Established imaging technologies such as widefield and confocal microscopy play key roles in elucidating structure and function in biological systems (Stephens and Allen, 2003
). However, as more detailed information has been needed, particularly in the context of in vivo
imaging, their limitations have become increasingly clear. One of these is the two order of magnitude gap between the level at which proteins interact within the cell, and the resolution limit of light microscopy. Another is the difficulty of imaging specimens in their three-dimensional (3D) entirety without sacrificing spatiotemporal information. Yet a third is the damage induced by light in living systems, particularly when higher resolution, larger volumes, and faster or longer imaging are required (Khodjakov and Rieder, 2006
; Schroeder, 2011
In response to these challenges, new technologies have arisen. In particular, several variants of superresolution microscopy have been developed that offer spatial resolution, depending on the method, from ~100 nm down to the near-molecular level (Schermelleh et al., 2010
). Additionally, plane illumination microscopy has emerged as a powerful tool for less invasive, long-term 3D imaging in developmental biology (Huisken and Stainier, 2009
). Unfortunately, the advantages of these methods are often mutually exclusive: superresolution is limited to specimens with sparsely distributed fluorescent features, either fixed or slowly evolving over only a few time points, while conventional plane illumination sacrifices resolution, particularly along the detection axis, to image at depth in multicellular specimens over broad fields of view. Nevertheless, with a sufficiently thin excitation light sheet, the combination of these methods could prove quite fruitful: the resulting suppression of out-of-focus background could extend widefield cellular superresolution approaches (Gustafsson, 2000
; Betzig et al., 2006
) to much thicker and/or densely fluorescent specimens, while the more efficient expenditure of the fluorescence photon budget in generating only in-focus signal could lead to improved spatiotemporal resolution and reduced phototoxicity.
Recently, we described the use of a scanned Bessel beam to confine the excitation in plane illumination microscopy more tightly to the detection focal plane than traditional Gaussian beams allow (Planchon et al., 2011
). We then introduced two modes of Bessel beam plane illumination to achieve near-isotropic, diffraction-limited 3D imaging of living cells. In the first, two-photon excitation (TPE) is used to eliminate unwanted excitation from side lobes of the Bessel beam, resulting in a thinner light sheet. This mode permits the imaging of living cells over dozens to hundreds of time points at up to 200 slice planes per second, given sufficiently dense fluorescent labeling. In the second mode, the Bessel beam is moved in discrete steps across the focal plane to create a periodic excitation pattern, and an optical sectioning algorithm (OS-SIM, Neil et al., 1997
) is applied to a series of images taken at different phases of the applied excitation pattern to computationally eliminate background fluorescence generated by the side lobes. This mode offers better axial resolution than the TPE mode, as well as simple multicolor capability.
Each of these modes has its own limitations. TPE introduces nonlinear photodamage mechanisms, and multicolor imaging is made difficult by the lower TPE photostability of red fluorescent proteins (FPs) compared to green ones, as well as the need for multiple, expensive ultra-fast lasers. On the other hand, the OS-SIM mode is slow and surprisingly far more phototoxic to live cells than the TPE mode, and thus is almost always restricted to imaging fixed cells. In addition, the mathematical operations employed in the OS-SIM algorithm call into question the strict validity of the resulting image reconstructions, a concern reinforced by variations in shape and intensity seen for ostensibly identical structures in such reconstructions.
Here we address all these issues by introducing Bessel beam superresolution structured plane illumination (Bessel plane SR-SIM), which combines the tightly confined planar illumination of a periodically stepped Bessel beam with the principles of widefield 3D superresolution structured illumination microscopy (widefield SR-SIM, Gustafsson et al., 2008
) to achieve non-invasive multicolor imaging of rapid 3D biological processes in thick living specimens. Although providing only modest gains in theoretical
resolution extension beyond the diffraction limit, we demonstrate, by comparison to Bessel plane OS-SIM and spinning disk confocal microscopy, substantial gains in the practical
resolvability, contrast, and signal-to-noise ratio (SNR) on samples ranging from single microtubules to D. melanogaster
embryos. Similarly, although the theoretical lateral resolution limits are slightly less than in widefield SR-SIM, we show the superiority of Bessel plane SR-SIM when imaging live samples ~10 µm thick or greater, such as mitotic cells and C. elegans
embryos. Furthermore, we demonstrate substantially reduced phototoxicity relative to the Bessel plane TPE mode and spinning disk confocal microscopy when imaging fast membrane dynamics both internally and at the surface of densely fluorescent COS-7 and light-sensitive D. discoideum
cells. Lastly, we show that SR-SIM can be extended to image portions of specimens hundreds of microns in size, such as the adult brain of D. melanogaster
and the entire C. elegans
L1 larval worm, by combining Bessel structured plane illumination with the greater depth penetration afforded by two-photon excitation.
In short, the theoretical resolution limits of microscopes are difficult to achieve in many biological samples, particularly as one pushes further and further beyond the diffraction limit. In addition, the successful imaging of dynamics in living systems, particularly in 3D, involves tradeoffs of spatial resolution, temporal resolution, and phototoxicity that render irrelevant a single minded focus on spatial resolution alone. Through a unique combination of high speed, low phototoxicity, and volumetric resolution ~2–3× beyond the diffraction limit, Bessel plane SR-SIM permits the investigation of complex biological processes that require fast, high resolution 3D imaging to visualize subcellular detail over many time points, not only in the optically tractable but biologically artificial context of immortalized cultured cells, but also in the more physiologically relevant context of interacting cells within whole multicellular organisms. Among other examples, we use these capabilities to identify and track every chromosome in an aneuploid cell during mitosis, thus opening the door to direct visualization of stereotypical chromosome arrangements and deviations therefrom during cell division, and identify a diverse range of actin dynamics at different planes throughout the volume of motile cells, such as a correlation between the lateral movement of single D. discoideum cells and rapid flows of actin to and from the supporting substrate.