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.

Superpenetration optical microscopy by iterative multiphoton adaptive compensation technique

“…immunology | neuron imaging | nonlinear imaging | lymphocyte imaging | nonlinear iterative feedback O ptical microscopy has revolutionized biomedical research in the past few decades (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Recent advances in spatial resolution (1,16), labeling techniques (2,11), imaging speed (7,10), and new contrast mechanisms (4,15,17) have greatly expanded the capabilities of optical microscopy; however, a major drawback of this technique is the limited imaging penetration depth because biological tissues are rarely transparent (3,6,18,19).…”

“…Recent advances in spatial resolution (1,16), labeling techniques (2,11), imaging speed (7,10), and new contrast mechanisms (4,15,17) have greatly expanded the capabilities of optical microscopy; however, a major drawback of this technique is the limited imaging penetration depth because biological tissues are rarely transparent (3,6,18,19). Little advance has been made in imaging depth since the invention of multiphoton microscopy (MPM) (3) about two decades ago.…”

How the Internet of cells has biologists buzzing

“…Smith is developing methods to visualize membrane nanotubes using lattice light-sheet microscopy, which monitors planes of light to build up 3D images. He hopes that the technique will be able to capture the process of material transfer from one cell to another, from start to finish 8 . Smith admits that he's taking a career risk: a colleague recently warned him this area was 'fringey' .…”

Unleashing Optics and Optoacoustics for Developmental Biology

“…How one generates this light sheet will determine how sensitive the setup is to scattering. The original setups made use of cylindrical lenses to generate the light sheet, while further developments employed structured illumination such as digitally scanned light sheet microscopy (DSLM) [47] and DSLM-SI [48], sometimes combined with multi-photon excitation to improve resolution even further [49][50][51] (Figure 2A shows high-speed volumetric imaging of chromosomes in mitosis with sub-micron resolution).…”