Coherent Phase Microscopy Research Articles

Blood cell characterization based on deep learning and diffraction phase microscopy

Quantitative characterization of blood cells is very important for medical diagnosis and life science research. This study presents a novel label-free and high-throughput approach for analyzing flowing blood cells in a microfluidic channel by combining deep learning with partially coherent illumination-based diffraction phase microscopy (PCI-DPM). Diluted blood was injected into a microchannel and imaged with PCI-DPM, yielding high-contrast phase images for the blood cells. A modified YOLOv5-s neural network was used to perform automatic identification and analysis of blood cells in the phase image of PCI-DPM, revealing the diameter, dry mass, volumetric density, and speed of blood cells precisely. The proposed approach allows quick and accurate assessment of blood samples, providing valuable insights for patients in different pathological states, such as hematological disorders and infections.

Partially coherent broadband 3D optical transfer functions with arbitrary temporal and angular power spectra.

Optical diffraction tomography is a powerful technique to produce 3D volumetric images of biological samples using contrast produced by variations in the index of refraction in an unlabeled specimen. While this is typically performed with coherent illumination from a variety of angles, interest has grown in partially coherent methods due to the simplicity of the illumination and the computation-free axial sectioning provided by the coherence window of the source. However, such methods rely on the symmetry or discretization of a source to facilitate quantitative analysis and are unable to efficiently handle arbitrary illumination that may vary asymmetrically in angle and continuously in the spectrum, such as diffusely scattered or thermal sources. A general broadband theory may expand the scope of illumination methods available for quantitative analysis, as partially coherent sources are commonly available and may benefit from the effects of spatial and temporal incoherence. In this work, we investigate partially coherent tomographic phase microscopy from arbitrary sources regardless of angular distribution and spectrum by unifying the effects of spatial and temporal coherence into a single formulation. This approach further yields a method for efficient computation of the overall systems' optical transfer function, which scales with O(n 3), down from O(mn 4) for existing convolutional methods, where n 3 is the number of spatial voxels in 3D space and m is the number of discrete wavelengths in the illumination spectrum. This work has important implications for enabling partially coherent 3D quantitative phase microscopy and refractive index tomography in virtually any transmission or epi-illumination microscope.

Use of modulation interference microscopy in applied immunology

Objective: to determine the osmotic resistance of red blood cells using modulation interference microscopy technologies in the light microscopy mode of biological objects to identify the dynamics and determine the possibilities for continuing apitherapy in patients with autoimmune diseases. Methods. Methodological approaches to the use of modulation interference microscopy and computed tomography for the tasks of diagnostic medicine and applied immunology are described. The technology of vital computer dynamic phase metering, special methods for sample preparation of cytoobjects, as well as a computer-aided automated analysis of cytological images were used; recognition algorithms, measurement and identification of micro-objects; methods of statistical data processing. Results. Using the domestic innovative laser microscope MIM340, the osmotic resistance of erythrocytes was estimated using the method of modulation interference microscopy to identify the dynamics and determine the possibilities for continuing apitherapy in patients with rheumatoid arthritis and multiple sclerosis. Using computer methods of cytodiagnostics, new aspects of the functional morphology of living cells were revealed, clinical and morphological parallels were established. It was possible to evaluate the diagnostic and prognostic value of vital cell morphometry in various pathological processes and assess the effectiveness of therapeutic measures. A data bank of graphic images of red blood cells and blood lymphocytes of patients with diseases of the immune system has been created. Findings. The study of the structural features and functional usefulness of circulating blood cells is of great importance in addressing the pathogenesis, diagnosis, assessment of the severity of various pathological conditions and the effectiveness of the therapy. We believe that the study of living cytoobjects using the new method of coherent phase microscopy will provide the most objective data and increase the information content of the analysis, which is undoubtedly an urgent and promising task. The immediate plans include the refinement of mathematical, algorithmic, and software to support decision-making in computer-aided analysis of images of the epidermis and the surface of the dermis in neoplastic processes - malignant skin diseases. It is also necessary to create algorithmic and software tools for computerizing studies of cell models for quantitative and qualitative assessment of the selective accumulation of xenobiotics by laser microscopy.

Mechanobiological study of the dynamics and morphology of cell structures by laser microscopy and applications in oncology

The research is devoted to experimental and theoretical substantiation of the use of mechanobiological characteristics of cells as phenotypic markers of oncological pathologies. In the development of approaches related to the study of the mechanical state of cells in the «normal» and in the evolution of tumors, a comparative analysis has been carried out of the nonlinear dynamics of cell structures on the basis of the original data on the dynamics of «phase thicknesses» of cells obtained by coherent phase microscopy. The results of experimental studies have been generalized on the basis of epigenetic models reflecting mechanobiological features of cell and tissue structures in the development of cancer.

