Individual Cells In Tissue Research Articles

In Situ Magnetometry of Iron in Human Dopaminergic Neurons Using Superresolution MRI and Ion-Beam Microscopy

Paramagnetic transition metals play a crucial role as cofactors in various cellular catalytic processes. However, their high concentrations in reactive oxidation states can induce oxidative stress, resulting in cell dysfunction or death. Hence, it is vital to have methods to monitor metal concentrations and paramagnetic properties in cells for medicine and cell biology. Here we present a novel multimodal method for in-cell magnetometry enabling direct measurement of metal magnetic properties within individual cells in tissue, without prior isolation and at room temperature. Individual cell magnetic moments are measured using superresolution magnetic resonance imaging (MRI) microscopy at 9.4 T by detecting microscopic magnetic-field perturbations around the cells. The cellular metal content is quantified using ion-beam microscopy or synchrotron micro-x-ray fluorescence for the same cells. The metal magnetic susceptibility at 9.4 T is then obtained from the slope of the cell magnetic moments’ dependence on cell metal content. To estimate the susceptibility at lower fields, multifield MR relaxometry and biophysical modeling are employed, extrapolating the 9.4-T susceptibility values to fields as low as 3 T. We apply the new method to determine the susceptibility of iron accumulated in human dopaminergic neurons inside neuromelanin, the by-product of dopamine synthesis. The susceptibility of iron in neuromelanin is measured to be χρ=(2.98±0.19)×10−6 m3/kg providing unique insights into the biochemistry of iron inside dopaminergic neurons. The obtained value reveals a predominant monoatomic low-affinity iron-binding site within neuromelanin, indicating a higher neurotoxicity of iron than previously suggested. Furthermore, the measured susceptibility value establishes a quantitative relationship between cellular iron concentration and iron-sensitive MRI parameters, which can be noninvasively measured . This breakthrough paves the way for the detection of dopaminergic neuron density and iron load, requiring a standard clinical MRI scanner only. It promises to facilitate early diagnosis of Parkinson’s disease. In conclusion, our presented novel method enables the direct measurements of magnetic properties of paramagnetic metals within single cells with high sensitivity and across large cell groups within a macroscopic volume, providing invaluable information about the cellular biology of metals. Published by the American Physical Society 2024

Resampling approaches for the quantitative analysis of spatially distributed cells

ABSTRACT Image segmentation is a crucial step in various image analysis pipelines and constitutes one of the cutting-edge areas of digital pathology. The advent of quantitative analysis has enabled the evaluation of millions of individual cells in tissues, allowing for the combined assessment of morphological features, biomarker expression, and spatial context. The recorded cells can be described as a point pattern process. However, the classical statistical approaches to point pattern processes prove unreliable in this context due to the presence of multiple irregularly-shaped interstitial cell-devoid spaces in the domain, which correspond to anatomical features (e.g. vessels, lipid vacuoles, glandular lumina) or tissue artefacts (e.g. tissue fractures), and whose coordinates are unknown. These interstitial spaces impede the accurate calculation of the domain area, resulting in biased clustering measurements. Moreover, the mistaken inclusion of empty regions of the domain can directly impact the results of hypothesis testing. The literature currently lacks any introduced bias correction method to address interstitial cell-devoid spaces. To address this gap, we propose novel resampling methods for testing spatial randomness and evaluating relationships among different cell populations. Our methods obviate the need for domain area estimation and provide non-biased clustering measurements. We created the SpaceR software (https://github.com/GBertolazzi/SpaceR) to enhance the accessibility of our methodologies.

Inherency and agency in the origin and evolution of biological functions

AbstractAlthough discussed by 20th century philosophers in terms drawn from the sciences of non-living systems, in recent decades biological function has been considered in relationship to organismal capability and purpose. Bringing two phenomena generally neglected in evolutionary theory (i.e. inherency and agency) to bear on questions of function leads to a rejection of the adaptationist ‘selected effects’ notion of biological function. I review work showing that organisms such as the placozoans can thrive with almost no functional embellishments beyond those of their constituent cells and physical properties of their simple tissues. I also discuss work showing that individual tissue cells and their artificial aggregates exhibit agential behaviours that are unprecedented in the histories of their respective lineages. I review findings on the unique metazoan mechanism of developmental gene expression that has recruited, during evolution, inherent ancestral cellular functionalities into specialized cell types and organs of the different animal groups. I conclude that most essential functions in animal species are inherent to the cells from which they evolved, not selected effects, and that many of the others are optional ‘add-ons’, a status inimical to fitness-based models of evolution positing that traits emerge from stringent cycles of selection to meet external challenges.

