Total Internal Reflection Fluorescence Microscopy: Applications in Biophysics

Thompson, Nancy L.; Pero, Jamie K.

doi:10.1007/3-540-27004-3_6

Cited by 8 publications

(3 citation statements)

References 228 publications

(268 reference statements)

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“…The b parameter from the fits contains information about the propensity for GFP to photobleach, since b = κ d . For our experimental conditions, d ≈ 0.1 μm and b = 0.026 ± 0.001 μm 3 μW −1 s −1 . Taken together, these values yield κ ≈ 0.26 μm 2 μW −1 s −1 , which is comparable to the value (0.21 ± 0.01 μm 2 μW −1 s −1 ) for the immobilized GFP (Figure ).…”

Section: Resultsmentioning

confidence: 99%

Quantifying Green Fluorescent Protein Diffusion in Escherichia coli by Using Continuous Photobleaching with Evanescent Illumination

et al. 2009

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Fluorescence recovery after photobleaching and fluorescence correlation spectroscopy are the primary means for studying translational diffusion in biological systems. Both techniques, however, present numerous obstacles for measuring translational mobility in structures only slightly larger than optical resolution. We report a new method using through-prism total internal reflection fluorescence microscopy with continuous photobleaching (TIR-CP) to overcome these obstacles. Small structures, such as prokaryotic cells or isolated eukaryotic organelles, containing fluorescent molecules are adhered to a surface. This surface is continuously illuminated by an evanescent wave created by total internal reflection. The characteristic length describing the decay of the evanescent intensity with distance from the surface is smaller than the structures. The fluorescence decay rate resulting from continuous evanescent illumination is monitored as a function of the excitation intensity. The data at higher excitation intensities provide apparent translational diffusion coefficients for the fluorescent molecules within the structures because the decay results from two competing processes (the intrinsic photobleaching propensity and diffusion in the small structures). We present the theoretical basis for the technique and demonstrate its applicability by measuring the diffusion coefficient, 6.3 ± 1.1 µm2/sec, of green fluorescent protein (GFP) in Escherichia coli cells.

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Section: Resultsmentioning

confidence: 99%

Quantifying Green Fluorescent Protein Diffusion in Escherichia coli by Using Continuous Photobleaching with Evanescent Illumination

et al. 2009

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“…Apparatus. A laser beam is internally reflected at a substrate/solution interface and creates an evanescent intensity that penetrates exponentially with characteristic depth d into the solution adjacent to the surface , (Figure ). The evanescent intensity, I ( z ), is given by where z is the distance in solution from the interface.…”

Section: Theoretical Backgroundmentioning

confidence: 99%

Size Dependence of Protein Diffusion Very Close to Membrane Surfaces: Measurement by Total Internal Reflection with Fluorescence Correlation Spectroscopy

2006

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The diffusion coefficients of nine fluorescently labeled antibodies, antibody fragments, and antibody complexes have been measured in solution very close to supported planar membranes by using total internal reflection with fluorescence correlation spectroscopy (TIR-FCS). The hydrodynamic radii (3-24 nm) of the nine antibody types were determined by comparing literature values with bulk diffusion coefficients measured by spot FCS. The diffusion coefficients very near membranes decreased significantly with molecular size, and the size dependence was greater than that predicted to occur in bulk solution. The observation that membrane surfaces slow the local diffusion coefficient of proteins in a size-dependent manner suggests that the primary effect is hydrodynamic as predicted for simple spheres diffusing close to planar walls. The TIR-FCS data are consistent with predictions derived from hydrodynamic theory. This work illustrates one factor that could contribute to previously observed nonideal ligand-receptor kinetics at model and natural cell membranes.

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“…At the point of TIR, an evanescent wave penetrates into the medium. The evanescent wave which penetrates from a higher into a lower refractive index material under TIR has been used [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] to excite fluorophores selectively at the surfaces and interfaces with relatively little penetration into the bulk.…”

Section: Introductionmentioning

confidence: 99%

Time-resolved total internal reflection fluorescence spectroscopy : Part I. Photophysics of Coumarin 343 at liquid/liquid interface

Pant

2005

Phys. Chem. Chem. Phys.

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Pico-second time-resolved time-correlated single photon counting (TCSPC) technique under the total internal reflection (TIR) condition has been used to study the photophysical properties of Coumarin 343 (C343) dye molecules adsorbed at the water/1,2-dichloroethane (DCE) interface. The fluorescence decay profile of C343 under TIR condition at the water/DCE interface was non-exponential and fitted to the double exponential decay function with the fluorescence lifetimes 0.3 and 3.6 ns, which proved the existence of two different forms of C343 species having largely different lifetimes at the interface. The longer fluorescence lifetime component of C343 at the interface is attributed to the emission from the monomeric form of the dye molecules and the shorter lifetime component is due to the aggregation of dye molecules. The penetration depth dependence of decay curves indicated no change in the fluorescence lifetime components, however, the amplitude corresponding to the lifetime of aggregate increased and the amplitude corresponding to the lifetime of monomer decreased with the decrease in penetration depth of the aqueous phase from the interface. Aggregation is significant in the interfacial layer. The decrease in monomer lifetime at the interface compared to that in the bulk solution is interpreted in terms of excitation energy migration between the dye molecules.

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Total Internal Reflection Fluorescence Microscopy: Applications in Biophysics

Cited by 8 publications

References 228 publications

Quantifying Green Fluorescent Protein Diffusion in Escherichia coli by Using Continuous Photobleaching with Evanescent Illumination

Quantifying Green Fluorescent Protein Diffusion in Escherichia coli by Using Continuous Photobleaching with Evanescent Illumination

Size Dependence of Protein Diffusion Very Close to Membrane Surfaces: Measurement by Total Internal Reflection with Fluorescence Correlation Spectroscopy

Time-resolved total internal reflection fluorescence spectroscopy : Part I. Photophysics of Coumarin 343 at liquid/liquid interface

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