and in vivo animal models. The dual‐mode confocal microscope (DCM) has light sources at 488, 664 and 784 nm, a frame rate of 15 frames per second, a maximum field of view of 300 × 250 μm and a resolution limit of 0.31 μm laterally and 1.37 μm axially. The DCM can image tissue architecture and cellular morphology, as well as molecular properties of tissue, using reflective and fluorescent molecular‐specific optical contrast agents. Images acquired with the DCM demonstrate that the system has the sub‐cellular resolution needed to visualize the morphological and molecular changes associated with cancer progression and has the capability to image animal models of disease in vivo. In the hamster cheek pouch model of oral carcinogenesis, the DCM was used to image the epithelium and stroma of the cheek pouch; blood flow was visible and areas of dysplasia could be distinguished from normal epithelium using 6% acetic acid contrast. In human oral cavity tissue slices, DCM reflectance images showed an increase in the nuclear‐to‐cytoplasmic ratio and density of nuclei in neoplastic tissues as compared to normal tissue. After labelling tissue slices with fluorescent contrast agents targeting the epidermal growth factor receptor, an increase in epidermal growth factor receptor expression was detected in cancerous tissue as compared to normal tissue. The combination of reflectance and fluorescence imaging in a single system allowed imaging of two different parameters involved in neoplastic progression, providing information about both the morphological and molecular expression changes that occur with cancer progression. The dual‐mode imaging capabilities of the DCM allow investigation of both morphological changes as well as molecular changes that occur in disease processes. Analyzing both factors simultaneously may be advantageous when trying to detect and diagnose disease. The DCM's high resolution and near‐video‐rate image acquisition and the growing inventory of molecular‐specific contrast agents and disease‐specific molecular markers holds significant promise for in vivo studies of disease processes such as carcinogenesis.     

In vivo coherent anti-Stokes Raman scattering imaging of sciatic nerve tissue

We report in vivo nonlinear optical imaging of mouse sciatic nerve tissue by epidetected coherent anti‐Stokes Raman scattering and second harmonic generation microscopy. Following a minimally invasive surgery to open the skin, coherent anti‐Stokes Raman scattering imaging of myelinated axons and second harmonic generation imaging of the surrounding collagen fibres were demonstrated with high signal‐to‐background ratio, three‐dimensional spatial resolution, and no need for labelling. The underlying contrast mechanisms of in vivo coherent anti‐Stokes Raman scattering were explored by three‐dimensional imaging of fat cells that surround the nerve. The epidetected coherent anti‐Stokes Raman scattering signals from the nerve tissues were found to arise from interfaces as well as back reflection of forward coherent anti‐Stokes Raman scattering.     

Robust fluorescent labelling of micropipettes for use in fluorescence microscopy: application to the observation of a mosquito borne parasite infection

The ability to monitor micropipette injections with a high‐resolution fluorescent microscope has utility for a variety of applications. Herein, different approaches were tested for creating broad‐band fluorescently labelled glass micropipettes including: UV cured glass glues, baked glass enamel containing fluorescent dyes as well as nanodiamonds attached during pipette formation in the microforge. The most robust and simplest approach was to use labelled baked enamel on the exterior of the pipette. This approach was tested using pipettes designed to mimic a mosquito proboscis for the injection of the malaria parasite, Plasmodium spp., into the dermis of a living mouse ear. The pipette (～30 micron diameter) was easily detected in the microscopy field of view and tolerated multiple insertions through the skin. This simple inexpensive approach to fluorescently labelling micropipettes will aid in the development of procedures under the fluorescent microscope.     

Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome

Topological analysis of the three‐dimensional (3D) chromatin nanostructure and its function in intact cell nuclei implies the use of high resolution far field light microscopy, e.g. confocal laser scanning microscopy (CLSM). However, experimental evidence indicates that, in practice, under biologically relevant conditions, the spatial resolution of CLSM is limited to about 300 nm in the lateral direction and about 700 nm in the axial direction. To overcome this shortcoming, the use of a recently developed light microscopical approach, spectral precision distance microscopy (SPDM) is established. This approach is based on the precise localization of small labelling sites of a given target in spectrally differential images. By means of quantitative image analysis, the bary centres (intensity weighted centroid analogous to the centre of mass) of these independently registered labelling sites can be used as point markers for distance and angle measurements after appropriate calibration of optical aberrations (here, polychromatic shifts). In combination with specific labelling of very small chromatin target sites with dyes of different spectral signatures by fluorescence in situ hybridization (FISH), SPDM presently allows us to analyse the nuclear topology in three‐dimensionally conserved nuclei with a ‘resolution equivalent’, many times smaller than the conventional optical resolution. Chronic myelogeneous leukaemia (CML) is genetically characterized by the fusion of parts of the BCR and ABL genes on chromosomes 22 and 9, respectively. In most cases, the fusion leads to a translocation t(9; 22) producing the Philadelphia chromosome. SPDM was applied to analyse the 3D chromatin structure of the BCR region on the intact chromosome 22 and the BCR‐ABL fusion gene on the Philadelphia chromosome (Ph) by using a new triple‐colour FISH protocol: two different DNA probes were used to detect the BCR region and the third DNA probe was used to identify the location of the ABL gene. Consistent 3D distance measurements down to values considerably smaller than 100 nm were performed. The angle distributions between the three labelled sites on the Philadelphia chromosome territory were compared to two state‐of‐the‐art computer models of nuclear chromatin structure. Significant differences between measured and simulated angle distributions were obtained, indicating a complex and non‐random angle distribution.     

