Build and operation of a custom 3D, multicolor, single-molecule localization microscope.

Single-molecule localization microscopy (SMLM) enables imaging scientists to visualize biological structures with unprecedented resolution. Particularly powerful implementations of SMLM are capable of three-dimensional, multicolor and high-throughput imaging and can yield key biological insights. However, widespread access to these technologies is limited, primarily by the cost of commercial options and complexity of de novo development of custom systems. Here we provide a comprehensive guide for interested researchers who wish to establish a high-end, custom-built SMLM setup in their laboratories. We detail the initial configuration and subsequent assembly of the SMLM, including the instructions for the alignment of all the optical pathways, the software and hardware integration, and the operation of the instrument. We describe the validation steps, including the preparation and imaging of test and biological samples with structures of well-defined geometries, and assist the user in troubleshooting and benchmarking the system's performance. Additionally, we provide a walkthrough of the reconstruction of a super-resolved dataset from acquired raw images using the Super-resolution Microscopy Analysis Platform. Depending on the instrument configuration, the cost of the components is in the range US$95,000-180,000, similar to other open-source advanced SMLMs, and substantially lower than the cost of a commercial instrument. A builder with some experience of optical systems is expected to require 4-8 months from the start of the system construction to attain high-quality three-dimensional and multicolor biological images.

Image restoration of degraded time-lapse microscopy data mediated by near-infrared imaging.

Time-lapse fluorescence microscopy is key to unraveling biological development and function; however, living systems, by their nature, permit only limited interrogation and contain untapped information that can only be captured by more invasive methods. Deep-tissue live imaging presents a particular challenge owing to the spectral range of live-cell imaging probes/fluorescent proteins, which offer only modest optical penetration into scattering tissues. Herein, we employ convolutional neural networks to augment live-imaging data with deep-tissue images taken on fixed samples. We demonstrate that convolutional neural networks may be used to restore deep-tissue contrast in GFP-based time-lapse imaging using paired final-state datasets acquired using near-infrared dyes, an approach termed InfraRed-mediated Image Restoration (IR2). Notably, the networks are remarkably robust over a wide range of developmental times. We employ IR2 to enhance the information content of green fluorescent protein time-lapse images of zebrafish and Drosophila embryo/larval development and demonstrate its quantitative potential in increasing the fidelity of cell tracking/lineaging in developing pescoids. Thus, IR2 is poised to extend live imaging to depths otherwise inaccessible.

Compact, high-speed multi-directional selective plane illumination microscopy.

We present an elegant scheme for providing multi-directional illumination in selective plane illumination microscopy (SPIM). Light sheets can be delivered from one of two opposed directions at a time and pivoted about their center for efficient stripe artifact suppression using only a single galvanometric scanning mirror to perform both functions. The scheme results in a much smaller instrument footprint and allows multi-directional illumination with reduced expense compared with comparable schemes. Switching between the illumination paths is near instantaneous and the whole-plane illumination scheme of SPIM maintains the lowest rates of photodamage, which is often sacrificed by other recently reported destriping strategies. The ease of synchronization allows this scheme to be used at higher speeds than resonant mirrors typically used in this regard. We provide validation of this approach in the dynamic environment of the zebrafish beating heart, where imaging at up to 800 frames per second is demonstrated alongside efficient suppression of artifacts.

Oblique plane microscope for mesoscopic imaging of freely moving organisms with cellular resolution.

Several important questions in biology require non-invasive and three-dimensional imaging techniques with an appropriate spatiotemporal resolution that permits live organisms to move in an unconstrained fashion over an extended field-of-view. While selective-plane illumination microscopy (SPIM) has emerged as a powerful method to observe live biological specimens at high spatio-temporal resolution, typical implementations often necessitate constraining sample mounting or lack the required volumetric speed. Here, we report on an open-top, dual-objective oblique plane microscope (OPM) capable of observing millimeter-sized, freely moving animals at cellular resolution. We demonstrate the capabilities of our mesoscopic OPM (MesOPM) by imaging the behavioral dynamics of the sea anemone Nematostella vectensis over 1.56 × 1.56 × 0.25 mm at 1.5 × 2.8 × 5.3 µm resolution and 0.5 Hz volume rate.

Super-Resolution Microscopy of the Synaptonemal Complex within the Caenorhabditis elegans Germline.

