Kyle M. Douglass

Super-resolution imaging of multiple cells by optimized flat-field epi-illumination

An epi-illumination system based on microlens arrays enables field-independent imaging of multiple cells with nanoscale resolution and large field of views. Biological processes are inherently multi-scale, and supramolecular complexes at the nanoscale determine changes at the cellular scale and beyond. Single-molecule localization microscopy (SMLM)1,2,3 techniques have been established as important tools for studying cellular features with resolutions of the order of around 10 nm. However, in their current form these modalities are limited by a highly constrained field of view (FOV) and field-dependent image resolution. Here, we develop a low-cost microlens array (MLA)-based epi-illumination system—flat illumination for field-independent imaging (FIFI)—that can efficiently and homogeneously perform simultaneous imaging of multiple cells with nanoscale resolution. The optical principle of FIFI, which is an extension of the Kohler integrator, is further elucidated and modelled with a new, free simulation package. We demonstrate FIFIs capabilities by imaging multiple COS-7 and bacteria cells in 100 × 100 μm2 SMLM images—more than quadrupling the size of a typical FOV and producing near-gigapixel-sized images of uniformly high quality.

The telomeric DNA damage response occurs in the absence of chromatin decompaction

Telomeres are specialized nucleoprotein structures that protect chromosome ends from DNA damage response (DDR) and DNA rearrangements. The telomeric shelterin protein TRF2 suppresses the DDR, and this function has been attributed to its abilities to trigger t-loop formation or prevent massive decompaction and loss of density of telomeric chromatin. Here, we applied stochastic optical reconstruction microscopy (STORM) to measure the sizes and shapes of functional human telomeres of different lengths and dysfunctional telomeres that elicit a DDR. Telomeres have an ovoid appearance with considerable plasticity in shape. Examination of many telomeres demonstrated that depletion of TRF2, TRF1, or both affected the sizes of only a small subset of telomeres. Costaining of telomeres with DDR markers further revealed that the majority of DDR signaling telomeres retained a normal size. Thus, DDR signaling at telomeres does not require decompaction. We propose that telomeres are monitored by the DDR machinery in the absence of telomere expansion and that the DDR is triggered by changes at the molecular level in structure and protein composition.

Passive optical mapping of structural evolution in complex fluids

Self-assembling complex systems exhibit properties that involve a broad spectrum of thermal, structural, morphological, and optical transitions. Various techniques have been used to assess different aspects of the phase transitions in these complex systems. However, because of inherent technical constraints, structural information is usually provided only within narrow ranges of concentrations and temperatures. We show here that by effectively suppressing multiple scattering, low-coherence dynamic light scattering permits assessing the aggregation dynamics of self-assembling systems in a completely passive manner and over ranges of concentration and temperatures well beyond the limits of traditional approaches. The power spectral analysis of scattered intensity fluctuations permits a reliable characterization of multiple relaxation times. We demonstrate that the entire phase diagram can be covered in a consistent way and structural phase transitions can be mapped over a broad optical regime from weak to strong scattering.

Correction of a Depth-Dependent Lateral Distortion in 3D Super-Resolution Imaging

Three-dimensional (3D) localization-based super-resolution microscopy (SR) requires correction of aberrations to accurately represent 3D structure. Here we show how a depth-dependent lateral shift in the apparent position of a fluorescent point source, which we term `wobble`, results in warped 3D SR images and provide a software tool to correct this distortion. This system-specific, lateral shift is typically > 80 nm across an axial range of ~ 1 μm. A theoretical analysis based on phase retrieval data from our microscope suggests that the wobble is caused by non-rotationally symmetric phase and amplitude aberrations in the microscope’s pupil function. We then apply our correction to the bacterial cytoskeletal protein FtsZ in live bacteria and demonstrate that the corrected data more accurately represent the true shape of this vertically-oriented ring-like structure. We also include this correction method in a registration procedure for dual-color, 3D SR data and show that it improves target registration error (TRE) at the axial limits over an imaging depth of 1 μm, yielding TRE values of < 20 nm. This work highlights the importance of correcting aberrations in 3D SR to achieve high fidelity between the measurements and the sample.

Dipole–dipole interaction in random electromagnetic fields

We demonstrate that a nonvanishing interaction force exists between pairs of induced dipoles in a random, statistically stationary electromagnetic field. This new type of optical binding force leads to long-range interaction between dipolar particles even when placed in spatially incoherent fields. We also discuss several unique features of the dipole-dipole interaction in spatially incoherent Gaussian fields.

Measuring anisotropic cell motility on curved substrates

Schwann cell motility was observed on laminin-coated quartz cylinders with different curvatures over an 18 hour period. A new analysis based on difference images helped to determine the minimal radius of curvature, 46 μm, which restricted motility along the cylinder axis. The migration speed, measured by calculating differences between successive images in the time series, ranged between 0.3 to 0.8 μm per minute and is similar to previously reported rates for Schwann cells. Difference images provide a rapid and simple method for the analysis of cell motility on large populations of cells.

Fluctuations of scattered waves: going beyond the ensemble average

The interaction between coherent waves and random media is a complicated, deterministic process that is usually examined upon ensemble averaging. The result of one realization of the interaction process depends on the specific disorder present in an experimentally controllable interaction volume. We show that this randomness can be quantified and structural information not apparent in the ensemble average can be obtained. We use the information entropy as a viable measure of randomness and we demonstrate that its rate of change provides means for discriminating between media with identical mean characteristics.

Anomalous flow of light near a photonic crystal pseudo-gap

Two different transport regimes of light are observed in reflection from the same disordered photonic crystal. A model based on the scaling theory of localization explains the behavior of the path length-resolved reflection at two different probing wavelengths. Our results demonstrate the continuous renormalization of the photon diffusion coefficient measured in reflection from random media.

Multicolor single-particle reconstruction of protein complexes

Single-particle reconstruction (SPR) from electron microscopy (EM) images is widely used in structural biology, but it lacks direct information on protein identity. To address this limitation, we developed a computational and analytical framework that reconstructs and coaligns multiple proteins from 2D super-resolution fluorescence images. To demonstrate our method, we generated multicolor 3D reconstructions of several proteins within the human centriole, which revealed their relative locations, dimensions and orientations.A computational and analytical framework enables multicolor 3D particle reconstruction of protein complexes from 2D images. The authors demonstrate the power of the approach by reconstructing native proteins within the human centriole.

Super-resolution microscopy to decipher multi-molecular assemblies

Super-resolution fluorescence microscopy (SRM) is increasingly being applied as a complementary method to resolve the organization of large biomolecular assemblies. One of its main advantages is that it provides information on protein organization and identity simultaneously, within the native cellular milieu. It also extends the accessible range of structures up to the micrometer scale, offering complementary information relative to classical structural biology methods. Furthermore, SRM is capable of resolving the organization of some biomolecular assemblies not accessible to other methods. We highlight these advantages within the context of deciphering the structure of the centrosome and chromatin, and discuss how computational data post-processing has been adapted for SRM data. We also outline current limitations and potential approaches to overcome them.