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- Time-Resolved Polarization Imaging By Pump-Probe (Stimulated...Time-Resolved Polarization Imaging By Pump-Probe (Stimulated Emission) Fluorescence Microscopy
Biophysical Journal, Volume 79, Issue 1, 1 July 2000, Pages 536-549
Ch. Buehler, C.Y. Dong, P.T.C. So, T. French and E. Gratton
AbstractWe report the application of pump-probe fluorescence microscopy in time-resolved polarization imaging. We derived the equations governing the pump-probe stimulated emission process and characterized the pump and probe laser power levels for signal saturation. Our emphasis is to use this novel methodology to image polarization properties of fluorophores across entire cells. As a feasibility study, we imaged a 15-m orange latex sphere and found that there is depolarization that is possibly due to energy transfer among fluorescent molecules inside the sphere. We also imaged a mouse fibroblast labeled with CellTracker Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethyl-rhodamine). We observed that Orange CMTMR complexed with gluthathione rotates fast, indicating the relatively low fluid-phase viscosity of the cytoplasmic microenvironment as seen by Orange CMTMR. The measured rotational correlation time ranged from ∼30 to ∼150ps. This work demonstrates the effectiveness of stimulated emission measurements in acquiring high-resolution, time-resolved polarization information across the entire cell.
Abstract | Full Text | PDF (433 kb) - Dynamic Bending Rigidity of a 200-bp DNA in 4mM Ionic Streng...Dynamic Bending Rigidity of a 200-bp DNA in 4mM Ionic Strength: A Transient Polarization Grating Study
Biophysical Journal, Volume 78, Issue 3, 1 March 2000, Pages 1498-1518
Alexei N. Naimushin, Bryant S. Fujimoto and J. Michael Schurr
AbstractDNA may exhibit three different kinds of bends: 1) permanent bends; 2) slowly relaxing bends due to fluctuations in a prevailing equilibrium between differently curved secondary conformations; and 3) rapidly relaxing dynamic bends within a single potential-of-mean-force basin. The dynamic bending rigidity (), or equivalently the dynamic persistence length, = governs the rapidly relaxing bends, which are responsible for the flexural dynamics of DNA on a short time scale, ≤10 s. However, all three kinds of bends contribute to the total equilibrium persistence length, , according to ≅ ++, where is the contribution of the permanent bends and is the contribution of the slowly relaxing bends. Both and are determined for the same 200-bp DNA in 4mM ionic strength by measuring its optical anisotropy, () from 0 to 10s. Time-resolved fluorescence polarization anisotropy (FPA) measurements yield () for DNA/ethidium complexes (1 dye/200 bp) from 0 to 120ns. A new transient polarization grating (TPG) experiment provides () for DNA/methylene blue complexes (1 dye/100 bp) over a much longer time span, from 20ns to 10s. Accurate data in the very tail of the decay enable a model-independent determination of the relaxation time () of the end-over-end tumbling motion, from which =500Å is estimated. The FPA data are used to obtain the best-fit pairs of and torsion elastic constant () values that fit those data equally well, and which are used to eliminate as an independent variable. When the relevant theory is fitted to the entire TPG signal (()) the end-over-end rotational diffusion coefficient is fixed at its measured value and is eliminated in favor of . Neither a true minimum in chi-squared nor a satisfactory fit could be obtained for anywhere in the range 500–5000Å, unless an adjustable amplitude of azimuthal wobble of the methylene blue was admitted. In that case, a well-defined global minimum and a reasonably good fit emerged at =2000Å and 〈ζ〉=25°. The discrimination against values <1600Å is very great. By combining the values, =500Å and =2000Å with a literature estimate, =1370Å, a value =1300Å is estimated for the contribution of slowly relaxing bends. This value is analyzed in terms of a simple model in which the DNA is divided up into domains containing bp, each of which experiences an all-or-none equilibrium between a straight and a uniformly curved conformation. With an appropriate estimate of the average bend angle per basepair of the curved conformation, a lower bound estimate, = bp, is obtained for the domain size of the coherently bent state. Previous measurements suggest that this coherent bend is not directional, or phase-locked, to the azimuthal orientation of the filament.
Abstract | Full Text | PDF (289 kb) - Restrained Torsional Dynamics of Nuclear DNA in Living Proli...Restrained Torsional Dynamics of Nuclear DNA in Living Proliferative Mammalian Cells
Biophysical Journal, Volume 78, Issue 5, 1 May 2000, Pages 2614-2627
Marc Tramier, Klaus Kemnitz, Christiane Durieux, Jacques Coppey, Patrick Denjean, Robert B. Pansu and Maïté Coppey-Moisan
AbstractPhysical parameters, describing the state of chromatinized DNA in living mammalian cells, were revealed by in situ fluorescence dynamic properties of ethidium in its free and intercalated states. The lifetimes and anisotropy decays of this cationic chromophore were measured within the nuclear domain, by using the ultra-sensitive time-correlated single-photon counting technique, confocal microscopy, and ultra-low probe concentrations. We found that, in living cells: 1) free ethidium molecules equilibrate between extracellular milieu and nucleus, demonstrating that the cation is naturally transported into the nucleus; 2) the intercalation of ethidium into chromatinized DNA is strongly inhibited, with relaxation of the inhibition after mild (digitonin) cell treatment; 3) intercalation sites are likely to be located in chromatin DNA; and 4) the fluorescence anisotropy relaxation of intercalated molecules is very slow. The combination of fluorescence kinetic and fluorescence anisotropy dynamics indicates that the torsional dynamics of nuclear DNA is highly restrained in living cells.
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Copyright © 1995 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 69, Issue 6, 2234-2242, 1 December 1995
doi:10.1016/S0006-3495(95)80148-7
Research Article
Fluorescence lifetime imaging by asynchronous pump-probe microscopy
C.Y. Dong, P.T. So, T. French and E. Gratton
Abstract
We report the development of a scanning lifetime fluorescence microscope using the asynchronous, pump-probe (stimulated emission) approach. There are two significant advantages of this technique. First, the cross-correlation signal produced by overlapping the pump and probe lasers results in i) an axial sectioning effect similar to that in confocal and two-photon excitation microscopy, and ii) improved spatial resolution compared to conventional one-photon fluorescence microscopy. Second, the low-frequency, cross-correlation signal generated allows lifetime-resolved imaging without using fast photodetectors. The data presented here include 1) determination of laser sources' threshold powers for linearity in the pump-probe signal; 2) characterization of the pump-probe intensity profile using 0.28 microns fluorescent latex spheres; 3) high frequency (up to 6.7 GHz) lifetime measurement of rhodamine B in water; and 4) lifetime-resolved images of fluorescent latex spheres, human erythrocytes and a mouse fibroblast cell stained by rhodamine DHPE, and a mouse fibroblast labeled with ethidium bromide and rhodamine DHPE.
