Diffusion Coefficient Of Fluorescent Molecules Research Articles

Determination of diffusion coefficients in live cells using fluorescence recovery after photobleaching with wide-field fluorescence microscopy.

Fluorescence recovery after photobleaching (FRAP) enables characterization of quantitative dynamic properties such as diffusion coefficients of fluorescent molecules in living cells by analyzing the recovery of fluorescence intensity after photobleaching in a specific cellular compartment or area. To quantitatively determine high intracellular diffusion coefficients, a suitable optical system as well as an appropriate model for fast diffusion analysis is necessary. Here, we propose a procedure to quantify the diffusion coefficient of rapidly-diffusing fluorescent molecules that makes use of an epi-fluorescence microscope with a photobleaching laser in combination with established models for diffusion analysis. Analysis for the diffusion coefficients of tandemly oligomerized green flurescent proteins (GFPs) in living cells when changing the photobleaching times showed that photobleaching with shorter times than the diffusion speed indicated not the only way to obtain appropriate diffusion coefficients of fast-moving molecules. Our results also showed that the apparent spreading of the effective radius of the photobleached area works as a correction factor for determining appropriate diffusion coefficients of fast-moving molecules like monomeric GFPs. Our procedure provides a useful approach for quantitative measurement of diffusion coefficients in living cells. This procedure is relevant for characterizing dynamic molecular interactions, especially of fast-moving molecules, and is relevant for studies in many biological fields.

Translational diffusion of probe molecules under high pressure: A study by fluorescence recovery after photobleaching technique

We present fluorescence recovery measurements after photobleaching performed under high pressure in liquids that fill square-section fused silica micro-capillaries. These micro-capillaries withstand pressure up to 2500 bar for a wall thickness of about 140 μm and fit easily on the microscope stage. This technique allows the translational diffusion coefficient of fluorescent molecules in liquids to be measured as a function of pressure. When the liquid sample is far from its glass transition the translational diffusive coefficient is in agreement with the Stokes-Einstein equation. As the glass transition is approached by further increasing the pressure, decoupling of the measured diffusion coefficient from the Stokes-Einstein relation is observed. These are the first measurements that combine the fluorescence recovery technique and high hydrostatic pressures. This experimental setup can also be used either with diamond or sapphire anvil cells in order to span a larger pressure range.

Measuring diffusion with polarization-modulation dual-focus fluorescence correlation spectroscopy

We present a new technique, polarization-modulation dual-focus fluorescence correlation spectroscopy (pmFCS), based on the recently intro-duced dual-focus fluorescence correlation spectroscopy (2fFCS) to measure the absolute value of diffusion coefficients of fluorescent molecules at pico- to nanomolar concentrations. Analogous to 2fFCS, the new technique is robust against optical saturation in yielding correct values of the diffusion coefficient. This is in stark contrast to conventional FCS where optical saturation leads to an apparent decrease in the determined diffusion coefficient with increasing excitation power. However, compared to 2fFCS, the new technique is simpler to implement into a conventional confocal microscope setup and is compatible with cw-excitation, only needing as add-ons an electro-optical modulator and a differential interference contrast prism. With pmFCS, the measured diffusion coefficient (D) for Atto655 maleimide in water at 25?C is determined to be equal to (4.09 +/- 0.07) x 10(-6)cm(2)/s, in good agreement with the value of 4.04 x 10-6cm2/s as measured by 2fFCS.

Precise Measurement of Diffusion Coefficients using Scanning Fluorescence Correlation Spectroscopy

We have implemented scanning fluorescence correlation spectroscopy (sFCS) for precise determination of diffusion coefficients of fluorescent molecules in solution. The measurement volume where the molecules are excited, and from which the fluorescence is detected, was scanned in a circle with radius comparable to its size at frequencies 0.5–2 kHz. The scan radius R, determined with high accuracy by careful calibration, provides the spatial measure required for the determination of the diffusion coefficient D, without the need to know the exact size of the measurement volume. The difficulties in the determination of the measurement volume size have limited the application of standard FCS with fixed measurement volume to relative measurements, where the diffusion coefficient is determined by comparison with a standard. We demonstrate, on examples of several common fluorescent dyes, that sFCS can be used to measure D with high precision without a need for a standard. The correct value of D can be determined in the presence of weak photobleaching, and when the measurement volume size is modified, indicating the robustness of the method. The applicability of the presented implementation of sFCS to biological systems in demonstrated on the measurement of the diffusion coefficient of eGFP in the cytoplasm of HeLa cells. With the help of simulations, we find the optimal value of the scan radius R for the experiment.

