RESUMO
Super-resolution microscopies have become an established tool in biological research. However, imaging throughput remains a main bottleneck in acquiring large datasets required for quantitative biology. Here we describe multifocal flat illumination for field-independent imaging (mfFIFI). By integrating mfFIFI into an instant structured illumination microscope (iSIM), we extend the field of view (FOV) to >100 × 100 µm2 while maintaining high-speed, multicolor, volumetric imaging at double the diffraction-limited resolution. We further extend the effective FOV by stitching adjacent images for fast live-cell super-resolution imaging of dozens of cells. Finally, we combine our flat-fielded iSIM with ultrastructure expansion microscopy to collect three-dimensional (3D) images of hundreds of centrioles in human cells, or thousands of purified Chlamydomonas reinhardtii centrioles, per hour at an effective resolution of ~35 nm. Classification and particle averaging of these large datasets enables 3D mapping of posttranslational modifications of centriolar microtubules, revealing differences in their coverage and positioning.
Assuntos
Microscopia de Fluorescência/métodos , Animais , Células COS , Centríolos/ultraestrutura , Chlorocebus aethiops , Humanos , Imageamento Tridimensional , Iluminação , Microtúbulos/ultraestruturaRESUMO
Chromatin is organized into topologically associating domains (TADs) enriched in distinct histone marks. In cancer, gain-of-function mutations in the gene encoding the enhancer of zeste homolog 2 protein (EZH2) lead to a genome-wide increase in histone-3 Lys27 trimethylation (H3K27me3) associated with transcriptional repression. However, the effects of these epigenetic changes on the structure and function of chromatin domains have not been explored. Here, we found a functional interplay between TADs and epigenetic and transcriptional changes mediated by mutated EZH2. Altered EZH2 (p.Tyr646* (EZH2Y646X)) led to silencing of entire domains, synergistically inactivating multiple tumor suppressors. Intra-TAD gene silencing was coupled with changes of interactions between gene promoter regions. Notably, gene expression and chromatin interactions were restored by pharmacological inhibition of EZH2Y646X. Our results indicate that EZH2Y646X alters the topology and function of chromatin domains to promote synergistic oncogenic programs.
Assuntos
Cromatina/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigênese Genética/genética , Mutação/genética , Transcrição Gênica/genética , Animais , Linhagem Celular Tumoral , Metilação de DNA/genética , Epigenômica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica/fisiologia , Histonas/genética , Humanos , Camundongos , Regiões Promotoras Genéticas/genéticaRESUMO
Super-resolution fluorescence microscopy improves spatial resolution, but this comes at a loss of image throughput and presents unique challenges in identifying optimal acquisition parameters. Microscope automation routines can offset these drawbacks, but thus far have required user inputs that presume a priori knowledge about the sample. Here, we develop a flexible illumination control system for localization microscopy comprised of two interacting components that require no sample-specific inputs: a self-tuning controller and a deep learning-based molecule density estimator that is accurate over an extended range of densities. This system obviates the need to fine-tune parameters and enables robust, autonomous illumination control for localization microscopy.
RESUMO
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.
Assuntos
Centríolos/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica/instrumentação , Microscopia Eletrônica/métodos , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Imagem Individual de Molécula/métodos , Algoritmos , Centríolos/metabolismo , HumanosRESUMO
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.
Assuntos
Microscopia , Imagem Molecular , Cromatina/química , Cromatina/ultraestrutura , Cristalografia por Raios X , Microscopia/métodos , Microscopia Eletrônica , Microscopia de Fluorescência/métodos , Imagem Molecular/métodosRESUMO
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.
Assuntos
Dano ao DNA , Telômero/ultraestrutura , Cromatina/fisiologia , Imunofluorescência , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Microscopia de Fluorescência , Proteína 1 de Ligação a Repetições Teloméricas/análise , Proteína 1 de Ligação a Repetições Teloméricas/imunologia , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/fisiologiaRESUMO
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.
Assuntos
Percepção de Profundidade , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Proteínas de Bactérias/metabolismo , Calibragem , Caulobacter/metabolismo , Proteínas do Citoesqueleto/metabolismo , RotaçãoRESUMO
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.
RESUMO
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.
Assuntos
Movimento Celular , Imagem Molecular , Células de Schwann/citologia , Animais , Anisotropia , Adesão Celular , RatosRESUMO
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.
Assuntos
Manufaturas , Modelos Teóricos , Nefelometria e Turbidimetria/métodos , Refratometria/métodos , Simulação por Computador , Cristalização , Luz , Fótons , Espalhamento de RadiaçãoRESUMO
We introduce and demonstrate a method for expanding the field of view of a typical imaging system by multiplexing images encoded onto different polarization states and recovering them from a limited number of measurements.
RESUMO
Based on optical-path-length spectroscopy, we present a method for determining optical diffusion coefficients that does not require a priori knowledge about the physical properties of inherent interfaces. Comparisons are made with both standard theoretical estimates for bulk diffusion and with traditional approaches relying on surface corrections.