The lateral shearing phase shifting interference microscopy with low coherent illumination for tomography of living cells has been proposed. To create projections of a sample at different angles the angular scanning of the probe beam was used. The experimental results of erythrocyte tomogram reconstruction are presented.
A refractometric complex based on dynamic laser goniometer including in its structure a modernized State primary standard of refractive index unit is considered.
The authors have proposed and tested the new type of the microscope with superimposed interference and fluorescence images of the living cell. The fluorescence image gives the information about the distribution of drugs in different parts of the cell. Interference microscopy technique allows implementing the quantitative analysis of a living transparent cell, for example, to measure dry cell weight and cell density spatial distribution. Interference microscopy makes possibility the observation cell vital activity at dynamics. Computer-aided processing of interferograms using phase shift technique, allows carrying out continuous (over 12 hours and more) monitoring of living cells quantitative parameters with time interval between measurements less than one minute. RMS error for dry weight of non-living fixed test object using this microscope is about 1%.
Interference microscopy technique allows implementing the quantitative analysis of a living cell, for example, to measure dry cell weight and cell density spatial distribution. The authors have created the microscope for quantitative analysis of living cells and observation of the dynamics of cell vital activity. Computer-aided processing of interferograms using phase shift technique, allows carrying out continuous (over 12 hours and more) monitoring of quantitative parameters of living cells with time interval between measurements less than one minute. RMS error for dry weight of non-living fixed test object using this microscope is about 1%. Numerous experiments on living lymphocytes and erythrocytes resulted in detecting the oscillations of dry weight. Spectral analysis of obtained time series has allowed allocating harmonics with the period from 2 hours to 15 minutes. Morphometric measurements of the same cells and cross correlation analysis of these data with oscillations of a dry mass were carried out also.
Many biological objects, including blood cells, are optically transparent. They don't absorb light and only change optical path length by variations of object's thickness or its refractive index variations. The phase shifting interferometry is used for the visualization of the phase information and the obtaining quantitative properties of cells. Cells are fixed by glutaraldehyde and mounted into immersion liquid for influences of refraction reduction. Blood cell's interferogram contains the information about space distribution refractive index. The phase-shift method was used for its reconstruction. This method includes record of four interferograms used different values of phase-shift of the reference wave. Many blood cells' interferograms are obtained (lymphocytes and erythrocytes). The shape, size and density of cells are measurement. We suppose that these date will be ones of the early diagnostically criteria of hematological diseases. Experimental results for interferometric microscopy and tomography of blood cells are presented.
Fluorescence microscopy is a powerful instrument for the study of cells and other biological objects. One of the actual tasks of the modern microscopy is the study of drugs transportation in cells and it's affect on the different cell organelles. The authors have proposed and tested the new type of the microscope with superimposed interference and fluorescence images of the living cell. The optical setup of the fluorescence interference microscope is based on confocal Linnik's type interference microscope. The fluorescence image gives the information about the distribution of drugs in different parts of the cell. The interference image gives the information about the distribution of dry mass or protein in the different parts of the cell.
New method of a tomographic microscopy with spatially incoherent illumination is proposed. Earlier, in our works for reconstruction of a refractive index with spatial distribution of 3D phase samples, a method of tomographic microscopy with the coherent light was used. Main disadvantage of this scheme is use of a coherent light, which brings to appearance the phase noise in projection data. Spatially incoherent tomographic microscope is based on the Linnik phase-shifting interference microscope reflected type. Using interferometry with large source of the monochromatic light allows greatly improve a quality of phase projections by averaging of interference patterns. For tomographic mode of Linnik microscope working and obtaining the oblique illumination of sample method for displacing a light source is used. Observation angle range for 100 X oil-immersion objective, N.A. equals 1.25, is 90 degrees. This method allows realizing a two-dimensional scanning trajectory for sample observation. Particularities of tomography for the phase objects, placed near plane mirror, are considered. So, a viewing angle range can be reduced to 90 degree, and sample is complement by its mirror reflection. The iterative algorithms for limited-angle tomographic reconstruction were used. Experimental results on three-dimensional reconstruction of the single human blood cell (erythrocyte) are presented.
