Microscopic images of a living cell are the main source of information on its functional state. Modern interference microscopy techniques allow the numerical parameters of cell images to be obtained with an accuracy not available with other methods. Quantitative analysis of phase images of T lymphocytes (TCs) in different functional states demonstrated that variations of the properties of intracellular water should be taken into account. This conclusion agrees with the current view that the physical parameters of water, including the refractive index (RI) of a water layer, depend on the hydrophilicity and other characteristics of the adjacent surface. Application of this concept to phase images of TCs showed that the contribution of the fourth phase of water (4-water) or the structured water component, which has an increased RI, should be considered. The proportion of 4-water depends on the functional state of the cell determined by the culture medium composition. Normally, the proportion of 4-water in organelles is as high as 30%; it is considerably lower in organelles of cells with inhibited metabolism.
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.
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.
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.
Investigation of intracellular dynamics of Allium cepa inner epidermal cells are described. The applicability of the method for quantitative estimation of spatio-temporal phase fluctuations and the effect due to external factors is discussed. The analysis of time-sampled series allows one to detect the regions of various motility in cytoplasm. The intense Fourier-spectra harmonics in 0.2 - 8 Hz interval were observed inside a cell wall and cytoplasm. Regularly spaced 2- to 4-s long batches of 100-ms pulses at cell-wall sites are recorded. The phase-fluctuation intensity decreased and the frequencies of certain harmonics were shifted with lowering temperature. The advantages and specific features of the method are discussed.
Investigation of features and functions of some small biological objects (smaller than 500 nm), in particular, viruses, with conventional optical microscopy is practically impossible. Usually their images are obtained with methods of scanning electron microscopy (SEM), which precludes work with samples in a native state. We obtained images of different viruses including influenza A virus in native state with computer-aided phase microscope (CPM) Airyscan, in which an He-Ne laser is used as a light source. The main purpose of this work was to show the possibility to obtain adequate structure images of influenza viruses with diameter about 100 nm in conditions quite close to native and to investigate different stages of influenza virus budding. We suppose that these results may be considered as a basis for further studies of cell-virus interaction.
Traditionally viruses are studied with scanning electron microscopy (SEM) after complicated procedure of sample preparation without the possibility to study it under natural conditions. We obtained images of viruses (Vaccinia virus, Rotavirus) and rickettsias (Rickettsia provazekii, Coxiella burnetti) in native state with computer-aided phase microscope airyscan -- the interference microscope of Linnik layout with phase modulation of the reference wave with dissector image tube as coordinate-sensitive photodetector and computer processing of phase image. A light source was the He-Ne laser. The main result is coincidence of dimensions and shape of phase images with available information concerning their morphology obtained with SEM and other methods. The fine structure of surface and nuclei is observed. This method may be applied for virus recognition and express identification, investigation of virus structure and the analysis of cell-virus interaction.
Parameters of intrinsic cell motility is one of the cell activity characteristics which can be measured in real-time. For evaluation of certain organelles velocity we propose to use high sensitivity of computer-aided phase microscope airyscan to local phase changes connected with refractive index. This method is based on periodical scanning of cell profile in direction perpendicular to organelles movement. Analysis of the obtained 2-dimensional time-coordinate matrix allows us to define organelle velocity in quasi-real time and areas of cell activity. The experiments with onion cells confirm the method applicability for cell activity investigation.
Living cells investigation and cell response to external factors are of great interest for practical medicine and biology. The main advantages of computer aided phase microscope (CPM) Cytoscan which permits us to observe the cell surface and internal structure consists in superresolution and the possibility of the dynamic processes registering. We attempt to characterize some aspects of the morphofunctional status of human lymphocytes determining the dynamics of the selected points in normal or pathological cells. To evaluate the lymphocyte homeostasis donors and persons of autoimmune diseases were analyzed and the changes of optical and geometrical cell parameters registered by CPM Cytoscan. The dynamic process registration allowed us to perform the real-time quantitative analysis of the living lymphocyte activity in norm and pathology.
In recent years we have been working out a new method of treatment of steroid-resistant asthma -- extracorporeal He-Ne-laser blood irradiation. The procedures gave good clinical effect and allowed reduction of steroid dose in a majority of patients. The monocytes were investigated by luminol-dependent chemiluminescence and the method of living cell microcopy. Extracorporeal He-Ne laser blood irradiation normalized both monocytes chemiluminescence and cell oscillation in asthmatic patients significantly earlier than in cases of ordinary treatment, although the mechanisms of action of He-Ne-laser irradiation upon blood steroid-resistant asthmatics were not fully determined.
The main advantages of the computer-aided phase microscope (CPM) Airyscan which permits the user to observe the cell surface and internal structure consists in superresolution and the possibility of the dynamic processes registering. Immunocompetent cells in process of activation with different agents were studied. Several criterions for evaluation of the functional status of immunocompetent cells based on the shape changes were suggested on the basis of real-time immunology reaction monitoring. The perspectives of the proposed method for immunology testing are discussed.
