Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination

Abstract

A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 μm) better suited to three-dimensional (3D) subcellular imaging. As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to ∼0.3 μm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.

Figure 1. Challenges in fluorescence imaging with high spatiotemporal resolution

(a) Increasing volumetric spatial resolution demands smaller voxels (boxes) encompassing fewer signal-generating fluorescent molecules (spheres). (b) Increasing temporal resolution demands that fewer molecules be expended (black spheres) at each time point. (c) To obtain images of acceptable SNR, a certain minimum number of photons must be emitted from these molecules, no matter how few they may be. Scale bar, 5 μm.

Figure 2. Modes of Bessel beam plane illumination microscopy

(a) Wide-field illumination geometry (left) and maximum-intensity projection (MIP) in the x–z plane (right; z, detection axis) from a 3D image stack of a fixed human osteosarcoma cell (U2OS) transfected with plasmids encoding mEmerald fused to human pyruvate dehydrogenase alpha 1 (PDHA1). (b) Bessel sheet mode geometry (left), showing fluorescence excitation from Bessel side lobes (light green) as well as the central peak (dark green), and x–z plane MIP (right) from same cell as in a. (c,d) Bessel SI mode geometry, showing periodic Bessel beam excitation pattern (left) and x–z plane MIPs with single-harmonic (c) and multiharmonic (d) excitation (right). (e) Two-photon excitation (TPE) Bessel sheet mode geometry (left), showing infrared excitation (red) of fluorescence in the central peak (green), with negligible fluorescence in side lobes and x–z plane MIP from a cell (right) similar to those in a–d. (f) Volume rendering in the multiharmonic SI mode (9 phases, 2.4 μm period) of mEmerald-tagged microtubule associated protein 4 (MAP4) in a live U2OS cell. (g) Volume rendering in the TPE sheet mode of mEmerald-labeled mitochondria in a live pig kidney epithelial cell (LLC-PK1 cell line). Insets in f and g show MIPs along orthogonal axes of the cubical volumes shown. Scale bars, 10 μm except 3 μm in insets.

Figure 3. Comparisons of Bessel beam plane illumination to confocal microscopy and DSLM

(a–e) Comparative raw image slices in a plane orthogonal to the coverslip through antibody-labeled microtubules in fixed HeLa cells: point-scanning confocal microscopy (a; Zeiss LSM 510, NA 1.2, 1 Airy unit filtering); line-scanning confocal microscopy (b; Zeiss LSM 5 LIVE, same conditions as in a); DSLM (c; NA 0.2); Bessel single harmonic SI mode (d; 3 phases, 0.9-μm period), and Bessel TPE sheet mode (e). Scale bars, 10 μm (inset, 1 μm). (f) Averages of linecuts (as shown in insets in a–e) through 40 microtubules for each method, with 50% intensity level (dashed line) shown for estimation of the FWHM. (g) Bleaching rates obtained from repeated 3D imaging of mEmerald fused to nuclear histones in fixed HeLa cells, normalized to account for differences in SNR. In addition to modes described in a–e, Bessel linear sheet and Bessel SI multiharmonic 9-phase modes are included. Dashed lines represent a double exponential fit. (h) Maximum-intensity projections of mitochondria in live LLC-PK1 cells for four image volumes as numbered at top from a series of 300 such volumes acquired by Bessel TPE sheet mode (top; 321 images per volume), confocal LSM 5 LIVE (middle; 294 images per volume) and confocal LSM 5 LIVE (bottom; 68 images per volume). Scale bar, 10 μm. (i) Photobleaching curves extracted from the data in h, with 10 μm × 10 μm intensity-renormalized insets extracted from the boxed sub-region shown in the 300^th volume in each case, showing mitochondrial fragmentation under confocal imaging. Scale bar, 3 μm.

Figure 4. Three-dimensional isotropic imaging of live-cell dynamics

(a) Images of ER in a live U2OS cell, visualized in the Bessel multiharmonic 9 phase SI mode, over 45 image volumes: representation of a subset of the 321 image planes (brown lines) comprising each volume (top); images from three indicated image planes; and image volumes after 10 and 30 min of observation. (b) Filopodia at the apical surface of a live HeLa cell, visualized in the Bessel TPE sheet mode over three consecutive image volumes from 100 such volumes taken at 6-s intervals. Filaments that wave (magenta and yellow arrowheads), extend outward (cyan arrowhead) or retract inward (green arrowhead) are marked. (c) African green monkey kidney cell (COS-7) transfected with plasmids encoding mEmerald–c-Src, demonstrating retrograde flow of membrane ruffles (left) and vacuole formation by macropinocytosis (right; arrowheads) in an exemplary plane in a translucent cell view (top). All data were extracted from 73 image stacks taken at 12-s intervals. Scale bars, 5 μm (a,b) and 10 μm (c).

Figure 5. High-speed volumetric imaging of chromosomes in mitosis

(a) Eight image volumes from a series of 200 such volumes depicting mitosis in a LLC-PK1 cell transfected with plasmids encoding mEmerald–histone H2B and imaged in the Bessel TPE sheet mode. Each volume, composed of 200 image planes, was acquired in 1.0 s. The rest interval between stacks varied from 20 s in metaphase and telophase to no rest in early anaphase, to expend more of the photon budget at the points of most rapid evolution. Two chromatids (green and purple) are traced through the series. (b) Four consecutive image volumes from the series, during the fast imaging period in anaphase, in which the two chromatids separate (arrowheads). Times indicate min:s. Scale bars, 5 μm.

Figure 6. Three-dimensional isotropic imaging of protein pairs

(a) Two-color volume rendering acquired in the 9 phase multiharmonic SI mode of microtubules (green) and nuclei (red) in a pair of live U2OS cells transfected with plasmids encoding mEmerald-MAP4 and tdTomato–histone H2B. Images on the right show slices through the cell along the planes shown in the image on the left. (b) Evolution of the Golgi apparatus (magenta) during mitosis of a live LLC-PK1 cell, with views parallel (top) and perpendicular (bottom) to the mitotic plane, showing partial fragmentation in metaphase and anaphase and eventual recondensation in telophase. The Golgi and chromosomes (green) are visualized via mEmerald–Mann II and mEmerald–histone H2B fluorescence, respectively, and were manually segmented for these images. Scale bars, 5 μm.