Correlative Organelle Microscopy: Fluorescence Guided Volume Electron Microscopy of Intracellular Processes.

Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. Therefore, directly linking molecular to nanoscale ultrastructural information is crucial in understanding cellular physiology. Volume or three-dimensional (3D) correlative light and electron microscopy (volume-CLEM) holds unique potential to explore cellular physiology at high-resolution ultrastructural detail across cell volumes. However, the application of volume-CLEM is hampered by limitations in throughput and 3D correlation efficiency. In order to address these limitations, we describe a novel pipeline for volume-CLEM that provides high-precision (<100 nm) registration between 3D fluorescence microscopy (FM) and 3D electron microscopy (EM) datasets with significantly increased throughput. Using multi-modal fiducial nanoparticles that remain fluorescent in epoxy resins and a 3D confocal fluorescence microscope integrated into a Focused Ion Beam Scanning Electron Microscope (FIB.SEM), our approach uses FM to target extremely small volumes of even single organelles for imaging in volume EM and obviates the need for post-correlation of big 3D datasets. We extend our targeted volume-CLEM approach to include live-cell imaging, adding information on the motility of intracellular membranes selected for volume-CLEM. We demonstrate the power of our approach by targeted imaging of rare and transient contact sites between the endoplasmic reticulum (ER) and lysosomes within hours rather than days. Our data suggest that extensive ER-lysosome and mitochondria-lysosome interactions restrict lysosome motility, highlighting the unique capabilities of our integrated CLEM pipeline for linking molecular dynamic data to high-resolution ultrastructural detail in 3D.

Optical Tweezers Approaches for Probing Multiscale Protein Mechanics and Assembly.

Multi-step assembly of individual protein building blocks is key to the formation of essential higher-order structures inside and outside of cells. Optical tweezers is a technique well suited to investigate the mechanics and dynamics of these structures at a variety of size scales. In this mini-review, we highlight experiments that have used optical tweezers to investigate protein assembly and mechanics, with a focus on the extracellular matrix protein collagen. These examples demonstrate how optical tweezers can be used to study mechanics across length scales, ranging from the single-molecule level to fibrils to protein networks. We discuss challenges in experimental design and interpretation, opportunities for integration with other experimental modalities, and applications of optical tweezers to current questions in protein mechanics and assembly.

Integrated super resolution fluorescence microscopy and transmission electron microscopy.

In correlative light and electron microscopy (CLEM), the capabilities of fluorescence microscopy (FM) and electron microscopy (EM) are united. FM combines a large field of view with high sensitivity for detecting fluorescence, which makes it an excellent tool for identifying regions of interest. EM has a much smaller field of view but offers superb resolution that allows studying cellular ultrastructure. In CLEM, the potentials of both techniques are combined but a limiting factor is the large difference in resolution between the two imaging modalities. Adding super resolution FM to CLEM reduces the resolution gap between FM and EM; it offers the possibility of identifying multiple targets within the diffraction limit and can increase correlation accuracy. CLEM is usually carried out in two separate setups, which requires transfer of the sample. This may result in distortion and damage of the specimen, which can complicate finding back regions of interest. By integrating the two imaging modalities, such problems can be avoided. Here, an integrated super resolution correlative microscopy approach is presented based on a wide-field super resolution FM integrated in a Transmission Electron Microscope (TEM). Switching imaging modalities is accomplished by rotation of the TEM sample holder. First imaging experiments are presented on sections of Lowicryl embedded Human Umbilical Vein Endothelial Cells labeled for Caveolin both with Protein A-Gold, and Alexa Fluor®647. TEM and FM images were overlaid using fiducial markers visible in both imaging modalities with an overlay accuracy of 28 ± 11 nm. This is close to the optical resolution of ~50 nm.

High accuracy, fiducial marker-based image registration of correlative microscopy images.

