Real-time tracking of single RNA molecules

The ability to track fast moving particles in 3D is limited in classic light microscopy as image stacks are acquired as a series of two-dimensional planes. Here, we reconstruct data from multifocal-microscopy that instantaneously captures entire 3D image stacks of live cells and show that β-actin mRNAs freely access the entire nucleus.

HFSP Long-Term Fellow Stephan Preibisch and colleagues
authored on Mon, 22 June 2015

Imaging single proteins or RNAs in living cells is a powerful tool to study details of regulation and organization of cells at molecular level. Typically, in three-dimensional (3D) microscopy images are acquired by sequentially capturing a series of 2D images. However, the time required to step through the sample often interferes with the capability to image large numbers of rapidly moving particles in 3D space. We therefore applied multifocus microscopy (MFM) to instantaneously capture entire 3D single-molecule real-time (3D-SMRT) images of live cells with a rate of 10 volumes per second (Figure a,b).

Figure: (a) Schematic drawing of a multifocal microscope able to acquire 9 z-planes in parallel using a multi-focal grating (MFG). (b) Magnified exemplary images from (a) (red box) show three simultaneously acquired image planes of nuclear pores (white arrowheads). Note the invagination in the nucleus. (c) Illustration of 9 z-planes in two channels (blue, black) after acquisition with the MFM and their misalignment that we account for using geometric local descriptor matching (e). (d) Illustration of the computed position of all z-planes of two channels. (e) Final reconstructed 3D image after deconvolution showing nuclear pores (red), β-actin mRNA (green) and DNA (blue).

The acquired 3D snapshots require image reconstruction in order to reliably track individual RNA molecules. Based on a DNA stain that is excited at all imaged wavelengths we developed image registration tools to align the instantaneously captured image planes of the 3D volume (Figure c,e), and to accurately identify the location of each of the image planes in the 3rd dimension (axial – z, Figure d). The final image volumes are corrected for chromatic as well as sample-induced aberrations with a remaining error of 80nm (Figure f).

After a custom deconvolution step taking into account spherical aberration and wavelength-dependent defocus (Figure f), individual MS2-tagged β-actin mRNA molecules and nuclear pore complexes are tracked for up to 30 seconds in nuclei of mouse fibroblasts. We find that β-actin mRNAs freely access any region of the nucleus and, for example, do not exclude heterochromatin-rich regions. Taking into account the 3D spatial inhomogeneity of the nucleus we show that nevertheless most of the mRNA molecules are less than 0.5μm away from a nuclear pore.


Nuclear accessibility of β-actin mRNA measured by 3D single-molecule real time (3D-SMRT) microscopy. Carlas Smith*, Stephan Preibisch*, Aviva Joseph, Bernd Rieger, Sjoerd Stallinga, Eugene Myers, Robert H. Singer and David Grunwald (2015), Journal of Cell Biology 209(4), 609-619.


Video 1 - Lateral alignment of image panels 

Video 2 - Simultaneous 3D recording of β-actin mRNA mobility inside a living cell

Video 3 - 360° rotation of DNA–RNA interaction

Video 4 - 360° rotation of RNA–NPC interaction


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Commentary “A SMRTer way to track molecules”