Interpolated detailed diffusion map of a part of a nucleus of a HeLa cell.

<p>The figure shows the H2A-mRFP1 tagged nucleus of a HeLa cell. Only the region in the cartridge is mapped and enlarged 2 times. Data were sampled on 25 points (white crosses in the cartridge), distributed on 5 lines of 5 points, with an inter-point distance of about 1 µm and 0.6 µm for the inter-line distance, covering an area of about 11 µm². Each color step corresponds to a 10.0 µm² s⁻¹ range of the diffusion coefficient of eGFP. The white crosses mark the measurement points. Measurements were carried out at 37°C in 5% CO₂ atmosphere, using a water immersion objective with a NA of 1.2.</p

Highlight on temperature control.

<p>Normalized autocorrelation curves measured on Alexa 488 10 nM aqueous solution at 20°C (black curve) and 37°C (red curve) (A) and associated diffusion times for temperatures ranging from 19.5°C to 39.3°C with their standard deviation (B, squares) and diffusion times corrected for 25°C using temperature and water viscosity (B, circles). All measurements were done with the same laser power and a water immersion objective with a NA of 1.2. C and D: temperature measurements of room temperature (black circles), air temperature one centimeter behind the sample (blue crosses), air temperature at the incubator output (green squares) and temperature in the sample (red line). Temperature was measured from the incubator startup (C) or from the introduction of a 4°C sample introduction in a stabilized incubator (D).</p

Diffusion map in the nucleus of a HeLa cell.

<p>Only the nucleus of a HeLa cell is represented on this figure and the color map shows the diffusion coefficient of eGFP in the nucleus. (A) Fluorescence intensity of H2A-mRFP1. (B) Diffusion map of eGFP (colors) underlaid by H2A-mRFP1 fluorescence intensity. The white crosses show the 48 measurement points, corresponding to a total measurement time of 288 seconds. Measurements were carried out at 37°C in 5% CO₂ atmosphere, using a water immersion objective with a NA of 1.2.</p

Overview of the diffusion coefficients of every eGFP construct in each cell line.

<p>The figure shows all the measured diffusion coefficients of the eGFP oligomers in the cell nuclei plotted against the normalized chromatin density, given by the H2A-mRFP1 fluorescence intensity. Black squares: eGFP-monomer, white squares: dimer, black triangles: trimer, white triangles: tetramer.</p

Effect of beam parking on reference measurements.

<p>(A) Diffusion times measured as a function of the diagonal distance from the center of the scanning field. An area of 100×100 µm gives a diagonal of approximately 140 µm. (B) Diffusion time map of the 30×30 µm central region. Each color step corresponds to a range of 2.5 µs in diffusion time ι. Both measurements were performed on Alexa 488 10 nM aqueous solution, using a water immersion objective with a NA of 1.2.</p

Free diffusion of the eGFP-oligomers in solution.

<p>The squares and the solid curve (blue) show the measured diffusion coefficient for the different eGFP-oligomers, while the circles and the dotted curve (red) represent the theoretical values for a globular shaped molecule and the triangles and dashed curve (green) for a rod-shaped molecule, plotted against the molecular weight of the different oligomers. All measurements were carried out at 37°C using a water immersion objective with a NA of 1.2.</p

Effect of XY focus position on live cell measurements.

<p>Diffusion time of free eGFP was measured in the cytoplasm of living cells at various scanner X and Y positions as depicted by the white stars in (A). Several such acquisitions over a 100×100 µm area were performed and analyzed. The resulting diffusion times of 33 points in about 20 cells – after sorting out cells that had moved during the measurement – were color-coded as shown in (B) and subdivided in 3 distinct regions. Panel (C) presents the mean diffusion time and standard deviation obtained in the 3 regions shown in (B): a) for a central region of 30×30 µm b) for the whole scanning area of 100×100 µm and c) for a band of 30×100 µm on the left of the scanning area. All measurements were carried out at 37°C, with the same laser power, using a water immersion objective with a NA of 1.2.</p