RESUMO
Chromosome organization is crucial for genome function. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization. Using the super-resolution microscopy methods of OligoSTORM and OligoDNA-PAINT, we trace 8 megabases of human chromosome 19, visualizing structures ranging in size from a few kilobases to over a megabase. Focusing on chromosomal regions that contribute to compartments, we discover distinct structures that, in spite of considerable variability, can predict whether such regions correspond to active (A-type) or inactive (B-type) compartments. Imaging through the depths of entire nuclei, we capture pairs of homologous regions in diploid cells, obtaining evidence that maternal and paternal homologous regions can be differentially organized. Finally, using restraint-based modeling to integrate imaging and Hi-C data, we implement a method-integrative modeling of genomic regions (IMGR)-to increase the genomic resolution of our traces to 10 kb.
Assuntos
Passeio de Cromossomo/métodos , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 19/ultraestrutura , Modelos Genéticos , Células Cultivadas , Coloração Cromossômica/métodos , Estruturas Cromossômicas/química , Estruturas Cromossômicas/genética , Estruturas Cromossômicas/ultraestrutura , Cromossomos Humanos Par 19/química , Feminino , Corantes Fluorescentes , Humanos , Imageamento Tridimensional , Hibridização in Situ Fluorescente/métodos , Masculino , Sondas de Oligonucleotídeos , LinhagemRESUMO
The ability to localize precisely a single optical emitter is important for particle tracking applications and super resolution microscopy. It is known that for a traditional microscope the ability to localize such an emitter is limited by the photon count. Here we analyze the ability to improve such localization by imposing interference fringes. We show here that a simple grating interferometer can introduce such improvement in certain circumstances and analyze what is required to increase the localization precision further.
RESUMO
The ability to track single fluorescent particles within a three dimensional (3D) cellular environment can provide valuable insights into cellular processes. In this paper, we present a modified nonlinear image decomposition technique called K-factor that reshapes the 3D point spread function (PSF) of an XYZ image stack into a narrow Gaussian profile. The method increases localization accuracy by ~60% with compare to regular Gaussian fitting, and improves minimal resolvable distance between overlapping PSFs by ~50%. The algorithm was tested both on simulated data and experimentally.
Assuntos
Algoritmos , Rastreamento de Células/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Reconhecimento Automatizado de Padrão/métodos , Aumento da Imagem/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
We report a novel optical single-emitter-localization methodology that uses the phase induced by path length differences in a Mach-Zehnder interferometer to improve localization precision. Using information theory, we demonstrate that the localization capability of a modified Fourier domain signal generated by photon interference enables a more precise localization compared to a standard Gaussian intensity distribution of the corresponding point-spread function. The calculations were verified by numerical simulations and an exemplary experiment, where the centers of metal nanoparticles were localized to a precision of 3 nm.
Assuntos
Interferometria/instrumentação , Interferometria/métodos , Nanopartículas/ultraestrutura , Refratometria/instrumentação , Refratometria/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Aumento da Imagem/instrumentação , Aumento da Imagem/métodos , Luz , Nanopartículas/química , Reprodutibilidade dos Testes , Espalhamento de Radiação , Sensibilidade e EspecificidadeRESUMO
Localization of a single fluorescent particle with sub-diffraction-limit accuracy is a key merit in localization microscopy. Existing methods such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) achieve localization accuracies of single emitters that can reach an order of magnitude lower than the conventional resolving capabilities of optical microscopy. However, these techniques require a sparse distribution of simultaneously activated fluorophores in the field of view, resulting in larger time needed for the construction of the full image. In this paper we present the use of a nonlinear image decomposition algorithm termed K-factor, which reduces an image into a nonlinear set of contrast-ordered decompositions whose joint product reassembles the original image. The K-factor technique, when implemented on raw data prior to localization, can improve the localization accuracy of standard existing methods, and also enable the localization of overlapping particles, allowing the use of increased fluorophore activation density, and thereby increased data collection speed. Numerical simulations of fluorescence data with random probe positions, and especially at high densities of activated fluorophores, demonstrate an improvement of up to 85% in the localization precision compared to single fitting techniques. Implementing the proposed concept on experimental data of cellular structures yielded a 37% improvement in resolution for the same super-resolution image acquisition time, and a decrease of 42% in the collection time of super-resolution data with the same resolution.