Labelling quality and chromosome morphology after low temperature FISH analysed by scanning far-field and near-field optical microscopy

A non‐enzymatic, low temperature fluorescence in situ hybridization (LTFISH) procedure was applied to metaphase spreads and interphase cell nuclei. In this context ‘low temperature’ means that the denaturation procedure of the chromosomal target DNA usually applied by heat treatment and chaotropic agents such as formamide was completely omitted so that the complete hybridization reaction took place at 37 °C. For LTFISH, the DNA probe had to be single‐stranded, which was achieved by means of separate thermal denaturation of the DNA probe only. The DNA probe pUC1.77 was used for all LTFISH experiments. The labelling quality (number of binding sites, relative background intensity, relative intensity of major and minor binding sites) was analysed by confocal laser scanning microscopy (CLSM). An optimum in specificity and signal quality was obtained for 15 h hybridization time. For this hybridization condition of LTFISH, the chromosomal morphology was analysed by scanning near‐field optical microscopy (SNOM). The results were compared with the morphology of chromosomes after (a) labelling of all centromeres using the same chemical treatment in the FISH procedure but with the application of target denaturation, and (b) labelling of all centromeres using a standard FISH protocol including thermal denaturation of the DNA probe and the chromosomal target. Depending on the FISH‐procedure applied, SNOM images show substantial differences in the chromosome morphology. After LTFISH the chromosome morphology appeared to be much better preserved than after standard FISH. In contrast, the application of the LTFISH chemical treatment accompanied by heat denaturation had a very destructive influence on chromosomal morphology. The results indicate that, at least for certain DNA probes, specific chromosome labelling can be obtained without the usually applied heat and chemical denaturation of the DNA target, resulting in an apparently well preserved chromatin morphology as visualized by SNOM. LTFISH may be therefore a useful labelling technique whenever the chromosomal morphology had to be preserved after specific labelling of DNA regions. Binding mechanisms of single‐stranded DNA probes to double‐stranded DNA targets are discussed.     

RASAL2 acts as a tumor suppressor in cervical cancer cells

Background: This study was designed to investigate the roles of RASAL2 in cervical cancer (CC). Methods: Fifty-four CC tissues and 33 adjacent tissues were obtained from CC patients admitted to our hospital between March 2012 and June 2014. Real-time polymerase chain reaction and western blotting were performed to analyze the expression of RASAL2 mRNA and protein in these tissues, CC cell lines, and normal cervical cells. Over-expression and silencing of RASAL2 were induced after transfection, and the migration, invasion, and proliferation of the CC cell lines were examined. Results: RASAL2 mRNA and protein expressions were significantly down-regulated in CC tissues and cell lines than in adjacent tissues and normal cervical cells, respectively. While low RASAL2 expression correlated with advanced stage and metastasis of CC, its over-expression significantly inhibited proliferation and metastasis of CC cells and induced apoptosis. Under in vitro conditions, silencing of RASAL2 expression could significantly increase the proliferation, invasion, and migration of CC cells. Conclusion: RASAL2 functioned as a tumor suppressor in CC, and was down-regulated in CC tissue samples and cell lines. tumor suppressor in CC, and was down-regulated in CC tissue samples and cell lines.     

Effect of ultrasonic activation on dentinal tubule penetration of calcium silicate‐based cements

This study investigated the dentinal tubule penetration of mineral trioxide aggregate (MTA), NeoMTA Plus and Biodentine placed by either manual condensation or ultrasonic activation in simulated open apex model. Standardized divergent open apex models were created using palatal roots of 60 human maxillary molars and divided into six groups according to the used cements and activation methods (n = 10): MTA‐manual condensation, MTA‐ultrasonic activation, NeoMTA Plus‐manual condensation, NeoMTA Plus‐ultrasonic activation, Biodentine‐manual condensation, Biodentine‐ultrasonic activation. For the measurement of penetration, the cements were mixed with 0.1% Rhodamin B and 6‐mm apical portions of each root canal were obturated in an orthograde direction. The roots were embedded into acrylic blocks, and 1‐mm‐thick sections were obtained at 3 mm from the apex. Specimens were mounted onto glass slides and scanned under a confocal laser scanning microscope (CLSM) and stereomicroscope. Dentinal tubule penetration areas, depth and percentage were measured using LSM and ImageJ software. The data were analyzed using two‐way analysis of variance (anova ) with Bonferroni correction (α = 0.05). No correlation was found between stereomicroscope and CLSM analyses (p > .05). CLSM analysis showed no significant differences between MTA, NeoMTA Plus, and Biodentine groups when manual condensation was used (p > .05). Ultrasonic activation did not increase the tubular penetration of MTA, NeoMTA Plus or Biodentine as compared to manual condensation of each material (p > .05). MTA, NeoMTA Plus and Biodentine showed similar tubular penetration when manual condensation was used. Ultrasonic activation of these cements had no effect on tubular penetration of each material as compared to the manual condensation counterparts.     

