Parallel Nanometric 3D Tracking of Intracellular Gold Nanorods Using Multifocal Two-Photon Microscopy

We report a novel technique for long-term parallel three dimensional (3D)-tracking of gold nanorods in live cells with nanometer resolution. Gold nanorods feature a strong plasmon-enhanced two-photon luminescence, can be easily functionalized, and have been shown to be nontoxic. These properties make gold nanorods very suitable for in vivo two-photon luminescence microscopy. By rapid multifocal scanning, we combine the advantages of 3D molecular tracking methods using wide-field imaging with the advantages of two-photon microscopy. Isolated gold nanorods can be localized with a resolution of 4 nm in the <i>xy</i>-plane and 8 nm in the <i>z</i>-direction. The polarization-dependence of the two-photon luminescence signal can be used to resolve the angular orientation, even when two gold nanorods are separated by less than the diffraction limit. Individual nanorods in live U2OS cells could be followed in 3 dimensions for over 30 min, with a photon noise limited accuracy, and a time resolution of 50 ms in 2D and 500 ms in 3D

Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy

<div><p>Single Molecule Localization super-resolution Microscopy (SMLM) has become a powerful tool to study cellular architecture at the nanometer scale. In SMLM, single fluorophore labels are made to repeatedly switch on and off (“blink”), and their exact locations are determined by mathematically finding the centers of individual blinks. The image quality obtainable by SMLM critically depends on efficacy of blinking (brightness, fraction of molecules in the on-state) and on preparation longevity and labeling density. Recent work has identified several combinations of bright dyes and imaging buffers that work well together. Unfortunately, different dyes blink optimally in different imaging buffers, and acquisition of good quality 2- and 3-color images has therefore remained challenging. In this study we describe a new imaging buffer, OxEA, that supports 3-color imaging of the popular Alexa dyes. We also describe incremental improvements in preparation technique that significantly decrease lateral- and axial drift, as well as increase preparation longevity. We show that these improvements allow us to collect very large series of images from the same cell, enabling image stitching, extended 3D imaging as well as multi-color recording.</p></div

pH in OxEA and Gloxy buffer.

<p>The pH in open dishes filled with 0.5 ml of OxEA (red squares) or Gloxy buffer (blue circles) is graphed at the indicated time points. Note the steep drop in pH in Gloxy buffer, which limits imaging to ~ 1 hour unless measures are taken to prevent oxygen influx.</p

Oxygen levels in OxEA and Gloxy buffers.

<p>O₂ levels were detected every two seconds using a FireSting fluorescence-lifetime based oxygen detector. Note that addition of Gloxy buffer (blue) causes a rapid drop in O₂ level to undetectable levels, whereas OxEA caused a more slow and less complete removal of O₂.</p

OTC enables near-unlimited blinking of Alexa-647.

<p>(A) Stitched image, composited of eight 1800x1800 pixel images (each based on 20k raw blinking images) showing the keratin cytoskeleton in a HUVEC cell. Despite partly overlapping acquisition areas, almost no loss in image quality was noticeable throughout the experiment. (B) Z-stack of keratin cytoskeleton in PA-JEB/β4 keratinocytes. Consecutive images were taken after refocusing the lens by 300 nm manually. The full-resolution images will be available at the associated data repository (<a href="https://osf.io/q684r/" target="_blank">https://osf.io/q684r/</a>).</p

Characterization of blinking in OxEA, Gloxy and MEA buffer.