Automatic wavelet-based 3D nuclei segmentation and analysis for multicellular embryo quantification

Identification of individual cells in tissues, organs, and in various developing systems is a well-studied problem because it is an essential part of objectively analyzing quantitative images in numerous biological contexts. We developed a size-dependent wavelet-based segmentation method that provides robust segmentation without any preprocessing, filtering or fine-tuning steps, and is robust to the signal-to-noise ratio. The wavelet-based method achieves robust segmentation results with respect to True Positive rate, Precision, and segmentation accuracy compared with other commonly used methods. We applied the segmentation program to zebrafish embryonic development IN TOTO for nuclei segmentation, image registration, and nuclei shape analysis. These new approaches to segmentation provide a means to carry out quantitative patterning analysis with single-cell precision throughout three dimensional tissues and embryos and they have a high tolerance for non-uniform and noisy image data sets.

Ex vivo MR microscopy of a human brain with multiple sclerosis: Visualizing individual cells in tissue using intrinsic iron

PurposeTo perform magnetic resonance microscopy (MRM) on human cortex and a cortical lesion as well as the adjacent normal appearing white matter. To shed light on the origins of MRI contrast by comparison with histochemical and immunostaining. Methods3D MRM at a nominal isotropic resolution of 15 and 18 µm was performed on 2 blocks of tissue from the brain of a 77-year-old man who had MS for 47 years. One block contained normal appearing cortical gray matter (CN block) and adjacent normal appearing white matter (NAWM), and the other also included a cortical lesion (CL block). Postmortem ex-vivo MRI was performed at 11.7T using a custom solenoid coil and T2*-weighted 3D GRE sequence. Histochemical and immunostaining were done after paraffin embedding for iron, myelin, oligodendrocytes, neurons, blood vessels, macrophages and microglia, and astrocytes. ResultsMRM could identify individual iron-laden oligodendrocytes with high sensitivity (70% decrease in signal compared to surrounding) in CN and CL blocks, as well as some iron-laden activated macrophages and microglia. Iron-deficient oligodendrocytes seemed to cause relative increase in MRI signal within the cortical lesion. High concentration of myelin in the white matter was primarily responsible for its hypointense appearance relative to the cortex, however, signal variations within NAWM could be attributed to changes in density of iron-laden oligodendrocytes. ConclusionChanges in iron accumulation within cells gave rise to imaging contrast seen between cortical lesions and normal cortex, as well as the patchy signal in NAWM. Densely packed myelin and collagen deposition also contributed to MRM signal changes. Even though we studied only one block each from normal appearing and cortical lesions, such studies can help better understand the origins of histopathological and microstructural correlates of MRI signal changes in multiple sclerosis and contextualize the interpretation of lower-resolution in vivo MRI scans.

Animal simulations facilitate smart drug design through prediction of nanomaterial transport to individual tissue cells.

Smart drug design for antibody and nanomaterial-based therapies allows optimization of drug efficacy and more efficient early-stage preclinical trials. The ideal drug must display maximum efficacy at target tissue sites, with transport from tissue vasculature to the cellular environment being critical. Biological simulations, when coupled with in vitro approaches, can predict this exposure in a rapid and efficient manner. As a result, it becomes possible to predict drug biodistribution within single cells of live animal tissue without the need for animal studies. Here, we successfully utilized an in vitro assay and a computational fluid dynamic model to translate in vitro cell kinetics (accounting for cell-induced degradation) to whole-body simulations for multiple species as well as nanomaterial types to predict drug distribution into individual tissue cells. We expect this work to assist in refining, reducing, and replacing animal testing, while providing scientists with a new perspective during the drug development process.

DEPENDENCE OF THE SPECKLE-PATTERNS SIZE AND THEIR CONTRAST ON THE BIOPHYSICAL AND STRUCTURAL PARAMETERS OF BIOLOGICAL TISSUES

