Credit: E. Betzig, T. Planchon & L. Gao

The damaging effects of prolonged exposure to light have long stymied analysis of dynamic processes in single living cells at high spatial resolution in real time. Photobleaching, which often results from light that illuminates parts of the sample outside the area in focus, further cuts into microscopists' so-called photon budget. Reporting in Nature Methods, Planchon et al.1 describe the use of Bessel beam plane-illumination microscopy to address these challenges. The ability to acquire two-dimensional image planes at rates approaching 200 frames s−1 enables them to assemble, within <10 s, three-dimensional images with resolutions down to ∼0.3 μm.

The approach is the first application of plane-illumination microscopy to single cells. Plane illumination confines excitation to the area in focus by shining a sheet of light through the side of the sample and illuminating only the specific plane that the objective above the sample is focused on. However, the broad sheets of light produced by the Gaussian beams used to image multicellular structures at single-cell resolution are too thick to monitor subcellular events.

To circumvent this limitation, Planchon et al. turned to Bessel beams—very narrow nondiffracting light rays each surrounded by concentric rings of less intense light. The key challenge lay in eliminating the phototoxicity, photobleaching and out-of-focus haze caused by the weaker peripheral light. The authors addressed this problem by using two-photon excitation and an approach called structured illumination, either alone or together. Structured illumination involves rapidly switching the beam on and off instead of sweeping it across the sample continuously. The combination of both approaches is slightly superior to either alone, but comes at the expense of slower image acquisition than can be obtained using a continuously swept beam.

As depicted in this montage, the authors use the approach to image (clockwise from left) internal architecture and vacuoles in a monkey kidney cell (gold), membrane ruffles at the surface of a monkey kidney cell (orange), microtubules in a pig kidney cell (green), mitochondria in a human osteosarcoma cell (blue and blue-green) and chromosomes during mitosis of a pig kidney cell (orange). Two-color live imaging at the same level of four-dimensional spatiotemporal detail is possible for processes that occur at rates compatible with the time needed to collect frames at the desired spatial resolution. Proteins that participate in processes with faster dynamics can be monitored by labeling them with the same fluorescent tag, as long as they are known a priori to remain spatially segregated during analysis.

The authors envision that better resolution is within reach, by, for example, combining the approach with techniques used in super-resolution microscopy.