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  1. Article ; Online: Real-time imaging of decompression gas bubble growth in the spinal cord of live rats.

    Alvarado, Roman / Scheven, Ulrich M / Meiners, Jens-Christian

    Magnetic resonance in medicine

    2024  

    Abstract: Purpose: To observe the growth and resolution of decompression gas bubbles in the spinal cord of live rats in real time using MRI.: Methods: We constructed an MRI-compatible pressure chamber system to visualize gas bubble dynamics in deep tissues in ... ...

    Abstract Purpose: To observe the growth and resolution of decompression gas bubbles in the spinal cord of live rats in real time using MRI.
    Methods: We constructed an MRI-compatible pressure chamber system to visualize gas bubble dynamics in deep tissues in real time. The system pressurizes and depressurizes rodents inside an MRI scanner and monitors their respiratory rate, heart rate, and body temperature while providing gaseous anesthesia under pressure during the experiments.
    Results: We observed the formation of decompression gas bubbles in the spinal cord of rats after compression to 7.1 bar absolute and rapid decompression inside the MRI scanner while maintaining continuous gaseous anesthesia and vital monitoring.
    Conclusion: We have shown the direct observation of decompression gas bubble formation in real time by MRI in live, anesthetized rats.
    Language English
    Publishing date 2024-04-23
    Publishing country United States
    Document type Journal Article
    ZDB-ID 605774-3
    ISSN 1522-2594 ; 0740-3194
    ISSN (online) 1522-2594
    ISSN 0740-3194
    DOI 10.1002/mrm.30128
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  2. Article ; Online: Fluorescence Correlation Spectroscopy on Genomic DNA in Living Cells.

    Hodges, Cameron / Meiners, Jens-Christian

    Methods in molecular biology (Clifton, N.J.)

    2018  Volume 1814, Page(s) 415–424

    Abstract: Fluorescence correlation spectroscopy (FCS) is a powerful technique used to measure diffusion, fluctuations, and other transport processes in biomolecular systems. It is, however, prone to artifacts and subject to considerable experimental difficulties ... ...

    Abstract Fluorescence correlation spectroscopy (FCS) is a powerful technique used to measure diffusion, fluctuations, and other transport processes in biomolecular systems. It is, however, prone to artifacts and subject to considerable experimental difficulties when applied to living cells. In this chapter, we provide protocols to conduct quantitative FCS measurements on DNA inside living eukaryotic and prokaryotic cells. We discuss sample preparation, dye selection and characterization, FCS data acquisition, and data analysis, including a method to com pensate for photobleaching to obtain quantitatively meaningful spectra.
    MeSH term(s) Cell Survival ; Cells, Immobilized/cytology ; Coloring Agents/chemistry ; DNA/chemistry ; Data Analysis ; Escherichia coli/cytology ; Genome, Human ; HeLa Cells ; Humans ; Spectrometry, Fluorescence/methods ; Staining and Labeling
    Chemical Substances Coloring Agents ; DNA (9007-49-2)
    Language English
    Publishing date 2018-06-28
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-4939-8591-3_25
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  3. Article ; Online: Fluorescence Correlation Spectroscopy with Photobleaching Correction in Slowly Diffusing Systems.

    Hodges, Cameron / Kafle, Rudra P / Hoff, J Damon / Meiners, Jens-Christian

    Journal of fluorescence

    2018  Volume 28, Issue 2, Page(s) 505–511

    Abstract: Fluorescence correlation spectroscopy (FCS) is a powerful tool to quantitatively study the diffusion of fluorescently labeled molecules. It allows in principle important questions of macromolecular transport and supramolecular aggregation in living cells ...

    Abstract Fluorescence correlation spectroscopy (FCS) is a powerful tool to quantitatively study the diffusion of fluorescently labeled molecules. It allows in principle important questions of macromolecular transport and supramolecular aggregation in living cells to be addressed. However, the crowded environment inside the cells slows diffusion and limits the reservoir of labeled molecules, causing artifacts that arise especially from photobleaching and limit the utility of FCS in these applications. We present a method to compute the time correlation function from weighted photon arrival times, which compensates computationally during the data analysis for the effect of photobleaching. We demonstrate the performance of this method using numerical simulations and experimental data from model solutions. Using this technique, we obtain correlation functions in which the effect of photobleaching has been removed and in turn recover quantitatively accurate mean-square displacements of the fluorophores, especially when deviations from an ideal Gaussian excitation volume are accounted for by using a reference calibration correlation function. This allows quantitative FCS studies of transport processes in challenging environments with substantial photobleaching like in living cells in the future.
    Language English
    Publishing date 2018-03
    Publishing country Netherlands
    Document type Journal Article
    ZDB-ID 2016892-5
    ISSN 1573-4994 ; 1053-0509
    ISSN (online) 1573-4994
    ISSN 1053-0509
    DOI 10.1007/s10895-018-2210-y
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  4. Article: Fast position measurements with scanning line optical tweezers.