Fluorescence microscopy (FM) and electron microscopy (EM) are complementary techniques. FM affords examination of large fields of view and identifying regions of interest but has a low resolution. EM exhibits excellent resolution over a limited field of view. The combination of these two techniques, correlative microscopy, received considerable interest in the past years and has proven its potential in biology and material science. Accurate correlation of FM and EM images is, however, challenging due to the differences in contrast mechanism, size of field of view and resolution. We report an accurate, fast and robust method to correlate FM and EM images using low densities of fiducial markers. Here, 120 nm diameter fiducial markers consisting of fluorescently labelled silica coated gold nanoparticles are used. The method relies on recording FM, low magnification EM and high magnification EM images. Two linear transformation matrices are constructed, FM to low magnification EM and low magnification EM to high magnification EM. Combination of these matrices results in a high accuracy transformation of FM to high magnification EM coordinates. The method was tested using two different transmission electron microscopes and different Tokuyasu and Lowicryl sections. The overall accuracy of the correlation method is high, 5-30 nm.

Fluorescently Labelled Silica Coated Gold Nanoparticles as Fiducial Markers for Correlative Light and Electron Microscopy.

In this work, gold nanoparticles coated with a fluorescently labelled (rhodamine B) silica shell are presented as fiducial markers for correlative light and electron microscopy (CLEM). The synthesis of the particles is optimized to obtain homogeneous, spherical core-shell particles of arbitrary size. Next, particles labelled with different fluorophore densities are characterized to determine under which conditions bright and (photo)stable particles can be obtained. 2 and 3D CLEM examples are presented where optimized particles are used for correlation. In the 2D example, fiducials are added to a cryosection of cells whereas in the 3D example cells are imaged after endocytosis of the fiducials. Both examples demonstrate that the particles are clearly visible in both modalities and can be used for correlation. Additionally, the recognizable core-shell structure of the fiducials proves to be very powerful in electron microscopy: it makes it possible to irrefutably identify the particles and makes it easy to accurately determine the center of the fiducials.

The Role of a Phonon Bottleneck in Relaxation Processes for Ln-Doped NaYF4 Nanocrystals.

The localized inner 4f shell transitions of lanthanide ions are largely independent of the local surroundings. The luminescence properties of Ln3+ ions doped into nanocrystals (NCs) are therefore similar to those in bulk crystals. Quantum size effects, responsible for the unique size-dependent luminescence of semiconductor NCs, are generally assumed not to influence the optical properties of Ln3+-doped insulator NCs. However, phonon confinement effects have been reported to hamper relaxation between closely spaced Stark levels in Ln3+-doped NCs. At cryogenic temperatures emission and excitation from higher Stark levels was observed for Ln3+ ions in NCs only and were explained by a cutoff in the acoustic phonon spectrum. Relaxation would be inhibited as no resonant low energy (long wavelength) acoustic phonon modes can exist in nanometer sized crystals, and this prevents relaxation by direct phonon emission between closely spaced Stark levels. This phenomenon is known as a phonon bottleneck. Here, we investigate the role of phonon confinement in Ln-doped NCs. High resolution emission spectra at temperatures down to 2.2 K are reported for various Ln3+ ions (Er3+, Yb3+, Eu3+) doped into monodisperse 10 nm NaYF4 NCs and compared with spectra for bulk (microcrystalline) material. Contrary to previous reports, we find no evidence for phonon bottleneck effects in the emission spectra. Emission from closely spaced higher Stark levels is observed only at high excitation powers and is explained by laser heating. The present results indicate that previously reported effects in NCs may not be caused by phonon confinement.

Probing the Influence of Disorder on Lanthanide Luminescence Using Eu-Doped LaPO4 Nanoparticles.

Lanthanide-doped nanocrystals (NCs) differ from their bulk counterparts due to their large surface to volume ratio. It is generally assumed that the optical properties are not affected by size effects as electronic transitions occur within the well-shielded 4f shell of the lanthanide dopant ions. However, defects and disorder in the surface layer can affect the luminescence properties. Trivalent europium is a suitable ion to investigate the subtle influence of the surface, because of its characteristic luminescence and high sensitivity to the local environment. Here, we investigate the influence of disorder in NCs on the optical properties of lanthanide dopants by studying the inhomogeneous linewidth, emission intensity ratios, and luminescence decay curves for LaPO4:Eu3+ samples of different sizes (4 nm to bulk) and core-shell configurations (core, core-isocrystalline shell, and core-silica shell). We show that the emission linewidths increase strongly for NCs. The ratio of the intensities of the forced electric dipole (ED) and magnetic dipole (MD) transitions, a measure for the local symmetry distortion around Eu3+ ions, is higher for samples with a large fraction of Eu3+ ions close to the surface. Finally, we present luminescence decay curves revealing an increased nonradiative decay rate for Eu3+ in NCs. The effects are strongest in core and core-silica shell NCs and can be reduced by growth of an isocrystalline LaPO4 shell. The present systematic study provides quantitative insight into the role of surface disorder on the optical properties of lanthanide-doped NCs. These insights are important in emerging applications of lanthanide-doped nanocrystals.