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures the interaction of homologous chromosomes is the assembly of the synaptonemal complex (SC) in meiotic prophase I. Even though there is little sequence homology between protein components within the SC among different species, the general structure of the SC has been highly conserved during evolution. In electron micrographs, the SC appears as a tripartite, ladder-like structure composed of lateral elements or axes, transverse filaments, and a central element. However, precisely identifying the localization of individual components within the complex by electron microscopy to determine the molecular structure of the SC remains challenging. By contrast, fluorescence microscopy allows for the identification of individual protein components within the complex. However, since the SC is only ~100 nm wide, its substructure cannot be resolved by diffraction-limited conventional fluorescence microscopy. Thus, determining the molecular architecture of the SC requires super-resolution light microscopy techniques such as structured illumination microscopy (SIM), stimulated-emission depletion (STED) microscopy, or single-molecule localization microscopy (SMLM). To maintain the structure and interactions of individual components within the SC, it is important to observe the complex in an environment that is close to its native environment in the germ cells. Therefore, we demonstrate an immunohistochemistry and imaging protocol that enables the study of the substructure of the SC in intact, extruded Caenorhabditis elegans germline tissue with SMLM and STED microscopy. Directly fixing the tissue to the coverslip reduces the movement of the samples during imaging and minimizes aberrations in the sample to achieve the high resolution necessary to visualize the substructure of the SC in its biological context.

Dynamic and non-contact 3D sample rotation for microscopy.

Precise sample orientation is crucial for microscopy but is often performed with macroscopic tools and low accuracy. In vivo imaging of growing and developing samples even requires dynamic adaptation of the sample orientation to continuously achieve optimal imaging. Here, we present a method for freely positioning a sample in 3D by introducing magnetic beads and applying a magnetic field. We demonstrate magnetic orientation of fixed mouse embryos and artemia, and live zebrafish embryos and larvae on an epi-fluorescence microscope and on a light-sheet system for optimal imaging.

Adaptable, illumination patterning light sheet microscopy.

Minimally-invasive optical imaging requires that light is delivered efficiently to limit the detrimental impact of photodamage on delicate biological systems. Light sheet microscopy represents the exemplar in tissue specific optical imaging of small and mesoscopic samples alike. However, further gains towards gentler imaging require a more selective imaging strategy to limit exposure to multiple yet discrete tissues without overexposing the sample, particularly where the information content is sparse or particularly optically sensitive tissues are present. The development of sample-adaptive imaging techniques is crucial in pursuit of the next generation of smart, autonomous microscopes. Herein, we report a microscope capable of performing 4D (x, y, z, t) light patterning to selectively illuminate multiple, rapidly reconfigurable regions of interest while maintaining the rapid imaging speed and high contrast associated with light sheet microscopy. We illustrate this utility in living zebrafish larvae and phantom samples.

A guide to light-sheet fluorescence microscopy for multiscale imaging.

The impact of light-sheet fluorescence microscopy (LSFM) is visible in fields as diverse as developmental and cell biology, anatomical science, biophysics and neuroscience. Although adoption among biologists has been steady, LSFM has not displaced more traditional imaging methods despite its often-superior performance. One reason for this is that the field has largely conformed to a do-it-yourself ethic, although the challenges of big image data cannot be overstated. With the most powerful implementations of LSFM available to only a few groups worldwide, the scope of this technique is unnecessarily limited. Here we elucidate the key developments and define a simple set of underlying principles governing LSFM. In doing so, we aim to clarify the decisions to be made for those who wish to develop and use bespoke light-sheet systems and to assist in identifying the best approaches to apply this powerful technique to myriad biological questions.

Diffusion and reactivity in ultraviscous aerosol and the correlation with particle viscosity.

The slow transport of water, organic species and oxidants in viscous aerosol can lead to aerosol existing in transient states that are not solely governed by thermodynamic principles but by the kinetics of gas-particle partitioning. The relationship between molecular diffusion constants and particle viscosity (for example, as reflected in the Stokes-Einstein equation) is frequently considered to provide an approximate guide to relate the kinetics of aerosol transformation with a material property of the aerosol. We report direct studies of both molecular diffusion and viscosity in the aerosol phase for the ternary system water/maleic acid/sucrose, considering the relationship between the hygroscopic response associated with the change in water partitioning, the volatilisation of maleic acid, the ozonolysis kinetics of maleic acid and the particle viscosity. Although water clearly acts as a plasticiser, the addition of minor fractions of other organic moieties can similarly lead to significant changes in the viscosity from that expected for the dominant component forming the organic matrix (sucrose). Here we highlight that the Stokes-Einstein relationship between the diffusion constant of water and the viscosity of the particle may be more than an order of magnitude in error, even at viscosities as low as 1 Pa s. We show that the thermodynamic relationships of hygroscopic response that underpin such kinetic determinations must be accurately known to retrieve accurate values for diffusion constants; such data are often not available. Further, we show that scaling of the diffusion constants of organic molecules of similar size to those forming the matrix with particle viscosity may be well represented by the Stokes-Einstein equation, suppressing the apparent volatility of semi-volatile components. Finally, the variation in uptake coefficients and diffusion constants for oxidants and small weakly interacting molecules may be much less dependent on viscosity than the diffusion constants of more strongly interacting molecules such as water.