DNA Breathers and Cell Dynamics

Molecular-morphological signs of oncogenesis can be linked to multiscale collective effects in molecular and cell ensembles. It was shown that nonlinear behavior of biological systems can be associated with the generation of characteristic collective modes representing the open states in molecular and cell organization as the mechanism of the coherent expression dynamics. The mechanical DNA model is developed to study the nonlinear dynamics of the helicoidal geometry DNA molecule. To construct the model of DNA the Peyrard–Bishop–Barbi approach has been applied. The analytical small localized solutions as the discrete breather and the antikink have been obtained by multiple scale expansion method for multicomponent lattices. The set of collective open states (breathers) in the molecular ensembles provides the collective expression dynamics to attract cells toward a few preferred global states. This result allows the formulation of the experimental strategy to analyze the qualitative changes in cell dynamics induced by mentioned collective modes. The biomechanical changes have been shown experimentally using the original data of Coherent Phase Microscopy analyzing the time series of phase thickness fluctuations. Study of the mechanical aspects of the behavior of single cells is a prerequisite for the understanding of cell functions in the case of qualitative changes in diseases affecting the properties of cells and tissues morphology to develop diagnostic and treatment design methodology.

Evaluation of the morphofunctional state of peripheral blood platelets in sepsis

Background: Sepsis is a severe life-threatening condition characterized by combined dysfunction of the inner organs and systems and by dysregulation of the platelet and coagulation compartments of hemostasis, in particular. Aim: To assess the character of abnormalities in morphofunctional state of platelets in the pathophysiology of sepsis and the multiple organ failure syndrome by means of coherent interference microscopy. Materials and methods: This single center, prospective, observational study included 78 hospitalized patients with abdominal sepsis; in 40 of them, the syndrome of multiple organ failure was diagnosed. The causes of sepsis were peritonitis of various etiologies in 55 patients, acute necrotic pancreatitis with suppurative and septic complications in 23. The control group included 25 physically healthy subjects. Coherent interference microscopy was used for vital assessment of the platelet morphofunctional state, with simultaneous analysis of routine coagulogram and platelet aggregation parameters. Results: The group of patients with sepsis (n = 38) had higher fbrinogen levels, compared to that in the control (4.5 ± 1 g/L, p = 0.0001), with mild thrombocytopenia (238 ± 12 × 109/L, p = 0.0001). Platelet aggregation was also increased (48 ± 3%, p = 0.0001). Assessment of circulating platelets by coherent interference microscopy showed a progressive increase of the mean populational morphodensitometrical parameters, such as the diameter (p = 0.0002), perimeter (p < 0.0001), and area (p = 0.0002), as well as a decrease in mean values of the phase height (p = 0.0002) and volume (p = 0.0002). There was an increase in the proportion of activated cells to 41% (vs. 33% in healthy control) and the proportion of degenerative cells up to 8%. The patients with severe sepsis complicated by multiple organ failure (n = 40), compared to the control ones, demonstrated exhausted of compensatory mechanisms with the development of consumption coagulopathy: prolonged activated partial thromboplastin time (57 ± 2 s, p = 0.0001), increased prothrombin index (117 ± 4, p = 0.0025), decreased fbrinogen level (1.6 ± 2 g/L, p = 0.0001) and platelet counts (110 ± 9 × 109/L, p = 0.0001), and their lower aggregation (24 ± 3%, p = 0.0001). According to the results of the coherent phase microscopy, the types of the morphodensitometrical abnormalities were similar to that in the main group, albeit they were more signifcant. The proportion of the activated cells was 43%, while that of the degenerated and functionally incompetent platelets, 15%. There were signifcant correlations between the morphodensitometrical parameters of the platelets and basic parameters of their aggregation induced by adenosine diphosphate and collagen. Conclusion: Sepsis is associated with advanced abnormalities in the morphofunctional state of peripheral blood platelets progressing concomitantly to the deterioration of the patient's state. Coherent interference microscopy allows for a real-time assessment of the morphological particulars and functioning of circulating thrombocytes. Further studies are required to validate the results of this method.

Dynamic changes in the cell membrane on three dimensional low coherent quantitative phase microscopy (3D LC-QPM) after treatment with the near infrared photoimmunotherapy.

