Light-sheet microscopy: a tutorial

This paper is intended to give a comprehensive review of light-sheet (LS) microscopy from an optics perspective. As such, emphasis is placed on the advantages that LS microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled several biomedical applications. Then, the basics are presented for understanding how a LS microscope works. This is followed by a presentation of a tutorial for LS microscope designs, each working at different resolutions and for different applications. Then, based on a numerical Fourier analysis and given the multiple possibilities for generating the LS in the microscope (using Gaussian, Bessel, and Airy beams in the linear and nonlinear regimes), a systematic comparison of their optical performance is presented. Finally, based on advances in optics and photonics, the novel optical implementations possible in a LS microscope are highlighted.Peer ReviewedPostprint (published version

Computational localization microscopy with extended axial range

A new single-aperture 3D particle-localization and tracking technique is presented that demonstrates an increase in depth range by more than an order of magnitude without compromising optical resolution and throughput. We exploit the extended depth range and depth-dependent translation of an Airy-beam PSF for 3D localization over an extended volume in a single snapshot. The technique is applicable to all bright-field and fluorescence modalities for particle localization and tracking, ranging from super-resolution microscopy through to the tracking of fluorescent beads and endogenous particles within cells. We demonstrate and validate its application to real-time 3D velocity imaging of fluid flow in capillaries using fluorescent tracer beads. An axial localization precision of 50 nm was obtained over a depth range of 120μm using a 0.4NA, 20× microscope objective. We believe this to be the highest ratio of axial range-to-precision reported to date

Super-resolution and super-localization microscopy: a novel tool for imaging chemical and biological processes

Optical microscopy imaging of single molecules and single particles is an essential method for studying fundamental biological and chemical processes at the molecular and nanometer scale. The best spatial resolution (~ λ/2) achievable in traditional optical microscopy is governed by the diffraction of light. However, single molecule-based super-localization and super-resolution microscopy imaging techniques have emerged in the past decade. Individual molecules can be localized with nanometer scale accuracy and precision for studying of biological and chemical processes. The obtained spatial resolution for plant cell imaging is not yet as good as that achieved in mammalian cell imaging. Numerous technical challenges, including the generally high fluorescence background due to significant autofluorescence of endogenous components, and the presence of the cell wall (\u3e 250 nm thickness) limit the potential of super-resolution imaging in studying the cellular processes in plants. Here variable-angle epi-fluorescence microscopy (VAEM) was combined with localization based super-resolution imaging, direct stochastic optical reconstruction microscopy (dSTORM), to demonstrate imaging of cortical microtubule (CMT) network in the Arabidopsis thaliana root cells with 20 – 40 nm spatial resolution for the first time. With such high spatial resolution, the subcellular organizations of CMTs within single cells, and different cells in many regions along the root, were analyzed quantitatively. Nearly all of these technical advances in super-localization and super-resolution microscopy imaging were originally developed for biological studies. More recently, however, efforts in super-resolution chemical imaging started to gain momentum. New discoveries that were previously unattainable with conventional diffraction-limited techniques have been made, such as a) super-resolution mapping of catalytic reactions on single nanocatalysts and b) mechanistic insight into protein ion-exchange adsorptive separations. Furthermore, single molecules and single particles were localized with nanometer precision for resolving the dynamic behavior of single molecules in porous materials. This work uncovered the heterogeneous properties of the pore structures. In this dissertation, the coupling of molecular transport and catalytic reaction at the single molecule and single particle level in multilayer mesoporous nanocatalysts was elucidated. Most previous studies dealt with these two important phenomena separately. A fluorogenic oxidation reaction of non-fluorescent amplex red to highly fluorescent resorufin was tested. The diffusion behavior of single resorufin molecules in aligned nanopores was studied using total internal reflection fluorescence microscopy (TIRFM). To fully understand the working mechanisms of biological processes such as stepping of motor proteins requires resolving both the translational movement and the rotational motions of biological molecules or molecular complexes. Nanoparticle optical probes have been widely used to study biological processes such as membrane diffusion, endocytosis, and so on. The greatly enhanced absorption and scattering cross sections at the surface plasmon resonance (SPR) wavelength make nanoparticles an ideal probe for high precision tracking. Furthermore, gold nanorods (AuNRs) were used for resolving orientation changes in all three dimensions. The translational and rotational motions of AuNRs in glycerol solutions were tracked with fast imaging rate up to 500 frames per second (fps) in reflected light sheet microscopy (RLSM). The effect of imaging rates on resolving details of single AuNR motions was studied