3D-printed external light trap for solar cells.

We present a universally applicable 3D-printed external light trap for enhanced absorption in solar cells. The macroscopic external light trap is placed at the sun-facing surface of the solar cell and retro-reflects the light that would otherwise escape. The light trap consists of a reflective parabolic concentrator placed on top of a reflective cage. Upon placement of the light trap, an improvement of 15% of both the photocurrent and the power conversion efficiency in a thin-film nanocrystalline silicon (nc-Si:H) solar cell is measured. The trapped light traverses the solar cell several times within the reflective cage thereby increasing the total absorption in the cell. Consequently, the trap reduces optical losses and enhances the absorption over the entire spectrum. The components of the light trap are 3D printed and made of smoothened, silver-coated thermoplastic. In contrast to conventional light trapping methods, external light trapping leaves the material quality and the electrical properties of the solar cell unaffected. To explain the theoretical operation of the external light trap, we introduce a model that predicts the absorption enhancement in the solar cell by the external light trap. The corresponding calculated path length enhancement shows good agreement with the empirically derived value from the opto-electrical data of the solar cell. Moreover, we analyze the influence of the angle of incidence on the parasitic absorptance to obtain full understanding of the trap performance. © 2015 The Authors. Progress in Photovoltaics: Research and Applications published by John Wiley & Sons, Ltd.

Incorporation of Ln-Doped LaPO4 Nanocrystals as Luminescent Markers in Silica Nanoparticles.

Lanthanide ions are promising for the labeling of silica nanoparticles with a specific luminescent fingerprint due to their sharp line emission at characteristic wavelengths. With the increasing use of silica nanoparticles in consumer products, it is important to label silica nanoparticles in order to trace the biodistribution, both in the environment and living organisms.In this work, we synthesized LaPO4 nanocrystals (NCs) with sizes ranging from 4 to 8 nm doped with europium or cerium and terbium. After silica growth using an inverse micelle method, monodisperse silica spheres were obtained with a single LaPO4 NC in the center. We demonstrate that the size of the silica spheres can be tuned in the 25-55 nm range by addition of small volumes of methanol during the silica growth reaction. Both the LaPO4 core and silica nanocrystal showed sharp line emission characteristic for europium and terbium providing unique optical labels in silica nanoparticles of variable sizes.

Jammed elastic shells - a 3D experimental soft frictionless granular system.

We present a new experimental system of monodisperse, soft, frictionless, fluorescent labeled elastic shells for the characterization of structure, universal scaling laws and force networks in 3D jammed matter. The elastic shells in a jammed packing are deformed in such a way that at each contact one of the shells buckles with a dimple and the other remain spherical, closely resembling overlapping spheres. Using confocal microscopy, we obtained 3D stacks of images of shells at different volume fractions which were subsequently processed in ImageJ software to find their coordinates. The determination of 3D coordinates involved three steps: locating the edges of shells in all 2D slices, analyzing their shape and subsequently finding their 2D coordinates, and finally determining their 3D centers by grouping the corresponding 2D coordinates. From this analysis routine we obtained particle coordinates with sub-pixel accuracy. In a contact pair we also identified the shell that underwent buckling forming a dimple by analyzing the intensity profile of a line that connects the centers of particle pairs. The amorphous structure of the packing was analyzed as a function of distance to the jamming threshold by investigating the radial distribution function, bond order parameters, contact numbers and the number of dimples per particle (buckling number), which is a unique property of this system. We find that the power law scaling of the contact number with excess volume fraction deviated from theoretical and computer simulation predictions. In addition, the buckling number also showed a similar scaling as that of the contact number with distance to the jamming transition.

Tethered particle motion reveals that LacI·DNA loops coexist with a competitor-resistant but apparently unlooped conformation.

