Smith et al. describe a microscopy technique that instantaneously captures 3D images of live cells, allowing them to track the movement of single mRNAs in the nucleus.
3D microscopy images are usually obtained by capturing a series of 2D images at different focal planes, but this process is often too slow to follow the rapid movements of individual molecules within a particular region of the cell. One potential solution is to use multifocus microscopy, in which the detection light is split into nine different focal planes that can be imaged simultaneously. However, the acquired images need to be carefully realigned with each other in order to generate an accurate 3D snapshot of the region of interest.
Smith et al. developed a way to achieve this realignment that they called 3D single-molecule real-time (3D-SMRT) microscopy. The researchers used multifocus microscopy to image the nuclei of cultured fibroblasts expressing fluorescent proteins labeling the nuclear pores and β-actin mRNA. The nuclei were also stained with a vital DNA dye that emits light across a broad spectral range, allowing the researchers to precisely align the images obtained in each focal plane and color channel. The entire nucleus could thus be imaged 10 times per second, fast enough to track the diffusion of individual β-actin transcripts.
The researchers found that β-actin mRNAs could freely access every part of the nucleus; the transcripts weren’t excluded from heterochromatin-rich regions, for example. Nevertheless, because of the nucleus’s disc-like shape, most mRNAs were located within 0.5 µm of a nuclear pore. Senior author David Grunwald says that such precise spatiotemporal localization studies are just the beginning of what 3D microscopy will eventually be capable of.
Text by Ben Short