Is there section deformation resulting in differential change of nuclear numerical densities along the z axis of thick methacrylate or paraffin sections?

The optical disector, a three‐dimensional counting frame or probe in stereology, is often positioned in the middle (depth) of a thick section for unbiased nuclear counting. Using 30–40 μm thick methacrylate or paraffin sections for nuclear counting of neurons with the optical disector, however, some studies showed markedly higher nuclear densities at 10% of the section thickness near the top or bottom surface of the section, suggestive of deformation of section along its z axis and thus affecting the number estimation. To verify the findings, this study obtained two sets of 12–14 methacrylate sections (average thicknesses 21.7 and 29.4 μm) and two sets of 12 paraffin sections (average thicknesses 13.8 and 29.2 μm) from mature rat testes. Each section was used to count round spermatid nuclei in the seminiferous epithelium densely packed with the cells, using 3–4 consecutive disectors placed vertically (along the z axis of the section) from the top surface of the section, through the whole section thickness (two sets of methacrylate and paraffin sections) or in 80–83% of the thickness (other sections). The results demonstrated that, overall, there were no considerable nonuniform changes of the nuclear densities along the z axis of the sections.     

Direct evaluation of fluorescence in single renal epithelial cells using a mitochondrial probe (DASPMI)

The study of distribution and quantitation of a fluorescent probe in living epithelia with the aid of an inverted microscope requires that individual cells can be analysed without optical interference from adjacent cells. This report describes the application of fluorescence microscopy and fluorometry to a recently developed in vitro culture system of renal epithelial cells. Epithelial cells derived from the mammalian renal cortical collecting tubule (CT) and the thick ascending loop of Henle (TAL) are cultivated as continuous monolayers in serum-free, hormone-supplemented media. A specific mitochondrial marker (DASPMI) is added to the medium and incorporated into the cytoplasm. The microscopic image reveals that the mitochondrial fluorescence distribution differs between CT and TAL cultures. The fluorometric quantitation shows a normally distributed histogram of medium-range intensity in TAL cell cultures while CT cultures exhibit a two-peak pattern of mitochondrial fluorescence distribution among epithelial cells.     

High-resolution scanning electron microscopy of immunogold-labelled cells by the use of thin plasma coating of osmium

The feasibility of plasma coating of a thin osmium layer for high‐resolution immuno‐scanning electron microscopy of cell surfaces was tested, using Drosophila embryonic motor neurones as a model system. The neuro‐muscular preparations were fixed with formaldehyde and labelled with a neurone‐specific antibody and 10 or 5 nm colloidal gold‐conjugated secondary antibodies. The specimens were post‐fixed with osmium tetroxide and freeze‐dried. Then they were coated with a 1–2 nm thick layer of osmium using a hollow cathode plasma coater. The thin and continuous coating of amorphous osmium gave good signals of gold particles and fine surface structures of neurites in backscattered electron images simultaneously. This method makes it possible to visualize the antigen distribution and the three‐dimensionally complex surface structures of cellular processes with a resolution of several nanometres.     

Estimating volumes from common carp hepatocytes using design‐based stereology and examining correlations with profile areas: Revisiting a nutritional assay and unveiling guidelines to microscopists

Assessing fish liver status is common in aquaculture nutrition assays. This often implies determining hepatocytes profile areas in routine thin (5–7 μm) histological sections. However, there are theoretical problems using planar morphometry in thin sections: inherent sampling cells biases, too small numbers of sampled cells, under/overestimation of size, measuring size as areas when cells are three‐dimensional (3D) entities. The gold standard for assessing/validate cell size is stereology using thick sections (20–40 μm). Here, we estimated the volume of hepatocytes and their nuclei by the nucleator and optical disector stereological probes (in thick sections), and, innovatively, in thin sections too (using single‐section disectors). The liver of common carp eating feed containing either low or high level of lipids was targeted. Results were compared with prior profile areas from planar morphometry using thin sections, and with profile areas estimated here with the two‐dimensional (2D) nucleator. Ratios between nucleus and cell/cytoplasm (N/C) areas and volumes were calculated and compared. There was high positive correlation between volumes in thin and thick sections (r = .85 to .89; p < .001), empirically validating the single‐section disector. Strong correlations existed between profile‐derived versus 2D‐nucleator areas (r = .74 to .83; p < .001). There was systematic underestimation of cells and nucleus size using planar morphometry. The N/C ratios derived from the 2D‐nucleator data were higher than those from planar morphometry. Despite theoretical premises for using simple planar morphometry in thin sections are flawed, our results support that such morphometry on carp/fish hepatocytes may offer some valid biological conclusions. Anyway, we advanced guidelines for implementing proper methods.