The lac repressor protein (LacI) efficiently represses transcription of the lac operon in Escherichia coli by binding to two distant operator sites on the bacterial DNA and causing the intervening DNA to form a loop. We employed single-molecule tethered particle motion to observe LacI-mediated loop formation and breakdown in DNA constructs that incorporate optimized operator binding sites and intrinsic curvature favorable to loop formation. Previous bulk competition assays indirectly measured the loop lifetimes in these optimized DNA constructs as being on the order of days; however, we measured these same lifetimes to be on the order of minutes for both looped and unlooped states. In a range of single-molecule DNA competition experiments, we found that the resistance of the LacI-DNA complex to competitive binding is a function of both the operator strength and the interoperator sequence. To explain these findings, we present what we believe to be a new kinetic model of loop formation and DNA competition. In this proposed new model, we hypothesize a new unlooped state in which the unbound DNA-binding domain of the LacI protein interacts nonspecifically with nonoperator DNA adjacent to the operator site at which the second LacI DNA-binding domain is bound.

Phasor based analysis of FRET images recorded using spectrally resolved lifetime imaging.

The combined analysis of spectral and lifetime images has the potential to provide more accurate and more detailed information about Förster resonance energy transfer (FRET). We have developed a novel FRET analysis method to analyze images recorded by multispectral lifetime imaging. The new method is based on a phasor approach and facilitates the simultaneous analysis of decay kinetics of donor and acceptor molecules. The method is applicable to both molecules that exhibit a mono-exponential decay and a bi-exponential decay. As an example we show the possibility of extracting the energy transfer efficiency and the fraction of interacting molecules even in the presence of non-interacting molecules. The reliability of the method is investigated by comparing it with conventional FRET-FLIM analyses. We show that, with the same number of detected photons, the spectrally resolved phasor approach provides higher accuracy than other analysis methods; the confidence interval is improved and the FRET efficiency is closer to the real value.

Monitoring the metabolic state of fungal hyphae and the presence of melanin by nonlinear spectral imaging.

Label-free nonlinear spectral imaging microscopy (NLSM) records two-photon-excited fluorescence emission spectra of endogenous fluorophores within the specimen. Here, NLSM is introduced as a novel, minimally invasive method to analyze the metabolic state of fungal hyphae by monitoring the autofluorescence of NAD(P)H and flavin adenine dinucleotide (FAD). Moreover, the presence of melanin was analyzed by NLSM. NAD(P)H, FAD, and melanin were used as biomarkers for freshness of mushrooms of Agaricus bisporus (white button mushroom) that had been stored at 4°C for 0 to 17 days. During this period, the mushrooms did not show changes in morphology or color detectable by eye. In contrast, FAD/NAD(P)H and melanin/NAD(P)H ratios increased over time. For instance, these ratios increased from 0.92 to 2.02 and from 0.76 to 1.53, respectively, at the surface of mushroom caps that had been harvested by cutting the stem. These ratios were lower under the skin than at the surface of fresh mushrooms (0.78 versus 0.92 and 0.41 versus 0.76, respectively), indicative of higher metabolism and lower pigment formation within the fruiting body. Signals were different not only between tissues of the mushroom but also between neighboring hyphae. These data show that NLSM can be used to determine the freshness of mushrooms and to monitor the postharvest browning process at an early stage. Moreover, these data demonstrate the potential of NLSM to address a broad range of fundamental and applied microbiological processes.

Blind unmixing of spectrally resolved lifetime images.

A method, is presented for blind unmixing spectrally resolved fluorescence lifetime images. The method is based on the combined analysis of spectral and lifetime phasors and allows unmixing of up to three components without any prior knowledge. Fractional intensities, spectra and decay curves of the individual components can be extracted with this new technique. The reliability and sensitivity are investigated and the possibility of extending the method to unmix more components is discussed. The method is evaluated on mixtures of fluorescent dyes and labeled cells.

Time-dependent motor properties of multipedal molecular spiders.

