3D Nanometer Images of Biological Fibers by Directed Motion of Gold Nanoparticles

Using near-infrared femtosecond pulses, we move single gold nanoparticles (AuNPs) along biological fibers, such as collagen and actin filaments. While the AuNP is sliding on the fiber, its trajectory is measured in three dimensions (3D) with nanometer resolution providing a high-resolution image of the fiber. Here, we systematically moved a single AuNP along nanometer-size collagen fibers and actin filament inside chinese hamster ovary K1 living cells, mapping their 3D topography with high fidelity

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

Raster scan images of a single gold nanoparticle fixed on a glass coverslip.

<p>(A)The sample is excited at 790 nm with 3 different orientations of linear polarized light. The emission is collected through a bandpass filter 520/30 nm. (B) Emission light collected with the spectral camera. The blue line in the spectrum is the raw data. The red line is the corrected intensity for the instrument response in the range 420–670 nm. Fig 2 shows 3 different polarization angles of excitation: 0°, 45° and 90°. The spectrum is characterized by a broad emission with a maximum around 600nm. The wavelength of the maximum is orientation dependent. There is a narrow feature at 395nm which is due to SHG.</p

Increment of internal calcium after HlyA addition.

<p>Histograms of the phasor distributions that are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021127#pone-0021127-g005" target="_blank">Figure 5A</a>, for RRBC (<b>A</b>). The maximum of the histograms corresponds to the average of calcium concentration, which is plotted as function of time in RRBC (<b>C</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

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

Spectrum of a AuNP excited at 4 different wavelengths and with an orientation of 45 degrees for the linear polarizer. This sample was added with a solution of 100nM mCherry.

<p>(A) mCherry is excited at 740 nm and we see the typical spectrum of the mCherry. The contribution of mCherry is not distinguishable at other excitation wavelengths. (B) At 790nm we observe the broad emission of the nanoparticle. (C) At 840nm the SHG signal starts to be observed. (D) At 890 nm the fluorescence mCherry is not excited and we can only see a strong SHG in addition to the broad fluorescence from the nanoparticle.</p

Spectrum of a AuNP excited at 4 different wavelengths and with an orientation of 45 degrees for the linear polarizer.

<p><b>This sample was added with a solution of 100nM EGFP</b>. (A) EGFP is minimally excited at 740 nm and we see the typical spectrum of the metallic nanoparticle. (B) at 790nm we broad emission of the nanoparticle shifts toward the red and a band starts to appear in the 520nm region typical of EGFP. (C) at 840nm the SHG signal starts to be observed. (D) at 890 nm the fluorescence from the EGFP is strongly enhanced and this fluorescence is clearly distinguishable in the 529 nm region. At this excitation wavelength there is also a very strong SHG signal.</p