Single particle tracking of GPI-GFP in live CV1 cells using functionalized Quantum dots.
a. Schematic of the construct used to track single GPI- molecules with quantum dots. b. Exemplary raw images of single fluorescent Quantum Dots. Image dimension is 1.6 µm x 1.6 µm. c. Trajectories of several single molecules visualized on a top-light illuminated image of CV1 cells. Scale bar represents 10 µm. d. Selected trajectory of a single particle over 167 time-points. Sampling rate is 25 ms, pixel size 104 nm. e. Histogram of the diffusion coefficient of around 5000 tracks with 20 or more detected time points each. f. MSS (moment scaling spectrum) of the same population as plotted in e. Since we were readily able to detect single quantum dots, which are single emitters of few nanometers in size, we reasoned, it might be possible to perform single molecule localization microscopy (SMLM) experiments as well. We immuno-stained CV-1 cells against tubulin using AF647 coupled secondary antibodies and incubated coverslips in BME/GLOX buffer for effective pumping into the dark state. When we then illuminated our sample with high intensity laser light (in our case ∼ 1kW/cm2), we found that the intense staining quickly faded and gave way to the typically observed blinking in SMLM experiments. We took a timeseries of 30’000 frames and localized the detected emitters using Thunderstorm (Ovesný et al., 2014). Even though we were limited to widefield illumination, we achieved a drastic improvement in resolution in reconstructed SMLM micrographs (Figure 6a). When we measured the profile of individual microtubules, we could easily detect the typical “railroad track” pattern with a spacing of 38 – 43 nm, indicative of high quality SMLM imaging (Figure 6b,c). To quantify the achieved resolution, we used Fourier ring correlation (Nieuwenhuizen et al., 2013) and found that the overall achieved resolution was 93 nm, clearly below the optical resolution limit (Figure 6d).