Molecular spiders are synthetic biomolecular walkers that use the asymmetry resulting from cleavage of their tracks to bias the direction of their stepping motion. Using Monte Carlo simulations that implement the Gillespie algorithm, we investigate the dependence of the biased motion of molecular spiders, along with binding time and processivity, on tunable experimental parameters, such as number of legs, span between the legs, and unbinding rate of a leg from a substrate site. We find that an increase in the number of legs increases the spiders' processivity and binding time but not their mean velocity. However, we can increase the mean velocity of spiders with simultaneous tuning of the span and the unbinding rate of a spider leg from a substrate site. To study the efficiency of molecular spiders, we introduce a time-dependent expression for the thermodynamic efficiency of a molecular motor, allowing us to account for the behavior of spider populations as a function of time. Based on this definition, we find that spiders exhibit transient motor function over time scales of many hours and have a maximum efficiency on the order of 1%, weak compared to other types of molecular motors.

Positional stability of holographic optical traps.

The potential of digital holography for complex manipulation of micron-sized particles with optical tweezers has been clearly demonstrated. By contrast, its use in quantitative experiments has been rather limited, partly due to fluctuations introduced by the spatial light modulator (SLM) that displays the kinoforms. This is an important issue when high temporal or spatial stability is a concern. We have investigated the performance of both an analog-addressed and a digitally-addressed SLM, measuring the phase fluctuations of the modulated beam and evaluating the resulting positional stability of a holographic trap. We show that, despite imparting a more unstable modulation to the wavefront, our digitally-addressed SLM generates optical traps in the sample plane stable enough for most applications. We further show that traps produced by the analog-addressed SLM exhibit a superior pointing stability, better than 1 nm, which is comparable to that of non-holographic tweezers. These results suggest a means to implement precision force measurement experiments with holographic optical tweezers (HOTs).

Stretching single DNA molecules to demonstrate high-force capabilities of holographic optical tweezers.

The well calibrated force-extension behaviour of single double-stranded DNA molecules was used as a standard to investigate the performance of phase-only holographic optical tweezers at high forces. Specifically, the characteristic overstretch transition at 65 pN was found to appear where expected, demonstrating (1) that holographic optical trap calibration using thermal fluctuation methods is valid to high forces; (2) that the holographic optical traps are harmonic out to >250 nm of 2.1 mum particle displacement; and (3) that temporal modulations in traps induced by the spatial light modulator (SLM) do not affect the ability of optical traps to hold and steer particles against high forces. These studies demonstrate a new high-force capability for holographic optical traps achievable by SLM technologies.

The Tumbleweed: towards a synthetic proteinmotor.

Biomolecular motors have inspired the design and construction of artificial nanoscale motors and machines based on nucleic acids, small molecules, and inorganic nanostructures. However, the high degree of sophistication and efficiency of biomolecular motors, as well as their specific biological function, derives from the complexity afforded by protein building blocks. Here, we discuss a novel bottom-up approach to understanding biological motors by considering the construction of synthetic protein motors. Specifically, we present a design for a synthetic protein motor that moves along a linear track, dubbed the "Tumbleweed." This concept uses three discrete ligand-dependent DNA-binding domains to perform cyclically ligand-gated, rectified diffusion along a synthesized DNA molecule. Here we describe how de novo peptide design and molecular biology could be used to produce the Tumbleweed, and we explore the fundamental motor operation of such a design using numerical simulations. The construction of this and more sophisticated protein motors is an exciting challenge that is likely to enhance our understanding of the structure-function relationship in biological motors.

Stretching submicron biomolecules with constant-force axial optical tweezers.

Optical tweezers have become powerful tools to manipulate biomolecular systems, but are increasingly difficult to use when the size of the molecules is <1 microm. Many important biological structures and processes, however, occur on the submicron length scale. Therefore, we developed and characterized an optical manipulation protocol that makes this length scale accessible by stretching the molecule in the axial direction of the laser beam, thus avoiding limiting artifacts from steric hindrances from the microscope coverslip and other surface effects. The molecule is held under constant mechanical tension by a combination of optical gradient forces and backscattering forces, eliminating the need for electronic feedback. We demonstrate the utility of this method through a measurement of the force-extension relationship of a 1298 bp ds-DNA molecule.

Label-free optical imaging of mitochondria in live cells.

The far-field optical imaging of mitochondria of live cells without the use of any label is demonstrated. It uses a highly sensitive photothermal method and has a resolution comparable to confocal fluorescence setups. The morphological states of mitochondria were followed under different physiological treatments, and the role of cytochrome c was ruled out as the main origin of the photothermal signals. This label free optical method provides a high contrast imaging of live mitochondria and should find many applications in biosciences.