    Nambiar, Rajalakshmi / Meiners, Jens-Christian

    Optics letters

    2007  Volume 27, Issue 10, Page(s) 836–838

    Abstract: Scanning line optical tweezers are a powerful tool for the study of colloidal or biomolecular systems in the low-force regime. We present a fast, high-resolution particle position measurement scheme that extends the capabilities of these instruments into ...

    Abstract Scanning line optical tweezers are a powerful tool for the study of colloidal or biomolecular systems in the low-force regime. We present a fast, high-resolution particle position measurement scheme that extends the capabilities of these instruments into the realm of dynamic measurements. The technique is based on synchronous detection of forward-scattered laser light during a line scan. We demonstrate a position resolution of better than 50 nm for bandwidths of as much as 40 kHz for pairs of microspheres trapped in a flat line potential at center-to-center separations of 1.7-6 microm.
    Language English
    Publishing date 2007-03-16
    Publishing country United States
    Document type Journal Article
    ISSN 0146-9592
    ISSN 0146-9592
    DOI 10.1364/ol.27.000836
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  5. Article ; Online: Stretching short sequences of DNA with constant force axial optical tweezers.

    Raghunathan, Krishnan / Milstein, Joshua N / Meiners, Jens-Christian

    Journal of visualized experiments : JoVE

    2011  , Issue 56, Page(s) e3405

    Abstract: Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA, occur on this ... ...

    Abstract Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs. The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension. Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces. We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.
    MeSH term(s) DNA/chemistry ; Optical Tweezers
    Chemical Substances DNA (9007-49-2)
    Language English
    Publishing date 2011-10-13
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. ; Video-Audio Media
    ZDB-ID 2259946-0
    ISSN 1940-087X ; 1940-087X
    ISSN (online) 1940-087X
    ISSN 1940-087X
    DOI 10.3791/3405
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  6. Article: Stretching short sequences of dna with constant force axial optical tweezers

    Raghunathan, Krishnan / Milstein, Joshua N / Meiners, Jens -Christian

    Journal of visualized experiments. 2011 Oct. 13, , no. 56

    2011  

    Abstract: Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2, occur on this ... ...

    Abstract Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs. The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6, (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension. Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces. We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.
    Keywords DNA ; asymmetry ; geometry ; histones ; light scattering ; mechanical properties ; microparticles ; nucleosomes ; optical traps ; packaging ; torque ; trapping ; wavelengths
    Language English
    Dates of publication 2011-1013
    Size p. e3405.
    Publishing place Journal of Visualized Experiments
    Document type Article
    ZDB-ID 2259946-0
    ISSN 1940-087X
    ISSN 1940-087X
    DOI 10.3791/3405
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  7. Article ; Online: Femtonewton entropic forces can control the formation of protein-mediated DNA loops.

    Chen, Yih-Fan / Milstein, J N / Meiners, Jens-Christian

    Physical review letters

    2010  Volume 104, Issue 4, Page(s) 48301

    Abstract: We show that minuscule entropic forces, on the order of 100 fN, can prevent the formation of DNA loops-a ubiquitous means of regulating the expression of genes. We observe a tenfold decrease in the rate of LacI-mediated DNA loop formation when a tension ... ...

    Abstract We show that minuscule entropic forces, on the order of 100 fN, can prevent the formation of DNA loops-a ubiquitous means of regulating the expression of genes. We observe a tenfold decrease in the rate of LacI-mediated DNA loop formation when a tension of 200 fN is applied to the substrate DNA, biasing the thermal fluctuations that drive loop formation and breakdown events. Conversely, once looped, the DNA-protein complex is insensitive to applied force. Our measurements are in excellent agreement with a simple polymer model of loop formation in DNA, and show that an antiparallel topology is the preferred LacI-DNA loop conformation for a generic loop-forming construct.
    MeSH term(s) DNA, Bacterial/chemistry ; DNA, Bacterial/metabolism ; Entropy ; Escherichia coli/metabolism ; Kinetics ; Lac Repressors/metabolism ; Nucleic Acid Conformation ; Operator Regions, Genetic
    Chemical Substances DNA, Bacterial ; Lac Repressors
    Language English
    Publishing date 2010-01-29
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 208853-8
    ISSN 1079-7114 ; 0031-9007
    ISSN (online) 1079-7114
    ISSN 0031-9007
    DOI 10.1103/PhysRevLett.104.048301
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  8. Article ; Online: Protein-mediated DNA loop formation and breakdown in a fluctuating environment.