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer therapy that relies on the binding of a near-infrared antibody photoabsorber conjugate (APC) to a cancer cell. Subsequent exposure to NIR light selectively induces rapid necrotic cell death on target-expressing cells with minimal off-target effects. When treated with NIR-PIT, targeted cells become swollen, develop blebs and burst within minutes of light exposure. Detailed spatial and temporal morphological changes of the cellular membrane of targeted cells treated with NIR-PIT have not been fully explored with state-of-the-art microscopic methods. In this study, we investigated the morphologic and kinetic effects of PIT on two types of cells, a spindle-shaped 3T3/Her cell and a spheric-shaped MDA-MB468 cell, after NIR-PIT using three-dimensional low-coherent quantitative phase microscopy (3D LC-QPM). Adhesive cells treated with NIR-PIT demonstrated region-specific cell membrane rupture occurring first on the distal free edge of the cell near the site of adhesion, in a process that was independent of cell shape. The results show that the peripheral portions of the cell membrane near the site of adhesion are particularly vulnerable to the effects of NIR-PIT, likely because these sites exhibit higher baseline surface tension.

A technique for the assessment of the cytotoxic capacity of CD8+ lymphocytes based on phase images

A novel technique for the assessment of cytotoxic capacity, which is a characteristic of the state of immune-system cells, has been proposed. Using coherent phase microscopy, perforin granules have been detected and described in the cytoplasm of live CD8+ lymphocytes. According to the analysis of phase images, the parameters of perforin granules in the cell were quantitatively assessed. Their average values were calculated as follows: the projected area on the image plane, 15 ± 7 μm2; phase volume, 0.94 ± 0.24 μm3; and the dry weight of the granules, 0.52 ± 0.11 pg. Taking the advantage of this technique, it appears possible to determine cytotoxic capacity using a small number of live cells without employing fluorescent-labeled probes.

Quantitative phase imaging of living cells: application of the phase volume and area functions to the analysis of “nucleolar stress”

We applied coherent phase microscopy to develop a method of quantitative evaluation of functional state of eukaryotic cells using the coordinates of characteristic points (CP) in the functions of the phase volume W and area S. In a fragment of a single cell image (HCT116 human colon carcinoma cell line) with detectable nucleolus, the values of the phase thickness, area, and volume were calculated. These values dramatically changed within the initial minutes of cell exposure to the transcriptional inhibitor actinomycin D. The positions of CP in the graphs of S and W functions allowed for monitoring the time-dependent decrease of nucleolar contrast, a major optical hallmark of "nucleolar stress." Given that the area and volume functions reflect optical heterogeneity of the cell and are independent of its optical model, these functions can be applicable as general mathematical tools for the analysis of cell morphology and physiology.

Dissecting eukaryotic cells by coherent phase microscopy: quantitative analysis of quiescent and activated T lymphocytes.

We present a concept for quantitative characterization of a functional state of an individual eukaryotic cell based on interference imaging. The informative parameters of the phase images of quiescent and mitogen-activated T lymphocytes included the phase thickness, phase volume, the area, and the size of organelles. These parameters were obtained without a special hypothesis about cell structure. Combinations of these parameters generated a "phase portrait" of the cell. A simplified spherical multilayer optic model of a T lymphocyte was used to calculate the refractivity profile, to identify structural elements of the image with the organelles, and to interpret the parameters of the phase portrait. The values of phase image parameters underwent characteristic changes in the course of mitogenic stimulation of T cells; thereby, the functional state of individual cells can be described using these parameters. Because the values of the components of the phase portrait are measured in absolute units, it is possible to compare the parameters of images obtained with different interference microscopes. Thus, the analysis of phase portraits provides a new and perspective approach for quantitative, real-time analysis of subcellular structure and physiologic state of an individual cell.

Interference Microscopy in Cell Biophysics. 2. Visualization of Individual Cells and Energy-Transducing Organelles

The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this article, we consider the applications of the CPM method to imaging different cells and energy-transducing intracellular organelles (mitochondria and chloroplasts). Experimental data presented below demonstrate that the optical path length difference of the object, which is the basic optical parameter measured by the CPM method, can serve as an indicator of metabolic states of different biological objects at cellular and subcellular levels of structural organization.

Interference Microscopy in Cell Biophysics. 1. Principles and Methodological Aspects of Coherent Phase Microscopy

The purpose of our tandem publications is to review the applications of the coherent phase microscopy to cell biophysics. In this article, we briefly consider the fundamentals and methodological aspects of the coherent phase microscopy (CPM). One of important advantages of this method is a high sensitivity of CPM images to changes in physical-chemical properties of biological samples. The optical path length difference (OPLD), measured with CPM instruments in different domains of an object, serves as the informative optical parameter used for visualization of individual cells or intracellular organelles. Metabolically dependent variations of CPM images reflect changes in the functional state of biological objects. In the next article, we review results of CPM studies of different cells and intracellular energy-transducing organelles.

