High-Speed Optical Sectioning Microscope for Cell Biology

Carnegie Mellon University, Pittsburgh PA

Investigators

Abstract

ABSTRACT / #9987393 / High-speed optical sectioning fluorescence microscope for cell biology. Many exciting directions in cell and molecular biology today are converging on the use of high-resolution fluorescence microscopy for both structural and functional studies of living cells. In addition to the intensive use of hundreds of types of fluorescent molecular probes and indicators, genetic engineering has opened the door to widespread use of green fluorescent proteins (GFP and variants) as direct fusion-protein tracers. This revolution has spanned research at all levels from yeast to mammalian cells and transgenic animals. Unlocking the vast potential of quantitative image analysis in these experiments depends upon "optical sectioning microscopy"; obtaining images in which out-of-focus zones of the specimen are strongly attenuated and in-focus features are accurately retained. In living brain sections, for example, measurements of neuronal calcium transients with fluorescent indicator dyes is meaningful only if the emission background due to cells in out-of-focus strata is eliminated. In yeast, which are small but never thin, the out-of-focus component in images of the cytoskeleton or of organelles is often brighter than the in-focus structures in any plane of focus. The need for improved resolution, including optical sectioning, under conditions compatible with specimen viability and the rapid time scale of cell dynamics places stringent requirements on the speed and efficiency of computerized microscope systems. The aim of this project is the development of a versatile, fast and reliable optical sectioning fluorescence microscope as a widely-useful instrument for this work. The unique feature of the high-speed microscope is an incoherent grating imager, a relatively new concept in biological fluorescence microscopy. In brief, a moveable grating mask (a Ronchi Ruling) within the incident-light illuminator is projected with finite depth-of-field into the specimen; With high numerical aperture optics and a fine mask, the depth-of-focus of the projected grating image can be less than 0.5 micron. Transverse movement of the grating therefore modulates the fluorescence of exclusively in-focus features, so that true optical sections can be derived by very simple and fast digital image processing operations. For a single plane of focus, the grating need only be shifted between three defined positions to get sufficient data to subtract away the out-of-focus image component and demodulate the in-focus features. Only low-cost components are needed in addition to a digital imaging microscope system. In preliminary work, the grating imager has outperformed confocal scanning in terms of speed of image acquisition for comparable optical sections in cellular specimens. The technical plan of the proposal is to incorporate the grating imager into an automated multi-mode microscope equipped with a fast, high-precision cooled CCD camera. Research will focus on design and automation for speed, improvement of precision and accuracy, and development of image processing and analysis capability. Key component and system designs will be disseminated so that other research groups can duplicate the instrument. The high-speed grating imager located in the NSF Center for Light Microscope Imaging and Biotechnology will have an immediate impact on the research of a number of Principal Investigators. Experiments range from time-lapse imaging of fibroblast dynamics in a collagen-based extracellular matrix gel to Golgi fragmentation and reassociation dynamics during mitosis in mammalian cells. More than a dozen PIs at Carnegie Mellon University and at University of Pittsburgh carry out basic research in molecular cell biology and cell physiology in which there is a need for high-speed, high-resolution fluorescence optical sectioning microscopy. Several in this group of PIs work in the area of tissue engineering, where imaging living cells clearly in polymeric scaffold materials is of fundamental importance. Because of its speed, simplicity, and versatility, the grating imager will make possible many cell-biological experiments that cannot be carried out at present. It will displace confocal microscopy in living-cell applications where speed is an important constraint, and will quickly become the optical system of choice for computational deconvolution (computational optical sectioning microscopy). Overall, the field of cell biology has lacked a robust method for optical sectioning that is camera-based, therefore able to capture image data in a massively-parallel manner. The high-speed microscope based on the incoherent grating imager is a solution to this problem.