    Chen, Yih-Fan / Milstein, J N / Meiners, Jens-Christian

    Physical review letters

    2010  Volume 104, Issue 25, Page(s) 258103

    Abstract: Living cells provide a fluctuating, out-of-equilibrium environment in which genes must coordinate cellular function. DNA looping, which is a common means of regulating transcription, is very much a stochastic process; the loops arise from the thermal ... ...

    Abstract Living cells provide a fluctuating, out-of-equilibrium environment in which genes must coordinate cellular function. DNA looping, which is a common means of regulating transcription, is very much a stochastic process; the loops arise from the thermal motion of the DNA and other fluctuations of the cellular environment. We present single-molecule measurements of DNA loop formation and breakdown when an artificial fluctuating force, applied to mimic a fluctuating cellular environment, is imposed on the DNA. We show that loop formation is greatly enhanced in the presence of noise of only a fraction of k_{B}T, yet find that hypothetical regulatory schemes that employ mechanical tension in the DNA-as a sensitive switch to control transcription-can be surprisingly robust due to a fortuitous cancellation of noise effects.
    MeSH term(s) Biomechanical Phenomena ; DNA/chemistry ; DNA/metabolism ; DNA/physiology ; Gene Expression Regulation ; Models, Biological ; Motion ; Nucleic Acid Conformation ; Proteins/metabolism ; Stochastic Processes ; Temperature ; Thermodynamics
    Chemical Substances Proteins ; DNA (9007-49-2)
    Language English
    Publishing date 2010-06-25
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 208853-8
    ISSN 1079-7114 ; 0031-9007
    ISSN (online) 1079-7114
    ISSN 0031-9007
    DOI 10.1103/PhysRevLett.104.258103
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  9. Article ; Online: Two-color DNA nanoprobe of intracellular dynamics.

    Milstein, Joshua N / Chu, Mike / Raghunathan, Krishnan / Meiners, Jens-Christian

    Nano letters

    2012  Volume 12, Issue 5, Page(s) 2515–2519

    Abstract: We have developed a correlation microscopy technique to follow the dynamics of quantum dot labeled DNA within living cells. The temporal correlation functions of the labels reflect the fluctuations of the DNA nanoprobe as a result of its interactions ... ...

    Abstract We have developed a correlation microscopy technique to follow the dynamics of quantum dot labeled DNA within living cells. The temporal correlation functions of the labels reflect the fluctuations of the DNA nanoprobe as a result of its interactions with the cellular environment. They provide a sensitive measure for the length of the probe on the scale of a persistence length (∼50 nm) and reveal strong nonthermal dynamics of the cell. These results pave the way for dynamic observations of DNA conformational changes in vivo.
    MeSH term(s) Color ; DNA Probes ; Nanotechnology
    Chemical Substances DNA Probes
    Language English
    Publishing date 2012-04-05
    Publishing country United States
    Document type Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
    ISSN 1530-6992
    ISSN (online) 1530-6992
    DOI 10.1021/nl300683p
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  10. Article ; Online: Stretching submicron biomolecules with constant-force axial optical tweezers.

    Chen, Yih-Fan / Blab, Gerhard A / Meiners, Jens-Christian

    Biophysical journal

    2009  Volume 96, Issue 11, Page(s) 4701–4708

    Abstract: Optical tweezers have become powerful tools to manipulate biomolecular systems, but are increasingly difficult to use when the size of the molecules is <1 microm. Many important biological structures and processes, however, occur on the submicron length ... ...

    Abstract Optical tweezers have become powerful tools to manipulate biomolecular systems, but are increasingly difficult to use when the size of the molecules is <1 microm. Many important biological structures and processes, however, occur on the submicron length scale. Therefore, we developed and characterized an optical manipulation protocol that makes this length scale accessible by stretching the molecule in the axial direction of the laser beam, thus avoiding limiting artifacts from steric hindrances from the microscope coverslip and other surface effects. The molecule is held under constant mechanical tension by a combination of optical gradient forces and backscattering forces, eliminating the need for electronic feedback. We demonstrate the utility of this method through a measurement of the force-extension relationship of a 1298 bp ds-DNA molecule.
    MeSH term(s) Algorithms ; Calibration ; DNA/chemistry ; Elasticity ; Nanotechnology/methods ; Optical Tweezers
    Chemical Substances DNA (9007-49-2)
    Language English
    Publishing date 2009-01-21
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 218078-9
    ISSN 1542-0086 ; 0006-3495
    ISSN (online) 1542-0086
    ISSN 0006-3495
    DOI 10.1016/j.bpj.2009.03.009
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