scholarly journals Correlative all-optical quantification of mass density and mechanics of sub-cellular compartments with fluorescence specificity

eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Raimund Schlüßler ◽  
Kyoohyun Kim ◽  
Martin Nötzel ◽  
Anna Taubenberger ◽  
Shada Abuhattum ◽  
...  
Keyword(s):  
Mass Density ◽  
Optical Diffraction ◽  
Physical Parameters ◽  
Implicit Assumption ◽  
Optical Diffraction Tomography ◽  
Quantitative Measurements ◽  
All Optical ◽  
Novel Method ◽  
Cellular Compartments ◽  
Longitudinal Modulus

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples - so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fluorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample - a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

scholarly journals Combined fluorescence, optical diffraction tomography and Brillouin microscopy

2020 ◽  
Author(s):  
Raimund Schlüßler ◽  
Kyoohyun Kim ◽  
Martin Nötzel ◽  
Anna Taubenberger ◽  
Shada Abuhattum ◽  
...  
Keyword(s):  
Optical Diffraction ◽  
Physical Parameters ◽  
Diffraction Tomography ◽  
Implicit Assumption ◽  
Optical Diffraction Tomography ◽  
Quantitative Measurements ◽  
Absolute Density ◽  
Molecular Specificity ◽  
Novel Method ◽  
Longitudinal Modulus

ABSTRACTQuantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples — so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with molecular specificity. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the cell nucleus, we find that it has lower density but higher longitudinal modulus. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample — a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

scholarly journals Intracellular mass density increase is accompanying but not sufficient for stiffening and growth arrest of yeast cells

2018 ◽  
Author(s):  
Shada Abuhattum ◽  
Kyoohyun Kim ◽  
Titus M. Franzmann ◽  
Anne Eßlinger ◽  
Daniel Midtvedt ◽  
...  
Keyword(s):  
Mass Density ◽  
Physical And Mechanical Properties ◽  
Optical Diffraction ◽  
Yeast Cells ◽  
Cellular Mechanisms ◽  
Optical Diffraction Tomography ◽  
Long Time ◽  
Metabolic Arrest ◽  
Macromolecular Architecture ◽  
Physical And Chemical

AbstractMany organisms, including yeast cells, bacteria, nematodes and tardigrades, endure harsh environmental conditions, such as nutrient scarcity, or lack of water and energy for a remarkably long time. The rescue programs that these organisms launch upon encountering these adverse conditions include reprogramming their metabolism in order to enter a quiescent or dormant state in a controlled fashion. Reprogramming coincides with changes in the macromolecular architecture and changes in the physical and mechanical properties of the cells. However, the cellular mechanisms underlying the physical-mechanical changes remain enigmatic. Here, we induce metabolic arrest of yeast cells by lowering their intracellular pH. We then determine the differences in the intracellular mass density and stiffness of active and metabolically arrested cells using optical diffraction tomography and atomic force microscopy. We show that an increased intracellular mass density is associated with an increase in stiffness when the growth of yeast is arrested. However, increasing the intracellular mass density alone is not sufficient for maintenance of the growth-arrested state in yeast cells. Our data suggest that the cytoplasm of metabolically arrested yeast displays characteristics of solid. Our findings constitute a bridge between the mechanical behavior of the cytoplasm and the physical and chemical mechanisms of metabolically arrested cells with the ultimate aim of understanding dormant organisms.

scholarly journals Physicochemical Properties of Nucleoli in Live Cells Analyzed by Label-Free Optical Diffraction Tomography

Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 699 ◽  
Author(s):  
Kim ◽  
Lee ◽  
Fujii ◽  
Kim ◽  
Pack
Keyword(s):  
Physicochemical Properties ◽  
Nuclear Bodies ◽  
Laser Scanning Microscopy ◽  
Atp Depletion ◽  
Optical Diffraction ◽  
Physical Parameters ◽  
Diffraction Tomography ◽  
Label Free ◽  
Molecular Density ◽  
Optical Diffraction Tomography

The cell nucleus is three-dimensionally and dynamically organized by nuclear components with high molecular density, such as chromatin and nuclear bodies. The structure and functions of these components are represented by the diffusion and interaction of related factors. Recent studies suggest that the nucleolus can be assessed using various protein probes, as the probes are highly mobile in this organelle, although it is known that they have a densely packed structure. However, physicochemical properties of the nucleolus itself, such as molecular density and volume when cellular conditions are changed, are not yet fully understood. In this study, physical parameters such as the refractive index (RI) and volume of the nucleoli in addition to the diffusion coefficient (D) of fluorescent probe protein inside the nucleolus are quantified and compared by combining label-free optical diffraction tomography (ODT) with confocal laser scanning microscopy (CLSM)-based fluorescence correlation spectroscopy (FCS). 3D evaluation of RI values and corresponding RI images of nucleoli in live HeLa cells successfully demonstrated varying various physiological conditions. Our complimentary method suggests that physical property of the nucleolus in live cell is sensitive to ATP depletion and transcriptional inhibition, while it is insensitive to hyper osmotic pressure when compared with the cytoplasm and nucleoplasm. The result demonstrates that the nucleolus has unique physicochemical properties when compared with other cellular components.