To obtain three-dimensional refractive index or density spatial distribution inside optically transparent living cells, it is necessary to combine computed tomography and interference microscopy. These methods use the oblique illumination and the sample viewing angles scanning for recording a number of the projections. A new application Linnik microscope reflected type for obtaining a high quality phase projections with spatially incoherent laser illumination is described. Phase-shifting technique for automated interferogram analysis is used. Interferometric computed- tomography Linnik laser microscope are fully automated integrated systems that incorporate CCD camera, frame grabber, computed operated PZT mirror as well as intelligible software for interferogram processing, projections preprocessing and tomographic reconstruction. Microscope measurement system and performance characterization are presented. There are some features in angle scanning, projections proceeding, and reconstruction for tomographic investigation of the phase samples that are placed near the mirror surface. For plane mirror the projections viewing angle range may be limited to 90-degree and the investigation object may be represented as a sum of the original object and its mirror reflection. The iterative algorithms for limited-angle tomographic reconstruction were used. Three-dimensional images of living blood cells (lymphocytes) were obtained.
Tomographic measurements of the 3D refractive index spatial distribution within optically transparent phase samples with computerized interferometric microscopes are proposed. Phase shifting interferometric microtomography applications for the 3D image reconstruction of the blood cells are represented. The immersion 100x, N.A. equals 1.25 objective was used to increase the spatial resolution and observation angle range to 90 degree. ART, combined ART and iterative Gerchberg-Papoulis 3D algorithm were used for the tomogram reconstruction. To determine the accuracy and spatial resolution of the blood cells image reconstruction by means of the interferometric microtomographic method the numerical simulations were implemented.
We offer to use the 3D surface profile real-time measurement using phase-shifted interference fringe projection technique for the cranioficial identification. Our system realizes the profile measurement by projecting interference fringe pattern on the object surface and by observing the deformed fringe pattern at the direction different from the projection. Fringes are formed by a Michelson interferometer with one mirror mounted on a piezoelectric translator. Four steps self- calibration phase-shift method was used.
Many biological objects, include blood cells, are optically transparent. They do not absorb light, and only change optical path length by variations of object's thickness or its refractive index variations. For visualization of phase information and obtaining quantitative properties of cells we use phase-shift interferometry. Cells are fixed by glutaraldehyde and mounted in immersion liquid for reduction the influences of refraction. Characteristic peculiarity of interferometric microscope are two channels of optical scheme: object and reference. Blood cell's interferogram contain the information about space distribution refractive index. For its reconstruction we use phase-shift method. This method includes record of four interferograms used different values of phase-shift of the reference wave. Many blood cells interferogram are obtained. The shape, size and density of cells are measured.
Measurements of refractive index spatial distribution within optically transparent phase 3-D samples with computerized interferometric microscopes is proposed. For the reconstruction of the phase information the phase shifting interferometry is used. Interferometric computed-tomography microscopes are fully automated integrated systems that incorporate CCD camera, frame grabber, computed operated PZT mirror as well as intelligible software for interferogram processing, tomographic reconstruction and refractive index distribution are displayed in 2-D and 3-D, sections, isolines, etc. Experimental results for interferometric microscopy and tomography of blood cells are presented.
The nature of 3D imaging with confocal optical system using principles of tomography and geometrical optics is analyzed. Image intensity in any plane of image space is described by the 2D section of a 3D summary image of the sample is obtained.
Noninvasive methods of obtaining the real-time data of internal structure of light emitting objects from their parts are considered to be rather significant for investigation of various high-speed processes such as flames, exposition, plasma, and jets. The method of dynamic operation (DPO) should be based on the spectral emission tomography. The DPO method essentially consists in treating the substrate surface by a high-enthalpy complex configuration plasma flow. We have constructed a light emission computed tomography system (LECT) and a light emission integrative tomography system (LEIT) for obtaining the real-time data.
This paper deals with the analysis of an algebraic reconstruction technique version for the restoration of refractive index spatial distribution in the objects cross-sections, using the tomographic holographic interferometry data; it also includes the digital simulation results. An example of tomographic reconstruction using the experimentally obtained interferograms is presented.
The paper describes a new approach to studying the spectral-spatial characteristics of the objects, proposed by the authors, and the results of its experimental testing. This approach is based on application of tomographic principles to the spectral investigations of the objects. The results obtained show the great advantages of this method for the spectral analysis of spatially extended objects.
A new technique for fast obtaining the quantitative characteristics of light-emitting object cross-section geometry, using the method of moments, is proposed. Optical calculation of integrated projection data (namely, the moments) considerably reduces the data for digital processing, thus making the real-time tomographic analysis possible. Theoretical aspects of the method along with its optical implementation are discussed.
A one-step method for direct measurement of refractive index field is proposed. The method consists in combination of the optical probing step, i.e. obtaining projection data, with the following step of tomogram reconstruction, which is done by analog processing in the tomographic interferometer. Holographic interferometry technique is used to visualize the optical wave field that carries refractive index tomogram information. The real-time interferometric visualization of refractive index spatial distribution in either longitudinal or cross section of the objects under investigation has become possible.
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