Application of Fluorescence Correlation Spectroscopy to the Measurement of Local Temperature in Solutions under Optical Trapping Condition

Fluorescence correlation spectroscopy (FCS) was applied to the quantitative evaluation of the local heating in small domains <1 microm in solutions under the laser trapping condition in the presence of a near-infrared (NIR) laser beam at 1064 nm. On the basis of the translational diffusion coefficient of fluorescent molecules obtained by FCS, the relationship between temperature rise and the incident NIR laser power, DeltaT/DeltaP, were determined to be 62 +/- 6, 49 +/- 7, and 23 +/- 1 deg K/W in ethylene glycol, ethanol, and water, respectively, while no remarkable temperature increase was observed for deuterated water. The value of DeltaT/DeltaP linearly increased as a function of alpha/lambda (alpha is the extinction coefficient of solvent at the wavelength and lambda is the thermal conductivity of the medium). The validity and the applicability of the present method for the measurement of the local temperature increase were discussed by comparing the present results with previous ones by other various methods.

Measurements of diffusion coefficients in 1-D micro- and nanochannels using shear-driven flows

The present paper describes a method for measuring the molecular diffusion coefficient of fluorescent molecules in microfluidic systems. The proposed static shear-driven flow method allows one to perform diffusion measurements in a fast and accurate manner. The method also allows one to work in very thin (i.e. submicron) channels, hence allowing the investigation of diffusion in highly confined spaces. In the deepest investigated channels, the obtained results were comparable to the existing literature values, but when the channel size dropped below the micrometer range, a significant decrease (more than 30%) in molecular diffusivity was observed. The reduction of the diffusivity was most significant for the largest considered molecules (ssDNA oligomers with a size ranging between 25 to 100 bases), but the decrease was also observed for smaller tracer molecules (FITC). This decrease can be attributed to the interactions of the analyte molecules with the channel walls, which can no longer be neglected when the depth of the channel reaches a critical value. The change in diffusivity seems to become more explicit as the molecular weight of the analytes increases.

Measurement of diffusion of fluorescent molecules in living cells

The possibilities of the method of fluorescence correlation spectroscopy for studying the molecular dynamics in living cells are demonstrated. The method provides point measurements of extremely low concentrations of fluorescent molecules and their diffusion coefficients with a high time resolution in a microscopic volume, which is especially important in pharmacological investigations. A biological model of the interaction of liposomes with a cellular membrane is considered. The diffusion coefficients of fluorescent molecules are measured directly in the living cell cytoplasm.

Disaggregation kinetics of a peat humic acid: mechanism and pH effects.

Fluorescence correlation spectroscopy was used to study the disaggregation kinetics of a peat humic acid (PPHA) at several pH. FCS measures diffusion coefficients of fluorescent molecules and aggregates, thus allowing for the determination of disaggregation rates with a temporal resolution of seconds to minutes. Disaggregation was initiated by dilution of a peat concentrate consisting of a mixture containing 80% large aggregates (average hydrodynamic radius, rH, of about 300 nm) and free monomers (average rH of about 1 nm). Upon dilution at different pH values, aggregate size decreased, and the proportion of free monomers in solution increased until complete disaggregation occurred. The mechanism appeared to involve the release of monomers from the surface of the aggregates. The pH markedly affected the disaggregation rate. Complete disaggregation took 1 month at pH 3.6, took less than 1 h at pH 5.6, and was extremely rapid in alkaline solutions. The results suggested that at least two processes were operating in parallel with the overall rate being the sum of both processes. At pH higher than 4.5, the disaggregation rate increased more than 3 orders of magnitude per pH unit increase. For concentrations lower than 30 mg L(-1), the equilibrium condition for the PPHA was complete disaggregation even for a pH as low as 3.6.

Peer Reviewed: Applications of Fluorescence Recovery after Photobleaching

Determining the diffusion coefficients of fluorescent molecules provides details on molecular-scale structure.