Quantitative real-time analysis of nucleolar stress by coherent phase microscopy

We develop a method of coherent phase microscopy (CPM) for direct visualization of nonfixed, nonstained mammalian cells (both cultured cells and freshly isolated tumor biopsies) followed by computer-assisted data analysis. The major purpose of CPM is to evaluate the refractive properties of optically dense intracellular structures such as the nucleus and the nucleoli. In particular, we focus on quantitative real-time analysis of the nucleolar dynamics using phase thickness as an equivalent of optical path difference for optically nonhomogenous biological objects. Pharmacological inhibition of gene transcription leads to a dramatic decrease of the phase thickness of the nucleoli within the initial minutes of cell exposure. Furthermore, the acute depletion of intracellular ATP pool, depolymerization of microtubules and inhibition of DNA replication resulted in a rapid decrease of the nucleolar phase thickness. These optical effects were paralleled by segregation of nucleolar components as documented by electron microscopy. Thus, CPM detects early changes of nucleolar dynamics, in particular, the nucleolar segregation as part of general cellular response to cytotoxic stress, regardless of whether the nucleolus is or is not the primary target of the toxin. CPM is applicable for monitoring and quantitative analysis of the "nucleolar stress" in living mammalian cells.

Reduction of phase thickness is a characteristic reaction of the nucleoli to toxicity as shown by coherent phase microscopy

Optical parameters of human cell nucleoli (HCT116 colorectal cancer cells) in depolymerization of microtubules and depletion of intracellular ATP pool were studied by coherent phase microscopy. These influences were associated with a rapid (recorded within the first minutes) reduction of the phase thickness of the nucleoli. These changes are similar to the nucleolar response to direct inhibitors of transcription. Hence, quantitative parameters of coherent phase microscopy describe common reaction of the nucleolus to stress; reduction of optical thickness of the nucleolus is a component of this reaction.

Study of optical parameters of the nucleoli under the effect of transcription inhibitors by coherent phase microscopy

The use of coherent phase microscopy for online quantitative registration of nucleolar reaction to transcription inhibition is validated. Reduction of phase thickness of the nucleoli was detected during the first minutes of the experiment; 30 min after addition of the drug rarefaction zones predominated and areas of condensation were seen. These changes reflect the dynamics of disorders in the nucleolar ultrastructure during transcription inhibition.

Coherent Phase Microscopy, a Novel Approach to Study the Physiological State of the Nucleolus

Coherent Phase Microscopy, a Novel Approach to Study the Physiological State of the Nucleolus

Coherent phase microscopy in cell biology: visualization of metabolic states

Visualization of functional properties of individual cells and intracellular organelles still remains an experimental challenge in cell biology. The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this work, we report results of statistical analysis of CPM images of cyanobacterial cells ( Synechocystis sp. PCC 6803) and spores ( Bacillus licheniformis). It has been shown that CPM images of cyanobacterial cells and spores are sensitive to variations of their metabolic states. We found a correlation between one of optical parameters of the CPM image (‘phase thicknesses’ Δ h) and cell energization. It was demonstrated that the phase thickness Δ h decreased after cell treatment with the uncoupler CCCP or inhibitors of electron transport (KCN or DCMU). Statistical analysis of distributions of parameter Δ h and cell diameter d demonstrated that a decrease in the phase thickness Δ h could not be attributed entirely to a decrease in geometrical sizes of cells. This finding demonstrates that the CPM technique may be a convenient tool for fast and non-invasive diagnosis of metabolic states of individual cells and intracellular organelles.

Mitochondria optical parameters are dependent on their energy state: a new electrooptical effect?

The membrane potential of mitochondria determines, to a large extent, their functional state and the response to the changing conditions of the environment. A correlation was found between the changes in the optical characteristics of isolated mitochondria and the composition of the incubation medium, which determines the potential of the inner mitochondrial membrane. The measurements performed by the method of coherent phase microscopy made it possible to establish a linear dependence of the phase height on the value of the membrane potential and to calculate the electrooptical constant, K approximately equal to (3-4) x 10(-7) m/V.

Coherent phase microscopy of intracellular processes

Information encoded in DNA implicitly determines the sequence of processes at different organizational levels of living matter. Optical microscopy is a major source of knowledge about processes in living cells. Further progress in biology to a large extent depends on the perfection of microscopic methods based on the latest advances in laser and computer technologies. This review outlines modern functional imagingtechniques for living cell microscopy with particular emphasis on dynamic coherent phase microscopy based on recording local fluctuations of refractive index. The measurement results produced with a laser phase microscope are reported.

Kogerentnaya fazovaya mikroskopiya vnutrikletochnykh protsessov

Kogerentnaya fazovaya mikroskopiya vnutrikletochnykh protsessov