Mechanochemical delivery and dynamic tracking of fluorescent quantum dots in the cytoplasm and nucleus of living cells - PubMed (original) (raw)

Mechanochemical delivery and dynamic tracking of fluorescent quantum dots in the cytoplasm and nucleus of living cells

Kyungsuk Yum et al. Nano Lett. 2009 May.

Abstract

Studying molecular dynamics inside living cells is a major but highly rewarding challenge in cell biology. We present a nanoscale mechanochemical method to deliver fluorescent quantum dots (QDs) into living cells, using a membrane-penetrating nanoneedle. We demonstrate the selective delivery of monodispersed QDs into the cytoplasm and the nucleus of living cells and the tracking of the delivered QDs inside the cells. The ability to deliver and track QDs may invite unconventional strategies for studying biological processes and biophysical properties in living cells with spatial and temporal precision, potentially with molecular resolution.

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Figures

Figure 1

Nanoscale mechanochemical delivery of QDs into living cells. (a) Schematic of the mechanochemical delivery of QDs into living cells. Inset, optical microscope image of a typical nanoneedle. (b) Procedure for functionalization of the nanoneedle and surface attachment of QDs. A NH2-terminated SAM was formed on the Au-coated nanoneedle by the chemisorption of thiols on gold. The nanoneedle was biotinylated by reacting the NH2 surface group with N - Hydroxysulfosuccinimide (sulfo-NHS) esters of biotin (sulfo-NHS-SS-biotin), forming a stable amide bond. Streptavidin-coated QDs were attached onto the nanoneedle by the specific binding of streptavidin and biotin. Scale bar, 5 μm

Figure 2

Delivery of QDs into the cytoplasm of living HeLa cells. (a-c) Optical microscope images of a nanoneedle functionalized with QDs during the QD delivery experiment, showing the nanoneedle penetrating through the cell membrane. The whole process was monitored in situ in the bright-field (a), the fluorescence (b), or the combined bright-field and fluorescence (c) image mode. The QDs attached on the nanoneedle are shown in red in (b) and bright white in (c). The gradually unfocused dark shade on the right side in (a) and (c) is the tip of the macroscopic needle on which the nanoneedle is attached. (d-f) Bright-field (d) and fluorescence (e) images of the cell after the QD delivery, and the overlay image (f) of (d )and (e) acquired in one focal plane. (g) and (h) the fluorescence and overlay images acquired in two additional focal planes. The arrows indicate QDs (red). Scale bars, 10 μm

Figure 3

Delivery of QDs into the nucleus of living HeLa cells. (a) Overlay of bright-filed and fluorescence images of the cell after the nuclear delivery. (b) Enlarged fluorescence image of the region marked in (a). (c) Overlay of bright-field and fluorescence images of the region marked in (a). (d-e) Delivery of QDs into the nucleus of a living HeLa cell expressing GFP on the nuclear envelope. The nuclear envelope (d) of the cell (green) was identified with the GFP filter and the cell (e) was imaged at the same focus with the QD filter. (f) Overlay of (d) and (e). The dotted lines locate the boundary of the nucleus. The arrows indicate QDs (red). Scale bars, 10 μm in (a) and (d), and 5 μm in (b).

Figure 4

Time trace of fluorescence intensity and tracking of QDs inside living HeLa cells. (a) Typical time trace of the fluorescence intensity of stationary QDs (red) in living cell plotted with the background signal of neighboring areas (black), showing the blinking pattern (see also Supplementary Videos 1 and 2). For comparison, the intensity variation of a single QD on a glass slide prepared by spreading a highly diluted QD dispersion (0.1 nM) on a glass slide is shown as labeled. (b) A fluorescence image sequence showing the QD in the living cell during the time period of 19-25 s in (a). (c) Tracking of a QD inside living cells (see also Supplementary Video 3). A series of fluorescence images shows the trajectory (the green line) of a tracked QD. (d) Mean-square displacement (MSD) versus time data show three types of characteristic motion of QDs: free diffusive (red square), confined (blue square), and stationary (black square). The solid red line and blue line are the line fit on the data based on a free diffusion model and a confined diffusion model, respectively. (e) Diffusion of QDs in the cytoplasm. The diffusion coefficient D (0.08-3.8 μm2/s, mean 0.8 μm2/s, n = 20) was determined by fitting a free diffusion model MSD(t) = 4D_t_ to the initial few data points of each MSD versus time curve acquired from tracking of different QDs. The fitted lines are shown in solid red. The dashed blue line indicates the MSD expected for freely diffusing QDs in aqueous solution (_D_0 = 17 μm2/s). The dashed green lines are reference lines for D of 1.0 and 4.0 μm2/s as labeled. Scale bars, 500 nm in (b) and 1 μm in (c).

Figure 5

Confined diffusion of a QD in the nucleus of a living HeLa cell. (a-b) Confined diffusion of a QD in the region marked in (a) inside the nucleus of a living HeLa cell and the trajectory of the QD (b) (see also Supplementary Video 4). (c) Corresponding MSD versus time curve (blue) for this QD (A). The data for other stationary QDs (B) (black) are also shown for comparison. The solid line (blue) in (c) is the fit based on a confined diffusion model. The fit estimates the diffusion coefficient of the QD (A) to be 0.01 μm2/s and the size of the confined domain LA to be ∼300 nm, consistent with the typical size of the nuclear microdomains.

References

1. Weiss S. Fluorescence spectroscopy of single biomolecules. Science. 1999;283:1676–1683. - PubMed
1. Xie XS, Yu J, Yang WY. Living cells as test tubes. Science. 2006;312:228–230. - PubMed
1. Evanko D. Watching single molecules in cells. Nature Methods. 2008;5:25.
1. Moerner WE. New directions in single-molecule imaging and analysis. Proc. Natl Acad. Sci. USA. 2007;104:12596–12602. - PMC - PubMed
1. Neuman KC, Nagy A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nature Methods. 2008;5:491–505. - PMC - PubMed

Mechanochemical delivery and dynamic tracking of fluorescent quantum dots in the cytoplasm and nucleus of living cells - PubMed (original) (raw)