Development of optical systems and imaging analyses for hyperspectral microscopy and 3D super-resolution imaging.

摘要

Many of the unanswered questions in cellular biology involve the functionality and structural composition of different cells. Generally, most of the unanswered questions that are of interest require information regarding protein-protein interactions or information on the general structure of the cell. Most of these features exist on size scales of 10 to 200 nm, of which conventional fluorescence microscopy is incapable of resolving details less than 200 nm. Therefore, many advanced fluorescence microscopy techniques have been developed over the past few decades to address the demands of higher resolutions (both spatial and temporal) as well as multicolor imaging in order to further the efforts of biological and medical science. Super-resolution microscopy techniques are capable of achieving resolutions several factors greater than conventional microscopy. Spectral imaging techniques have been developed towards generating hyper-spectral movies at 30 frames per second. Many new techniques are in development to meet the growing demand for better biological information.;In this work, we first demonstrate a high-speed hyperspectral line-scanning microscope (HSM) that is capable of recording 128 spectral channels in 27 frames per second. To reduce the aberrations of the spectral images, a spherical prism spectrometer is implemented in HSM. The design and calibrations of the spectrometer are given in detail. Next, we describe a 3D supper-resolution localization algorithm based on the phase-retrieved point spread function (PSF) model, which gives better fitting accuracy and less artifacts than a Gaussian PSF model. The phase-retrieval process, PSF generation, 3D single-emitter localization algorithm, and the results from localizing various samples are discussed in the first part of this section. The second part discusses the theoretical estimation precisions of various multiemitter models in 3D localization and describes a 3D multiemitter localization algorithm as well as the reconstruction results of microtubules. In the end, we demonstrate a new configuration of a light sheet microscope that is capable of reconstructing a high-contrast 3D whole-cell image with the use of a single objective lens. The optical layout of this light sheet microscope, the optical alignment procedures, the instrumentation, and the system calibration are given in this section.