Speckle fields are widely used in optical diagnostics of biotissues and evaluation of the functional state of bioobjects. The speckle field is formed by laser radiation scattered from the object under study. It bears information about the average dimensions of the scatterers, the degree of surface roughness makes it possible to judge the structural and biophysical characteristics of individual tissue cells (particles), on the one hand, and the integral optical characteristics of the entire biological tissue. The aim of the study was – the determination of connections between the biophysical and structural characteristics of the biotissue and the light fields inside the biotissues.The model developed of the medium gives a direct relationship between the optical and biophysical parameters of the biotissue. Calculations were carried out using known solutions of the radiation transfer equation, taking into account the multilayer structure of the tissue, multiple scattering in the medium, and multiple reflection of irradiation between the layers.With the increase wavelength, the size of speckles formed by the non-scattered component (direct light) of laser radiation increases by a factor of 2 from 400 to 800 μm in the stratum corneum and 5 times from 0.6 to 3 μm for the epidermis and from 0.27 to 1.4 μm to the dermis. Typical values of sizes of speckles formed by the diffraction component of laser radiation for the stratum corneum and epidermis range from 0.02 to 0.15 μm. For the dermis typical spot sizes are up to 0.03 μm. The speckle-spot size of the diffusion component in the dermis can vary from ±10 % at 400 nm and up to ±23 % for 800 nm when the volume concentration of blood capillaries changes. Characteristic dependencies are obtained and biophysical factors associated with the volume concentration of blood and the degree of it’s oxygenation that affect the contrast of the speckle structure in the dermis are discussed.The of speckles׳ size in the layers of tissue varies from a share of micrometer to millimeter. The established dependence makes it possible to determine the depth of penetration of light into the biotissue based on the dimensions of speckles. Calculation of the contrast of the speckle structure of scattered light in visible spectral range at different depths in the biotissue made it possible to establish the dependence of the contrast value of the interference pattern on the degree of oxygenation of the blood and the volume concentration of capillaries in the dermis.

Molecular stretching modulates mechanosensing pathways.

For individual cells in tissues to create the diverse forms of biological organisms, it is necessary that they must reliably sense and generate the correct forces over the correct distances and directions. There is considerable evidence that the mechanical aspects of the cellular microenvironment provide critical physical parameters to be sensed. How proteins sense forces and cellular geometry to create the correct morphology is not understood in detail but protein unfolding appears to be a major component in force and displacement sensing. Thus, the crystallographic structure of a protein domain provides only a starting point to then analyze what will be the effects of physiological forces through domain unfolding or catch-bond formation. In this review, we will discuss the recent studies of cytoskeletal and adhesion proteins that describe protein domain dynamics. Forces applied to proteins can activate or inhibit enzymes, increase or decrease protein-protein interactions, activate or inhibit protein substrates, induce catch bonds and regulate interactions with membranes or nucleic acids. Further, the dynamics of stretch-relaxation can average forces or movements to reliably regulate morphogenic movements. In the few cases where single molecule mechanics are studied under physiological conditions such as titin and talin, there are rapid cycles of stretch-relaxation that produce mechanosensing signals. Fortunately, the development of new single molecule and super-resolution imaging methods enable the analysis of single molecule mechanics in physiologically relevant conditions. Thus, we feel that stereotypical changes in cell and tissue shape involve mechanosensing that can be analyzed at the nanometer level to determine the molecular mechanisms involved.

Quantitative digital in situ senescence-associated β-galactosidase assay

BackgroundCellular senescence plays important roles in the aging process of complex organisms, in tumor suppression and in response to stress. Several markers can be used to identify senescent cells, of which the most widely used is the senescence-associated β-galactosidase (SABG) activity. The main advantage of SABG activity over other markers is the simplicity of the detection assay and the capacity to identify in situ a senescent cell in a heterogeneous cell population. Several approaches have been introduced to render the SABG assay quantitative. However none of these approaches to date has proven particularly amenable to quantitative analysis of SABG activity in situ. Furthermore the role of cellular senescence (CS) in vivo remains unclear mainly due to the ambiguity of current cellular markers in identifying CS of individual cells in tissues.ResultsIn the current study we applied a digital image analysis technique to the staining generated using the original SABG assay, and demonstrate that this analysis is highly reproducible and sensitive to subtle differences in staining intensities resulting from diverse cellular senescence pathways in culture. We have further validated our method on mouse kidney samples with and without diabetes mellitus, and show that a more accurate quantitative SABG activity with a wider range of values can be achieved at a pH lower than that used in the conventional SABG assay.ConclusionsWe conclude that quantitative in situ SABG assay, is feasible and reproducible and that the pH at which the reaction is performed should be tailored and chosen, depending on the research question and experimental system of interest.

