Summary of GP measurements.

<p>GP measurements in hN2, HEK293, NIH3T3 and L6 cells at a) 12 h; b) 72 h and c) 92 h.</p

Laurdan GP analysis.

<p>A) Fluorescence-intensity images of three hN2 cells at 12 h observed in the blue channel (460–480). GP scale to pseudo color the intensity image is shown at the right. C) GP histogram from the corresponding image (membrane) in B). One Gaussian component is observed referring to the cell membrane after digital mask application. Average GP = 0.062.The width at half maximum is ~ 0.1.</p

Visualization 1: 3D fluorescence anisotropy imaging using selective plane illumination microscopy

Vosualization1 Originally published in Optics Express on 24 August 2015 (oe-23-17-22308

Calcium concentration changes after HlyA addition.

<p>Phasor representation of FLIM data (<b>A</b>) and the corresponding intensity images (<b>B</b>) of RRBC labeled with CaG-1 at 37°C after the addition of 0.82 nM of HlyA. Red and yellow concentration ranges are the same used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021127#pone-0021127-g004" target="_blank">Figure 4</a>, green concentration range goes from 80 to 490 nM. The black line and dots at the extremes represent the internal calibration curve determined for RRBC. The black arrow indicates the direction of movement experienced by the center of the phasor cluster after the addition of HlyA Numbers on the upper right of each image correspond to the time in seconds after HlyA addition.</p

Calibration of CaG-1 inside the RRBC.

<p>Phasor plot representation of FLIM data acquired from sequential images of RRBC containing CaG-1, after the addition of 0.82 nM of HlyA and before hemolysis occurrence. Black dots represent the phasor of the bound (p_b) and free (p_f) species of CaG-1 inside the cells. The line is the internal calibration curve determined by using the phasors at the extremities and the K_d determined for the dye (79 nM).</p

The phasor representation method.

<p>The phasor plot coordinates are S = M cos φ and G = M sin φ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021127#pone-0021127-g001" target="_blank">Figure 1A</a>), with φ being the phase delay between excitation and emission and M the demodulation (<b>A</b>). Each fluorescent species, independently of the number of exponentials needed to determine its decay, is represented by a point (phasor) with (S,G) coordinates. From an image, the lifetime will be determined at each pixel and the corresponding phasor will be represented on the phasor plot (<b>B</b>). The phasor analysis can be performed in any harmonic of the original modulation frequency. If two phasors are the linear combination of different pair of phasors, for example t₁-t₂ and t₃-t₄, they may overlap in the first harmonic (<b>C left</b>), but may be separated at higher harmonics (<b>C right</b>) <b>(see Methods)</b>.</p

Single cell increase in calcium.

<p>Single cell increase in calcium.</p

Separation of auto fluorescence from CaG-1 signal in RRBC.

<p>Phasor representation of FLIM images acquired on RRBC incubated with and without CaG-1. For comparison purposes, the same power and the same integration time were used to obtain the images from both samples. The auto fluorescence corresponds to the signal obtained from the RRBC without CaG-1, Data were analyzed calculating the phasors at the basic laser frequency (80 MHz) (<b>A</b>) and at the second harmonic of it (160 MHz) (<b>B</b>). Using the second harmonic the cluster of phasors from the auto fluorescence can be separated from the CaG-1 phasors and the concentration of Ca⁺² inside the RRBC can be determined.</p

DIC image of AUNPs.

<p>(A) DIC image. The large structure on the bottom is an impurity over the glass surface that was used to guide the recognition of the same area for the fluorescence image. (B) Fluorescence image of a small region of the slide with fixed AuNPs excited at 890 nm with emission band pass filter 560–650 nm. The image in B is strongly contrasted to make the AuNP visible. A threshold of 20 levels above the background was applied to the fluorescence image to select only the fluorescent AuNPs. (C) Overlay of the DIC and fluorescence image. All particles in B have a corresponding DIC signal in A, but not all dots in A have a corresponding fluorescence dot in B.</p

1 nM AuNP suspension in the presence of 100nM EGFP.

<p>The excitation wavelength was 890 nm. In all graph the vertical axis is in intensity units from the Andor camera. (A) Intensity trace of a small region of the overall data collection. The intensity is obtained by the integrating the spectrum from 420nm to 680nm. A total of 500,000 spectra were collected every 200 microseconds. Occasional burst are observed along the intensity trace above the average spectral intensity due to EGFP in solution. (B) The average spectrum of 144 intensity bursts. (C) Average spectrum of the regions of the trace without bursts. The spectra in B and C are very similar.</p