<p>(A) Raw blinking frames (10 ms each, i.e. non-merged results) taken at the indicated time points. Shown are data for Alexa-488 (A488), Alexa-555 (A555) and Alexa-647 (A647) in both fresh Gloxy and OxEA buffer. Imaging was started after a 2–5 second pumping period at full laser power. Note presence of significant structured background in Gloxy buffer. (B) Mean intensity of individual blinks (merged in consecutive frames). Data are mean +/- SEM. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158884#sec010" target="_blank">Methods</a> for further details. (C) Left panel, average number of blinks per frame (calculated in blocks of 1000 frames) of a preparation labeled with Alexa-488 and imaged in Gloxy or OxEA. Similar parts of cells with similar initial labeling density were selected based on the low-intensity wide-field image. Note the much larger number of blinks in OxEA for Alexa-488. Right panel, summary of blinks per frame data for Alexa-488, Alexa-555, Alexa-647 and FITC in three different buffers. Data are mean +/- SEM; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158884#sec010" target="_blank">Methods</a> for further details. (D) Number of blinks per frame, averaged over the full duration of the acquisition movie, in experiments carried out at the indicated times after applying the buffers. Within the hour, blinking has dropped dramatically in Gloxy whereas OxEA performs well for several hours. (E) Duration of individual blinks of Alexa-488 and Alexa-647 in fresh and ageing (90 minutes) Gloxy, and in fresh and ageing (120 min) OxEA. Multi-frame blinks are very common in ageing Gloxy buffer, as witnessed from the increased average duration of blinks and the enormous increase in duration spread (Data are mean +/- standard deviation). Increased blink duration adds to the appearance of structured background.</p

Minimal Jablonski diagram of fluorophore blinking.

<p>Simplified Jablonski diagram showing molecular states essential to STORM/GSDIM. For simplicity, neither vibrational levels nor possible additional dark states have been indicated. In the bright On-state, fluorophores can be excited (Ex) from the ground state (S₀) to the excited singlet state (S₁₎. From there, they may either relax to the ground state by emitting a quantum of light (hν) or alternatively, they may undergo intersystem crossing to the dark triplet state (T₁). From the triplet state, molecules may return to the ground state or progress to a second, long-lived dark state (D), e.g. through redox reactions. Direct transfer from the excited singlet state to the dark state has also been reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158884#pone.0158884.ref002" target="_blank">2</a>] (dashed arrow). Molecules in the dark state may return to the ground state by inverse redox reactions, or alternatively, by exposure to near-UV radiation (back-pumping). S₀ and S₁ are called On-states, while T₁ and D are Off-states. With most molecules in the Off state, it is possible to detect the few remaining bright fluorophores individually in the preparation. Dark states may last between milliseconds and minutes, whereas triplet states may last microseconds at ambient oxygen levels.</p

GSDIM imaging in OxEA buffer.

<p>(A) comparison of image quality in ageing Gloxy buffer (right) to that in OxEA buffer (left). Images of Ab-labeled vimentin intermediate filaments were collected ~ 2 hours after mounting the preparation in an open dish. (B) 3-color image of keratin (green, Alexa-555), plectin (blue, Alexa-488) and β4 integrin (red, Alexa-647). Approximately 12000 frames where collected for each color channel. Full resolution images are available at <a href="https://osf.io/q684r/" target="_blank">https://osf.io/q684r/</a>.</p

Sealing cell culture dishes to prevent oxygen influx.

<p>(A) O₂ levels, detected by daily fluorescence lifetime-based recording in a WillCo Well sealed with Twinsil glue (blue) or aluminum tape (red), respectively. At 48 days, the seal was broken to test responsiveness of the O₂ sensor (black arrow). (B) A fluorescent O₂ indicator pad was covered with Twinsil (blue) or aluminum tape (red) and submerged in buffer at ambient oxygen levels. O₂ levels were recorded continuously, and at t = 8 min oxygen scavenger was added from concentrated stock. Note the rapid drop in O₂ levels below the Twinsil seal, indicating its permeability to oxygen. (C) WillCo Wells #GWSB 3512-N dish shown with 24-mm coverslip lid (right) and sealed with pieces of aluminum tape (left).</p

Optimizing drift in SR preparations.

<p>(A) Example traces of drift quantifications during 30 min in #3512 dishes (blue), #3512-N dishes (red) and #3512-N dishes sealed with coverslip and adhesive-backed aluminum tape (pink). Shown is the mean displacement away from the origin of immobilized beads during 30 min. (B) Summary of 2D (lateral; square symbols) and 3D (lateral + focus, round symbols) drift experiments. Shown are endpoint drifts at 30 min and at 60 min for the indicated imaging dishes. The WillCo Wells optimized #3512-N dishes display significantly improved stability. Data are mean +/- SEM of >3 experiments each.</p