Three respiratory gases and the red cell: oxygen, nitrogen, and carbon dioxide

Three vital gases—oxygen, nitrogen, and carbon dioxide—intersect at the level of the human red blood cell. The delivery of oxygen to all tissues by red cells is essential to human life. Evolution has created a complex molecule, haemoglobin, designed for efficient uptake and off-loading of oxygen. Iron rests at the centre of the haem moiety and is critical for oxygen exchange. Although studied for over a century, some details of oxygen transport by the red cell remain uncertain. Recent research has focusedon the interactionofhaemoglobin with a complex of cell-membrane proteins centred on band 3. In addition, there is renewed interest in the question of whether or not stored red cells deliver oxygen to tissues as well as fresh red cells. Nitric oxide (NO) physiology is directly related to oxygen delivery by the red cell. NO serves a local vasodilator to increase blood flow to hypoxic tissue beds. NO binds strongly to haemoglobin which serves as an NO sink. Under normal conditions, plasma NO synthesised by endothelial cells is not consumed by red cell haemoglobin due to a diffusion blockade across the red cell membrane that results from membrane structures not fully identified. Under conditions of red cell lysis, free haemoglobin scavenges NO reducing local vasodilation. Scavenging of NO is now recognised as an important component of the physiological response to chronic haemolysis and is very likely to play an important role in the renal lesion of acute haemolysis. The interactions of CO 2 with the red cell have drawn far less research attention. Plasma CO 2 released by tissues serves as an essential trigger for oxygen release by haemoglobin via the Hal-dane effect. CO 2 transport depends on conversion of CO 2 to bicarbonate via red cell carbonic anhydrase in conjunction with chloride exchange across the red cell membrane. There is very little research on the effect of blood storage on CO 2 excretion although this aspect of respiratory physiology is every bit as important as oxygen delivery. The three RBC gases are also the three principal gases of the Earth’s atmosphere. While CO 2 is the least abundant of the three by far, it is also likely to be the most critical to the future survival of life on Earth because small further increases in the concentration of CO 2 will result in continued climate change and large increases will be deadly. Thus, the management of three vital gases by the collection of red cells found within us has broad similarities to the collective management of our atmosphere. Just as the survival of individual tissue cells depends upon proper balance of these three respiratory gases, so too will their proper balance be the key to survival of life on Earth.

Effects of Morphology vs. Cell–Cell Interactions on Endothelial Cell Stiffness

Biological processes such as atherogenesis, wound healing, cancer cell metastasis, and immune cell transmigration rely on a delicate balance between Cell-Cell and cell-substrate adhesion. Cell mechanics have been shown to depend on substrate factors such as stiffness and ligand presentation, while the effects of Cell-Cell interactions on the mechanical properties of cells has received little attention. Here, we use atomic force microscopy to measure the Young's modulus of live human umbilical vein endothelial cells (HUVECs). In varying the degree of Cell-Cell contact in HUVECs (single cells, groups, and monolayers), we observe that increased cell stiffness correlates with an increase in cell area. Further, we observe that HUVECs stiffen as they spread onto a glass substrate. When we weaken Cell-Cell junctions (i.e., through a low dose of cytochalasin B or treatment with a VE-cadherin antibody), we observe that cell-substrate adhesion increases, as measured by focal adhesion size and density, and the stiffness of cells within the monolayer approaches that of single cells. Our results suggest that while morphology can roughly be used to predict cell stiffness, Cell-Cell interactions may play a significant role in determining the mechanical properties of individual cells in tissues by careful maintenance of cell tension homeostasis.

Three respiratory gases and the red cell: oxygen, carbon dioxide and nitric oxide

Three vital gases – oxygen, carbon dioxide and nitrogen – intersect at the level of the human red blood cell. The delivery of oxygen to all tissues by red cells is essential to human life. Evolution has created a complex molecule, haemoglobin, designed for efficient uptake and off‐loading of oxygen. Iron rests at the centre of the haeme moiety and is critical for oxygen exchange. Although studied for over a century, some details of oxygen transport by the red cell remain uncertain. Recent research has focused on the interaction of haemoglobin with a complex of cell‐membrane proteins centred on band 3. In addition, there is renewed interest in the question of whether or not stored red cells deliver oxygen to tissues as well as fresh red cells. The interactions of CO2 with the red cell have drawn far less research attention. Plasma CO2 released by tissues serves as an essential trigger for oxygen release by haemoglobin via the Bohr effect. CO2 transport depends on conversion of CO2 to bicarbonate via red cell carbonic anhydrase in conjunction with chloride exchange across the red cell membrane. There is very little research on the effect of blood storage on CO2 excretion although this aspect of respiratory physiology is every bit as important as oxygen delivery. Nitric oxide (NO) physiology is directly related to oxygen delivery by the red cell. NO serves a local vasodilator to increase blood flow to hypoxic tissue beds. NO binds strongly to haemoglobin which serves as an NO sink. Under normal conditions, plasma NO synthesized by endothelial cells is not consumed by red cell haemoglobin because of a diffusion blockade across the red cell membrane that results from membrane structures not fully identified. Under conditions of red cell lysis, free haemoglobin scavenges NO reducing local vasodilation. Scavenging of NO is now recognized as an important component of the physiologic response to chronic haemolysis and is very likely to play an important role in the renal lesion of acute haemolysis. The three RBC gases are also the three principal gases of the Earth’s atmosphere. While CO2 is the least abundant of the three by far, it is also likely to be the most critical to the future survival of life on Earth – because small further increases in the concentration of CO2 will result in continued climate change, and large increases will be deadly. Thus, the management of three vital gases by the collection of red cells found within us has broad similarities to the collective management of our atmosphere. Just as the survival of individual tissue cells depends upon proper balance of these three respiratory gases, so too will their proper balance be the key to survival of life on Earth.

