Measurements of submicron structures with the Airyscan laser phase microscope

Tychinskii, V. P.; Kufal, Georgy E.; Vyshenskaya, T. V.; Perevedentseva, E.; Nikandrov, S L

doi:10.1070/qe1997v027n08abeh001023

Cited by 19 publications

(23 citation statements)

References 12 publications

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“…These results are close to the values (〈∆n〉 = 0.01-0.02) obtained for deenergized mitochondria [4,6], chloroplasts [5], and bacteria [3]. According to our assessments, the refraction index of the nucleus was only slightly greater than the index of the cytoplasm (〈∆n〉 = 〈n N 〉 -〈n 0 〉 ≤ 0.01).…”

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confidence: 90%

“…About twenty randomly selected cells were analyzed in each sample in different fields of view. The measurements were performed with the use of an Airyskan coherent phase microscope [2,8] using a helium-neon laser ( λ = 633 nm) as a light source, an LI-620 dissector to record the interference signal, and an electronic unit for computer presentation of phase images. The rate of image input was 1 ms per pixel.…”

mentioning

confidence: 99%

“…Hence, the intracellular structures that have different refraction indices will be present in the static phase images. The local changes in the refraction index may be a result of metabolic processes and can be measured as changes in the phase thickness of intracellular structures [2][3][4][5][6][7][8]. CPM was used to study the metabolic state of single large organelles (isolated mitochondria) [4 − 6], plant cells (bean chloroplasts) [7], cyanobacteria, and spores [3].…”

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

Tychinsky¹,

Kretushev²,

Klemyashov³

et al. 2005

Dokl Biochem Biophys

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The assessment of metabolism of microobjects is a topical problem of biology. Studies of cells and subcellular structures and their responses to extracellular stress stimuli are of fundamental importance. Important practical applications of such studies include the selection of biologically active substances and the diagnostics of metabolic disturbances. The solution of these problems requires methods that allow quantitative analysis of physiological processes in real time using nonfixed single cells, without utilization of fluorescent dyes or other invasive approaches which may introduce experimental artifacts. The heterogeneity of animal cells and microorganisms stimulates the development of novel methods for the analysis of physiological state of single cells and recording of individual response to changed microenvironment [1]. The method of coherent phase microscopy (CPM) developed by our group [2, 3] is based on representation of a real biological object as a spatially heterogeneous distribution of optical refraction index. The phase images obtained by interference techniques represent two-dimensional distributions of optical path difference (or phase thickness). Therefore, the structures with higher optical density are clearly distinguished in such phase images. Hence, the intracellular structures that have different refraction indices will be present in the static phase images. The local changes in the refraction index may be a result of metabolic processes and can be measured as changes in the phase thickness of intracellular structures [2][3][4][5][6][7][8]. CPM was used to study the metabolic state of single large organelles (isolated mitochondria) [4 − 6], plant cells (bean chloroplasts) [7], cyanobacteria, and spores [3]. A decrease in the phase thickness induced by pharmacologic uncoupling of mitochondrial transmembrane potential of protons or inhibition of electron transport (deenergization) was detected [3 − 7]. These studies showed that it is possible to assess the response of living biological objects to changes in homeostasis by CPM.The purposes of this work were (1) to identify the optically dense structures in the whole (unfractionated) cells by the CPM method and (2) to determine optical parameters of the disturbances of the state of nucleoli induced by suppression of gene transcription.The experiments were performed with the cell lines NIH 3T3 (immortalized murine fibroblasts) and HCT116 (transformed human colon epithelium) cultured in Dulbecco's modified Eagle's medium supplemented with 10% bovine serum, 2 mM glutamine, and gentamicin. The cells were maintained at 37°ë in 5% ëé 2 humidified atmosphere. The cells were seeded on cover slips 24 h before experiments; the monolayer density at the day of experiments was 50-75%. The cells were incubated with pharmacological reagents; the cover slips were placed on a polished silicon substrate and analyzed by CPM. Single cells were first studied using the optical channel of CPM; then, their topograms and phase thickness profiles were measured. Abou...

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mentioning

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

Tychinsky¹,

Kretushev²,

Klemyashov³

et al. 2005

Dokl Biochem Biophys

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“…Laser interference microscopy is based on the measurement of the local phase shifts of the laser beam transmitted through an object [27][28][29]. Figure 1 illustrates the principle of the interference microscope.…”

Section: Experimental Techniquementioning

confidence: 99%

Unraveling Cell Processes: Interference Imaging Interwoven with Data Analysis

Brazhe

Pavlov

et al. 2006

J Biol Phys

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The paper presents results on the application of interference microscopy and wavelet-analysis for cell visualization and studies of cell dynamics. We demonstrate that interference imaging of erythrocytes can reveal reorganization of the cytoskeleton and inhomogenity in the distribution of hemoglobin, and that interference imaging of neurons can show intracellular compartmentalization and submembrane structures. We investigate temporal and spatial variations of the refractive index for different cell types: isolated neurons, mast cells and erythrocytes. We show that the refractive dynamical properties differ from cell type to cell type and depend on the cellular compartment. Our results suggest that low frequency variations (0.1-0.6 Hz) result from plasma membrane processes and that higher frequency variations (20)(21)(22)(23)(24)(25)(26) are related to the movement of vesicles. Using double-wavelet analysis, we study the modulation of the 1 Hz rhythm in neurons and reveal its changes under depolarization and hyperpolarization of the plasma membrane. We conclude that interference microscopy combined with wavelet analysis is a useful technique for non-invasive cell studies, cell visualization, and investigation of plasma membrane properties.

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“…Methodological advantage of the method is that determination of phase is not limited by diffraction constrains peculiar to measurements of light intensity distribution. This approach provided the realization of super-resolution of samples [12,[27][28][29][30][31][32][33]. For this trend of interference microscopy, we use a term the ''coherent phase microscopy''.…”

Section: Introductionmentioning

confidence: 99%

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

Tychinsky

Тихонов

2010

Cell Biochem Biophys

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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.

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Measurements of submicron structures with the Airyscan laser phase microscope

Cited by 19 publications

References 12 publications

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

Unraveling Cell Processes: Interference Imaging Interwoven with Data Analysis

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

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