scholarly journals Optical quantification of intracellular mass density and cell mechanics in 3D mechanical confinement

Soft Matter ◽  
2021 ◽  
Author(s):  
Sadra Bakhshandeh ◽  
Hubert M. Taïeb ◽  
Raimund Schlüßler ◽  
Kyoohyun Kim ◽  
Timon Beck ◽  
...  
Keyword(s):  
Cell Mechanics ◽  
Mass Density ◽  
Optical Diffraction ◽  
Diffraction Tomography ◽  
Brillouin Spectroscopy ◽  
Optical Diffraction Tomography

Optical quantification of intracellular mass density using optical diffraction tomography (ODT) and cell mechanics using Brillouin spectroscopy under 3D mechanical confinement.

scholarly journals Quantitative imaging of Caenorhabditis elegans dauer larvae during cryptobiotic transition using optical diffraction tomography

2021 ◽  
Author(s):  
Kyoohyun Kim ◽  
Vamshidhar R. Gade ◽  
Teymuras V. Kurzchalia ◽  
Jochen Guck
Keyword(s):  
Caenorhabditis Elegans ◽  
Material Properties ◽  
Structural Changes ◽  
Mass Density ◽  
Quantitative Imaging ◽  
Optical Diffraction ◽  
Physical Analysis ◽  
Diffraction Tomography ◽  
Optical Diffraction Tomography ◽  
Dauer Larvae

Upon starvation or overcrowding, the nematode Caenorhabditis elegans enters diapause by forming a dauer larva. This larva can further transit into an anhydrobiotic state and survive harsh desiccation. We previously identified the genetic and biochemical pathways essential for survival — but without an accompanying physical model, the mechanistic understanding of this amazing phenomenon will remain inadequate. Neither microscopic investigation of structural changes upon entry into anhydrobiosis nor the most basic quantitative characterization of material properties of living desiccated larvae, however, have been feasible, due to lack of appropriate techniques. Here, we employed optical diffraction tomography (ODT) to quantitatively assess the internal mass density distribution of living larvae in the reproductive and diapause stages. More importantly, ODT allowed for the first time physical analysis of desiccated dauer larvae: their mass density was significantly increased in the anhydrobiotic state. We also applied ODT on different mutants that are sensitive to desiccation. Remarkably, one of them displayed structural abnormalities in the anhydrobiotic stage that could not be observed either by conventional light or electron microscopy. Our advance opens a door to quantitatively assessing fine differences in material properties and structure necessary to fully understanding an organism on the verge of life and death.

Total-shear grating based optical diffraction tomography

2021 ◽  
Author(s):  
Piotr Zdańkowski ◽  
Julianna Winnik ◽  
Paweł Gocłowski ◽  
Maciej Trusiak
Keyword(s):  
Optical Diffraction ◽  
Diffraction Tomography ◽  
Optical Diffraction Tomography ◽  
Total Shear

scholarly journals Feature of Echo Envelope Fluctuation and Its Application in the Discrimination of Underwater Real Echo and Synthetic Echo

2018 ◽  
Vol 8 (8) ◽  
pp. 1329
Author(s):  
Yunfei Chen ◽  
Sheng Li ◽  
Bing Jia ◽  
Guijuan Li ◽  
Zhenshan Wang
Keyword(s):  
Physical Parameters ◽  
Scaled Model ◽  
Theoretical Simulation ◽  
Active Sonar ◽  
Fluctuation Intensity ◽  
Characterization Model ◽  
Novel Method ◽  
Underwater Target ◽  
Echo Features ◽  
Synthetic Target

Discriminating a real underwater target echo from a synthetic echo is a key challenge to identifying an underwater target. The structure of an echo envelope contains information which closely relates to the physical parameters of the underwater target, and the characterization and extraction of echo features are problematic issues for active sonar target classification. In this study, firstly, the high-frequency envelope fluctuation of a complex underwater target echo was analyzed, the envelope fluctuation was characterized by the envelope fluctuation intensity, and a characterization model was established. The features of a benchmark model echo were extracted and analyzed by theoretical simulation and sea testing of a scaled model, and the result shows that the envelope fluctuation intensity varies with carrier frequency and azimuth of incident signal, but the echo envelope fluctuation of the synthetic target echo does not present these features. Then, based on the characteristics of echo envelope fluctuation, a novel method was developed for active sonar discrimination of a real underwater target echo from the synthetic echo. Through a sea experiment, the real target echo and synthetic echo were classified by their different echo envelope fluctuations, and the feasibility of the method was verified.

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