RESUMO
Our experimental results demonstrate that full-field hard-X-ray microscopy is finally able to investigate the internal structure of cells in tissues. This result was made possible by three main factors: the use of a coherent (synchrotron) source of X-rays, the exploitation of contrast mechanisms based on the real part of the refractive index and the magnification provided by high-resolution Fresnel zone-plate objectives. We specifically obtained high-quality microradiographs of human and mouse cells with 29 nm Rayleigh spatial resolution and verified that tomographic reconstruction could be implemented with a final resolution level suitable for subcellular features. We also demonstrated that a phase retrieval method based on a wave propagation algorithm could yield good subcellular images starting from a series of defocused microradiographs. The concluding discussion compares cellular and subcellular hard-X-ray microradiology with other techniques and evaluates its potential impact on biomedical research.
Assuntos
Estruturas Celulares/citologia , Processamento de Imagem Assistida por Computador/métodos , Microrradiografia/métodos , Microscopia/métodos , Algoritmos , Animais , Aorta/citologia , Desenho de Equipamento , Ouro , Células HeLa , Humanos , Nanopartículas Metálicas/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microrradiografia/instrumentação , Microscopia/instrumentação , Neoplasias Experimentais/patologia , Síncrotrons , Tomografia Computadorizada por Raios X/métodos , Raios X , Peixe-ZebraRESUMO
We report an experimental investigation of the optical transfer functions for an X-ray microscope operated in defocus phase-contrast mode. The results are compared with a theoretical model of partially coherent image formation and are found to be in excellent agreement.
RESUMO
The fabrication of gold Fresnel zone plates, by a combination of e-beam lithography and electrodeposition, with a 30 nm outermost zone width and a 450 nm-thick structure is described. The e-beam lithography process was implemented with a careful evaluation of applied dosage, tests of different bake-out temperatures and durations for the photoresist, and the use of a developer without methylisobutylketone. Electrodeposition with a pulsed current mode and with a specially designed apparatus produced the desired high-aspect-ratio nanostructures. The fabricated zone plates were examined by electron microscopy and their performances were assessed using a transmission X-ray microscope. The results specifically demonstrated an image resolution of 40 nm.