A Targeted Gain-of-Function Screen Identifies Genes Affecting Salivary Gland Morphogenesis/Tubulogenesis in Drosophila

During development individual cells in tissues undergo complex cell-shape changes to drive the morphogenetic movements required to form tissues. Cell shape is determined by the cytoskeleton and cell-shape changes critically depend on a tight spatial and temporal control of cytoskeletal behavior. We have used the formation of the salivary glands in the Drosophila embryo, a process of tubulogenesis, as an assay for identifying factors that impinge on cell shape and the cytoskeleton. To this end we have performed a gain-of-function screen in the salivary glands, using a collection of fly lines carrying EP-element insertions that allow the overexpression of downstream-located genes using the UAS-Gal4 system. We used a salivary-gland-specific fork head-Gal4 line to restrict expression to the salivary glands, in combination with reporters of cell shape and the cytoskeleton. We identified a number of genes known to affect salivary gland formation, confirming the effectiveness of the screen. In addition, we found many genes not implicated previously in this process, some having known functions in other tissues. We report the initial characterization of a subset of genes, including chickadee, rhomboid1, egalitarian, bitesize, and capricious, through comparison of gain- and loss-of-function phenotypes.

Laser scanning cytometry: understanding the immune system in situ

Flow cytometry allows quantitative analysis of the identity and effector function of individual cells. However, it cannot provide information on cellular responses that occur within physiological tissue microenvironments. Laser scanning cytometry is an emerging technology that allows imaging and quantitative analysis of individual cells in tissues in situ. This article describes the technology and its potential for delineating the molecular and cellular events underpinning the immune response in health and disease.

When the lung is stretched, could it be thrombospondin via TGFβ1 peptide activation?

When the lung is stretched, could it be thrombospondin via TGFβ1 peptide activation?

Whole cell segmentation in solid tissue sections

Understanding the cellular and molecular basis of tissue development and function requires analysis of individual cells while in their tissue context. We developed software to find the optimum border around each cell (segmentation) from two-dimensional microscopic images of intact tissue. Samples were labeled with a fluorescent cell surface marker so that cell borders were brighter than elsewhere. The optimum border around each cell was defined as the border with an average intensity per unit length greater that any other possible border around that cell, and was calculated using the gray-weighted distance transform. Algorithm initiation requiring the user to mark two points per cell, one approximately in the center and the other on the border, ensured virtually 100% correct segmentation. Thereafter segmentation was automatic. The method was highly robust, because intermittent labeling of the cell borders, diffuse borders, and spurious signals away from the border do not significantly affect the optimum path. Computer-generated cells with increasing levels of added noise showed that the approach was accurate provided the cell could be detected visually. We have developed a highly robust algorithm for segmenting images of surface-labeled cells, enabling accurate and quantitative analysis of individual cells in tissue.

