Fluorescence relaxation in 3D from diffraction-limited sources of PAGFP or sinks of EGFP created by multiphoton photoconversion.
ABSTRACT The relaxation of fluorescence from diffraction-limited sources of photoactivatable green fluorescent protein (PAGFP) or sinks of photobleached enhanced GFP (EGFP) created by multiphoton photo-conversion was measured in solutions of varied viscosity (eta), and in live, spherical Chinese hamster ovary (CHO) cells. Fluorescence relaxation was monitored with the probing laser fixed, or rapidly scanning along a line bisected by the photoconversion site. Novel solutions to several problems that hamper the study of PAGFP diffusion after multiphoton photoconversion are presented. A theoretical model of 3D diffusion in a sphere from a source in the shape of the measured multiphoton point-spread function was applied to the fluorescence data to estimate the apparent diffusion coefficient, D(ap). The model incorporates two novel features that make it of broad utility. First, the model includes the no-flux boundary condition imposed by cell plasma membranes, allowing assessment of potential impact of this boundary on estimates of D(ap). Second, the model uses an inhomogeneous source term that, for the first time, allows analysis of diffusion from sources produced by multiphoton photoconversion pulses of varying duration. For diffusion in aqueous solution, indistinguishable linear relationships between D(ap) and eta(-1) were obtained for the two proteins: for PAGFP, D(aq)= 89 +/- 2.4 microm2 s(-1) (mean +/- 95% confidence interval), and for EGFP D(aq)= 91 +/- 1.8 microm2 s(-1). In CHO cells, the application of the model yielded D(ap)= 20 +/- 3 microm2 s(-1) (PAGFP) and 19 +/- 2 microm2 s(-1) (EGFP). Furthermore, the model quantitatively predicted the decline in baseline fluorescence that accompanied repeated photobleaching cycles in CHO cells expressing EGFP, supporting the hypothesis of fluorophore depletion as an alternative to the oft invoked 'bound fraction' explanation of the deviation of the terminal fluorescence recovery from its pre-bleach baseline level. Nonetheless for their identical diffusive properties, advantages of PAGFP over EGFP were found, including an intrinsically higher signal/noise ratio with 488-nm excitation, and the requirement for approximately 1/200th the cumulative light energy to produce data of comparable signal/noise.
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ABSTRACT: The quantification of metabolite leakage from damaged mammalian cells to the surrounding medium is of high interest for the processing of samples for metabolomic analysis. It is also of relevance to know the typical time span which is required for a promoted metabolite release through a selectively permeabilized cell membrane. The real-time observation of such a process is difficult since small metabolites cannot be observed directly by optical methods and other more indirect assays can disturb the metabolite concentration itself. However, the diffusion based loss of metabolites from the cytoplasm can be predicted on the basis of reference measurements taken from an easy-to-detect molecule with known diffusion coefficient. In this work, we use green fluorescent protein (GFP) as a marker and model its release from damaged cells using the finite-element method. A correlation between the disrupted membrane area fraction, A d , the distribution of membrane ruptures and the rate of GFP efflux, k e , has been established. k e has been determined experimentally for Chinese hamster ovary cells, which have been damaged mechanically by passage through a micronozzle geometry in a microfluidic system. The immediate GFP release downstream of the micronozzles has been observed in real-time and the corresponding membrane damage has been predicted. On this basis, we calculated the expected times required for the drainage of freely diffusable cytosolic glucose and found a loss of ≈90% within 1 s for a disrupted membrane area fraction of ≈5%. Hence, even minimal membrane damage would lead to a rapid loss of cytosolic metabolites by diffusion unless membrane resealing processes take place.Metabolomics 12/2012; 8(6):1081-1089. · 3.97 Impact Factor
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ABSTRACT: The multiphoton fluorescence recovery after photobleaching (MP-FRAP) technique has been developed to measure the three-dimensional (3D) solute diffusion within biological systems. However, current 3D MP-FRAP models are based on isotropic diffusion and spatial domain analysis. The 3D anisotropic diffusion and frequency domain analysis for MP-FRAP measurements are rarely studied. In this study, a new technique is demonstrated for the quantitative and non-destructive determination of 3D anisotropic solute diffusion tensors within biological fibrosis tissues by multiphoton photobleaching and spatial Fourier analysis (SFA). Compared to the spatial domain analysis based MP-FRAP techniques, this SFA-based method has the capability for determining the 3D anisotropic diffusion tensors as well as the flexibility for satisfying initial and boundary conditions. First, a close-form solution of the 3D anisotropic diffusion equation is derived by solely using SFA. Next, this new method is validated by computer-simulated MP-FRAP experiments with pre-defined 3D anisotropic diffusion tensors as well as experimental diffusion measurements of FITC-Dextran (FD) molecules in aqueous glycerol solutions. Finally, this MP-FRAP technique is applied to the measurement of 3D anisotropic diffusion tensors of FD molecules within porcine tendon tissues. This study provides a new tool for complete determination of 3D anisotropic solute diffusion tensor in biological tissues.Annals of Biomedical Engineering 03/2014; 42(3):555-565. · 3.23 Impact Factor
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ABSTRACT: Our understanding of the mechanisms governing the response to DNA damage in higher eucaryotes crucially depends on our ability to dissect the temporal and spatial organization of the cellular machinery responsible for maintaining genomic integrity. To achieve this goal, we need experimental tools to inflict DNA lesions with high spatial precision at pre-defined locations, and to visualize the ensuing reactions with adequate temporal resolution. Near-infrared femtosecond laser pulses focused through high-aperture objective lenses of advanced scanning microscopes offer the advantage of inducing DNA damage in a 3D-confined volume of subnuclear dimensions. This high spatial resolution results from the highly non-linear nature of the excitation process. Here we review recent progress based on the increasing availability of widely tunable and user-friendly technology of ultrafast lasers in the near infrared. We present a critical evaluation of this approach for DNA microdamage as compared to the currently prevalent use of UV or VIS laser irradiation, the latter in combination with photosensitizers. Current and future applications in the field of DNA repair and DNA-damage dependent chromatin dynamics are outlined. Finally, we discuss the requirement for proper simulation and quantitative modeling. We focus in particular on approaches to measure the effect of DNA damage on the mobility of nuclear proteins and consider the pros and cons of frequently used analysis models for FRAP and photoactivation and their applicability to non-linear photoperturbation experiments.Frontiers in Genetics 01/2013; 4:135.