Precise, contactless measurements of the surface tension of picolitre aerosol droplets.

The surface composition and surface tension of aqueous droplets can influence key aerosol characteristics and processes including the critical supersaturation required for activation to form cloud droplets in the atmosphere. Despite its fundamental importance, surface tension measurements on droplets represent a considerable challenge owing to their small volumes. In this work, we utilize holographic optical tweezers to study the damped surface oscillations of a suspended droplet (<10 µm radius) following the controlled coalescence of a pair of droplets and report the first contactless measurements of the surface tension and viscosity of droplets containing only 1-4 pL of material. An advantage of performing the measurement in aerosol is that supersaturated solute states (common in atmospheric aerosol) may be accessed. For pairs of droplets starting at their equilibrium surface composition, surface tensions and viscosities are consistent with bulk equilibrium values, indicating that droplet surfaces respond to changes in surface area on microsecond timescales and suggesting that equilibrium values can be assumed for growing atmospheric droplets. Furthermore, droplet surfaces are shown to be rapidly modified by trace species thereby altering their surface tension. This equilibration of droplet surface tension to the local environmental conditions is illustrated for unknown contaminants in laboratory air and also for droplets exposed to gas passing through a water-ethanol solution. This approach enables precise measurements of surface tension and viscosity over long time periods, properties that currently are poorly constrained.

Probing the micro-rheological properties of aerosol particles using optical tweezers.

The use of optical trapping techniques to manipulate probe particles for performing micro-rheological measurements on a surrounding fluid is well-established. Here, we review recent advances made in the use of optical trapping to probe the rheological properties of trapped particles themselves. In particular, we review observations of the continuous transition from liquid to solid-like viscosity of sub-picolitre supersaturated solution aerosol droplets using optical trapping techniques. Direct measurements of the viscosity of the particle bulk are derived from the damped oscillations in shape following coalescence of two particles, a consequence of the interplay between viscous and surface forces and the capillary driven relaxation of the approximately spheroidal composite particle. Holographic optical tweezers provide a facile method for the manipulation of arrays of particles allowing coalescence to be controllably induced between two micron-sized aerosol particles. The optical forces, while sufficiently strong to confine the composite particle, are several orders of magnitude weaker than the capillary forces driving relaxation. Light, elastically back-scattered by the particle, is recorded with sub-100 ns resolution allowing measurements of fast relaxation (low viscosity) dynamics, while the brightfield image can be used to monitor the shape relaxation extending to times in excess of 1000 s. For the slowest relaxation dynamics studied (particles with the highest viscosity) the presence and line shape of whispering gallery modes in the cavity enhanced Raman spectrum can be used to infer the relaxation time while serving the dual purpose of allowing the droplet size and refractive index to be measured with accuracies of ±0.025% and ±0.1%, respectively. The time constant for the damped relaxation can be used to infer the bulk viscosity, spanning from the dilute solution limit to a value approaching that of a glass, typically considered to be >10(12) Pa s, whilst the frequencies of the normal modes of the oscillations of the particle can be used to infer surface properties. We will review the use of optical tweezers for studying the viscosity of aerosol particles and discuss the potential use of this micro-rheological tool for probing the fundamental concepts of phase, thermodynamic equilibrium and metastability.

Toward optical-tweezers-based force microscopy for airborne microparticles.

Optical tweezers have found widespread application in biological and colloidal physics for the measurement of pN forces over nanometer to micrometer length scales. Similar aerosol-phase measurements of interparticle force have not been reported in spite of the potential to better resolve particle coagulation kinetics. Various refractive index mismatches in the beam path as well as the need to explicitly account for gravity and inertial particle motion provide a number of challenges that must be overcome to make such measurements tractable. In this regard, we demonstrate schemes by which the particle position and trap stiffness may be unambiguously measured using bright-field microscopy with resolution comparable with analogous condensed-phase measurements. Moreover, some of the challenges of working with highly dynamic aqueous particles are introduced and exploited to observe size-dependent phenomena in aerosol optical tweezers. Notably, when combined with cavity-enhanced Raman spectroscopy, this provides a unique opportunity to explore trapping forces over a continuum of particle size and refractive index. It is expected that the methods developed will provide a basis for the measurement of pairwise interaction forces in aerosol optical tweezers while providing a probe of fundamental airborne particle trapping dynamics.