Urokinase and the Urokinase Receptor: Association With In Vitro Invasiveness of Human Bladder Cancer Cell Lines

The plasminogen activators urokinase and tissue plasminogen activator are enzymes that degrade proteins in tissue basement membranes and the extracellular matrix (a biomolecular complex surrounding individual cells in tissues that serves as a barrier between the cells and the vascular and lymph systems). The action of these enzymes allows tumor cells to escape their local environment and metastasize. Plasminogen activator activity can be influenced by the urokinase receptor, which is expressed on the surface of cells, and by the plasminogen activator inhibitors 1 and 2. Because bladder tumors differ in their propensity to invade local areas and distant sites, we studied the expression of both plasminogen activators, the two plasminogen activator inhibitors, and the urokinase receptor in four human bladder cancer cell lines (RT4, 253J, EJ, and T24) to see if there was an association between the expression of these proteins and tumor cell invasiveness in vitro. The expression of urokinase, tissue plasminogen activator, and the two inhibitors was measured by enzyme-linked immunosorbent assays of serum-free, cell-conditioned media (i.e., culture fluids). Cell surface expression of the urokinase receptor was assayed by flow cytometry, using an anti-receptor monoclonal antibody (Mab3936). The invasive capacity of untreated cells and of cells exposed to exogenous, high-molecular-weight urokinase was analyzed by use of Matrigel invasion chambers. The four bladder cancer cell lines demonstrated differential expression of both plasminogen activators and both inhibitors; three of the cell lines (T24, EJ, and 253J) expressed the urokinase receptor. The four cell lines differed in their invasive potential in vitro. Neither expression of tissue plasminogen activator nor production of the inhibitors appeared to influence Matrigel invasion. EJ cells and 253J cells produced the highest levels of urokinase and demonstrated the greatest propensity for invasion; T24 cells, which produced only small amounts of urokinase, exhibited a low invasive potential. Pretreatment of T24 cells with exogenous high-molecular-weight urokinase markedly increased their invasiveness. Similar pretreatment of EJ and 253J cells increased their invasiveness as well. RT4 cells, which lacked urokinase receptor expression but produced moderate amounts of urokinase, were not invasive and did not become so after exposure to exogenous high-molecular-weight urokinase. Binding of Mab3936 to urokinase receptors inhibited Matrigel invasion. To our knowledge, this is the first study demonstrating that bladder tumor cells express the urokinase receptor and that both receptor expression and urokinase expression are required for bladder tumor cell invasion in vitro.

FDTD analysis of the radiometric temperature measurement of a bilayered biological tissue using a body-contacting waveguide probe

Radiometric signal received by an open-ended rectangular waveguide probe in direct contact with a bilayered biological tissue is analyzed by the FDTD method. A two-layer tissue model consists of an outer thin skin layer over a semi-infinite fat layer is analyzed for a X-band total power radiometer. A spherical tumor with same permittivity as, but slightly higher temperature than, the surrounding normal fat tissue is assumed to exist inside the fat tissue. Active probe characteristics are analyzed first using the FDTD method for a propagating, sinusoidally time-varying, TE/sub 10/ type of excitation source. For the same waveguide probe operated as the radiometer antenna, radiometric weighting factors associated with individual tissue cells are then derived from the FDTD calculated field values. Near field radiating characteristics of the probe and radiometric signals determined for tumors of various size and depth are discussed. The presence of the skin is found to result in a lower power transmission coefficient across the probe aperture and significantly lower absorbed powers by fat; both of these contribute to the much lower radiometric signals observed. >

Combined immunocytochemical staining and image analysis for the study of lymphocyte specificity and function in situ

Immunocytochemical staining methods were combined with computer-aided image analysis to quantitate relative intracytoplasmic and cell membrane products within single lymphoid cells. Lymphoid cells fixed to microscope slides were immunocytochemically stained and the densities of immunocytochemical stains for individual lymphoid cells were determined on video-camera-captured microscopic images to quantitate synthesized and fully assembled antibody molecules detected by an anti-idiotypic antibody. Similarly, expression of a lymphoid cell membrane molecule, Ly-1, was comparatively quantitated in individual lymphoid cells. Densitometric analysis of tissue sections was utilized to measure tissue areas and quantitate cell nuclei in tissues. Two-color immunocytochemical staining and sequential image analyzer-determined locations of individual cells in tissues allowed unambiguous identification of doubly stained cells in their native tissue environment. This analysis was applied to the in situ identification of antibody forming cells producing the CRIc idiotype of the BALB/c anti-arsonate response and a determination of the mu chain usage by those individual cells. Applications of combined immunocytochemistry and computerized image analysis to studies of in vivo immunologic functions are discussed.

Detection of genes of interest in tissues and cells by in situ hybridization

In situ hybridization (ISH) is a powerful technique for assessing the expression of particular genes of interest within individual cells in tissues or cytospins. The only limitations of the technique are the availability of appropriate probes and tissues. When coupled with routine histology and DNA analysis, characterization and classification of tissues can be greatly extended. Future applications include the identification of tumor-specific markers that can be combined with ISH in diagnostic strategies.