RESUMEN
An optical projection tomography microscope (OPTM) can improve axial resolution by viewing a sample from different perspectives. Here, we report a dual-mode OPTM that can generate 3D images of single cancer cells in both absorption mode and polarization mode. Cancer cells were labeled with hematoxylin for absorption imaging and nanorods for polarization imaging. Absorption images can provide morphologic information, and polarization images can provide molecular information. The combination of molecular detection and 3D cytological cell analysis may help with early cancer diagnosis.
Asunto(s)
Oro , Hematoxilina , Aumento de la Imagen/instrumentación , Neoplasias Pulmonares/patología , Microscopía/instrumentación , Nanoestructuras , Tomografía/instrumentación , Línea Celular Tumoral , Medios de Contraste , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Aumento de la Imagen/métodos , Nanoestructuras/ultraestructura , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The practice of clinical cytology relies on bright-field microscopy using absorption dyes like hematoxylin and eosin in the transmission mode, while the practice of research microscopy relies on fluorescence microscopy in the epi-illumination mode. The optical projection tomography microscope is an optical microscope that can generate 3-D images of single cells with isometric high resolution both in absorption and fluorescence mode. Although the depth of field of the microscope objective is in the submicron range, it can be extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. Cells suspended in optical gel flow through a custom-designed microcapillary. Multiple pseudoprojection images are taken by rotating the microcapillary. After these pseudoprojection images are further aligned, computed tomography methods are applied to create 3-D reconstruction. 3-D reconstructed images of single cells are shown in both absorption and fluorescence mode. Fluorescence spatial resolution is measured at 0.35 microm in both axial and lateral dimensions. Since fluorescence and absorption images are taken in two different rotations, mechanical error may cause misalignment of 3-D images. This mechanical error is estimated to be within the resolution of the system.
Asunto(s)
Técnicas Citológicas/métodos , Microscopía Fluorescente/métodos , Tomografía Óptica/métodos , Animales , Línea Celular , Femenino , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Metafase/fisiología , Ciervo Muntjac , Coloración y Etiquetado/métodosRESUMEN
The Cell-CT is an optical projection tomography microscope (OPTM) developed for high resolution 3D imaging of single cells based on absorption stains and brightfield microscopy. In this study we demonstrate the use of the Cell-CT in multi-color mode for simultaneous imaging of cellular 3D morphology and the 3D distribution of nanoparticle clusters in the cytoplasm. The ability to image cellular processes in relation to cellular compartments with a non-fluorescence 3D technology opens new perspectives for molecular research.
Asunto(s)
Imagenología Tridimensional/métodos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Nanopartículas/análisis , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/patología , Tomografía Óptica/métodos , Algoritmos , Animales , Línea Celular Tumoral , Tamaño de la Célula , Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/métodos , Tamaño de la Partícula , Ratas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
A method is presented for imaging single isolated cell nuclei in 3D, employing computed tomographic image reconstruction. The system uses a scanning objective lens to create an extended depth-of-field (DOF) image similar to a projection or shadowgram. A microfabricated inverted v-groove allows a microcapillary tube to be rotated with sub-micron precision, and refractive index matching within 0.02 both inside and outside the tube keeps optical distortion low. Cells or bare cell nuclei are injected into the tube and imaged in 250 angular increments from 0 to 180 degrees to collect 250 extended DOF images. After these images are further aligned, the filtered backprojection algorithm is applied to compute the 3D image. To estimate the cutoff spatial frequency in the projection image, a spatial frequency ratio function is calculated by comparing the extended depth-of-field image of a typical cell nucleus to the fixed focus image. To assess loss of resolution from fixed focus image to extended DOF image to 3D reconstructed image, the 10-90% rise distance is measured for a dyed microsphere. The resolution is found to be 0.9 microm for both extended DOF images and 3D reconstructed images. Surface and translucent volume renderings and cross-sectional slices of the 3D images are shown of a stained nucleus from fibroblast and cancer cell cultures with added color histogram mapping to highlight 3D chromatin structure.
