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(Color online) Photoactivation dependence on imaging conditions. (a) The pre- and postactivation images were collected exciting only the square regions 1 and 2 at , respectively, at and of mean laser intensity delivered on the sample. The activation step was performed using the same parameters in both regions: , , pixel dwell , and pixel . (b) The histograms show the mean fluorescence intensity collected in the selected cell’s regions of the pre- and postimages. (c) By dividing the fluorescence intensity collected in the postactivation images and the preactivation image, respectively, for regions 1 and 2, we reported in the histogram the increase of fluorescence induced by photoactivation of paGFPs.
(Color online) Observation and characterization of activation. (a) Preactivation and postactivation images were collected at low excitation intensity at excitation wavelength ; the activation was performed in the region of under high excitation power at . The experiment was performed on fixed HeLa cells expressing paGFP-H2B fusion protein (b) Emission spectra of activated paGFP at as excitation wavelength , collected in three different samples: fixed HeLa cells expressing the vector paGFP-H2B, and fixed and unfixed sepharose beads with purified paGFP immobilized on the surface.
(Color online) Activation kinetics under illumination. We show six curves for different laser intensities within the range of . The ordinate values represent the mean fluorescence intensity emitted by paGFP molecules inside the nucleus as a function of the irradiation time. The curves are normalized to the highest intensity value obtained by illuminating at . Each activation pulse was performed with pixel dwell and pixel .
Characterization of visible activation respect to the UV-vis activation. (a) The mean fluorescence intensity collected in square regions of the cell nucleus observed at low intensity and activated at different laser intensities within the range of with sequential pulses with pixel dwell time of and pixel size of . On the time scale is shown the cumulative activation time per pixel. (b) Different maxima reached from each curve until an evidence of saturation. The maximum value reached activating at is normalized at 1 and used as reference in order to compare UV-vis irradiation photoproduct with respect to the visible one. (c) Recovery of fluorescence obtained in regions of the cell illuminated at several laser line intensities at several powers and subsequently irradiated with a short pulse at UV-vis. The visualization was performed at with an average intensity of . These values are shown in the histogram for all the activation visible power analyzed. (d) The graph shows the visible activation process dependence on the power. The mean fluorescence intensity values, collected on regions of the bead model system activated at different laser line intensities, were visualized in a bilogarithmic as function of laser intensity. Black line represents the linear fit of the data with a slope of .
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