Walter A. Carrington

Three-dimensional molecular distribution in single cells analysed using the digital imaging microscope

Cellular changes in molecular distribution are believed to underly a wide range of cell functions. In order to investigate changes in molecular distribution in single cells utilizing fluorescent probes we have developed a digital imaging microscope. The system, consisting of both hardware and software, automatically acquires 3‐D data sets consisting of optical sections and then processes such data to facilitate the analysis of molecular distribution in single cells. The first major step in processing reverses distortion introduced principally by the optics of the fluorescent microscope. Various procedures for accomplishing this task are compared and a method based on regularization theory is shown to give superior results for several different 3‐D images. Following this step features of interest are automatically extracted from 3‐D images utilizing an artificial 3‐D visual system. This artificial visual system utilizes a system of spatial filters to identify regional characteristics of images, the information obtained from these filters being used to identify and characterize clusters of molecules within the image. This information is then utilized to construct a 3‐D graphical model of molecular distribution in single cells. Such models are displayed in 3‐D and may be further analysed utilizing interactive 3‐D computer graphics. These methods are illustrated by results obtained regarding alpha‐actinin distribution in single smooth muscle cells.

Visualization of single molecules of mRNA in situ

Publisher Summary The chapter discusses methods and concepts that facilitate the detection and identification of single molecules of mRNA in situ using fluorescence in situ hybridization (FISH). FISH is a very widely used technique in cell biology. The sensitivity of detection is the major concern when implementing the FISH technology. The methodology employs stringent imaging requirements that include a carefully calibrated quantitative epifluorescence digital imaging microscope, three-dimensional (3D) optical sectioning, constrained iterative deconvolution, and 3D interactive analysis software. The analysis of point sources representing one hybridized probe has provided additional information concerning the nature of the imaging process. The pixel does not suffice as the unit for analysis of a digital image involving fluorescence in situ hybridization. The detailed characterization of biological molecules in situ is now possible using FISH. Unprecedented detail can be revealed from a hybridized cell that has been interrogated with carefully engineered, quantitatively accurate probes, followed by three-dimensional imaging and deconvolution.

Three-dimensional Imaging on Confocal and Wide-field Microscopes

The conventional (wide-field) light microscope accepts light from planes above and below the plane of focus. This lack of depth discrimination is the main limitation of the wide-field microscope for 3D imaging. The confocal microscope rejects this out-of-focus light with the confocal pinhole and provides greater resolution than the wide-field microscope. This depth discrimination of the confocal microscope makes it attractive for 3D optical sectioning microscopy. This advantage of the confocal microscope is balanced by inherent signal losses from the confocal pinhole (see also Chapter 2: Pinhole) and by the use of detectors in current commercial confocal microscopes that have substantially lower quantum efficiency than the cooled CCD cameras used in wide-field digital imaging microscopes. These additional losses of current commercial confocal instruments are especially important in 3D fluorescence imaging of single cells.

Image restoration in 3-D microscopy with limited data

We present an image restoration method that is suitable for images with missing or truncated data. This method, L2 regularization with a non-negativity constraint, is applied to image restoration of 3D optically sectioned microscope images of fluorescently labelled cells. Its ability to use axially truncated data is useful when applying it to 3D images from a conventional wide field microscope and to operate effectively on a small number of optical sections. Its ability to correctly place light originating from outside the field of view by making use of the out of focus information allows us to subdivide a large image into smaller pieces without a substantial edge effect. Large 3D data sets can be restored on a modestly priced computer workstation.

Three-Dimensional Analysis Of Molecular Distribution In Single Cells Using The Digital Imaging Microscope

The development and operation of the digital imaging microscope, a system capable of analyzing the 3D molecular distribution in singe cells is described. It is a system consisting of hardware and software that: 1. obtains 2D or 3D microscope images of the faint signals from fluorescent molecules introduced into single cells; 2. reverses the distortion in such images introduced by the optics; 3. automatically or semi-automatically extracts features of interest from such images, and; 4. displays 2D, 3D images and even higher order information for interactive analysis. The strategies used to accomplish these tasks are discussed and their application to the analysis of the molecular events underlying white blood cell chemotaxis is illustrated.