Some living biological objects were investigated with `AiryScan'--computer- aided Phase Microscope with spatial resolution up to 0.05 micrometers and time resolution 1 ms. Influence of ATP on the frequency and intensity of Infuzoria Paramecium Caudatum and Unio pictorium cilia beat were observed.
The main goal of this paper is to present a new approach based on computer-aided phase microscope Airyscan for submicron biological structures dynamic investigation. In our experiments micelial cell walls were used as the object with well known submicron structure. Two types of cilia beating specimens and cytoplasm movement inside the onion cell were chosen for dynamic processes registering. This paper peruses mainly methodological goals for demonstration of the new method possibilities.
A factor-of-ten over classical diffraction limit enhancement was achieved with a computer-aided phase microscope 'Airyscan'. It also permits dynamic processes registration at any selected point of the image. The theoretical backgrounds of the new approach to the problem of super-resolution are discussed. 'Airyscan' may be useful in a wide range of scientific and industrial applications. Special attention is paid to biophysics and medicine.
The question of informative character of special image points--wavefront dislocations, is discussed. They were observed in real phase images of microstructures, which had comparable and smaller linear dimensions than diffraction limit. According to the existing classification, phase patterns of the main types of wavefront dislocations were identified. Their influence on the phase imaging formation is investigated. The case of induced phase contrast due to considerable decrease, but not quite vanishing of the complex amplitude, is taken into consideration. The contour making and contrasting tendency in the presence of the combination of dislocations, is demonstrated. Observation of anomal small-size details in phase and polarization images and the possibility of their observation in general `functional' image, is discussed. The experimental pictures were obtained with the help of unique phase microscope `AiryScan' technique.
Some living biological objects were investigated with `AiryScan'-computer-aided Phase Microscope with spatial resolution up to 0.05 micrometers and time resolution 1 ms. Influence of ATP on the frequency and intensity of Infusoria Paramecium Caudatum and Unio pictorium cilia beat were observed.
A factor-of-ten over classical diffraction limit enhancement was achieved with a Computer- aided Phase Microscope `Airyscan.' It also permits dynamic processes registration at any selected point of the image. The theoretical backgrounds of the new approach to the problem of super-resolution are discussed. `Airyscan' may be useful in a wide range of scientific and industrial applications. Special attention is paid to biophysics and medicine.
KEYWORDS: Spatial resolution, Image resolution, 3D image processing, Microscopes, Data processing, Medicine, Biology, Microorganisms, Super resolution, Software development
Some biological structures like chromosomes, mitochondria, cell walls, and living cells were observed with the computer-aided phase microscope (CPM) `Airyscan' having spatial resolution 5 - 20 nm. We succeeded in obtaining new information about dynamic processes in living cells and micro-organisms by a new registering method. Some of the possible applications in the areas of medicine and biology are discussed.
KEYWORDS: Spatial resolution, Very large scale integration, Microscopes, Resolution enhancement technologies, Phase measurement, 3D metrology, Radio optics, Diffraction, Semiconductors, Super resolution
Phase object pattern analysis has shown the possibility of considerably increasing microscope spatial resolution. Experiments have revealed more than ten times resolution enhancement. Some phase object images inside an Airy disk are presented.
Phase object pattern analysis has shown the possibility of increasing considerable microscope spatial resolution. Experiment has revealed resolution enhancement of greater than a factor of ten. Some phase object images inside an Airy disk are presented.
The accessibility of the optical information inside the Airy disk is experimentally proved. A 3- D image of a 0.1 micrometers slit was obtained with an Airyscan microscope at (lambda) equals 0.63 micrometers .
The possibility of phase objects superresoluton imaging is theoretically justifieth The measurements with CPM " AIRYSCAN" showed the reality of O structures observations when the Airy disc di ameter i s 0 86 j. . m SUMMARY It has been known that the amount of information contained in the image of any object is mostly determined by the number of points measured i ndependentl y or by spati al resol uti on of the system. From the classic theory of the optical systems it follows that for noncoherent sources the -spatial resolution is limited by the aperture dd 6LX/N. A. ( Rayleigh criterion where X is wave length NA numerical aperture. ) The use of this criterion is equivalent tO the statement that any object inside the Airy disc of radius d that is the difraction image of a point is practical ly unresolved. However at the coherent illumination the intensity distribution in the image plane depends also upon the phase iq (r) of the wave scattered by the object and this is the basis of the Zernike method of phasecontrast microscopy differential interference contrast (DIC) and computer phase microscopy ( CPM ). In theoretical foundation of these methods there was no doubt in the correctness of Rayleigh criterion since the phase information is derived out of intensity distribution and as we know there were no experiments that disproved this
KEYWORDS: Microscopes, Phase measurement, Wavefronts, Point spread functions, Electronics, Very large scale integration, Inspection, Objectives, Sensors, Scientific research
Previous measurements with computer-aided phase microscope
using Linnik scheme with a dissector as coordinate sensitive
detector showed an opportunity to obtain superdiffractional
resolution d
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