Frederick Lanni - US grants

A video image memory system, acquistion procesor and digitizer, and a cursor generator will be acquired to complete a distributed, yet integrated, imaging facilty for the Center for Fluorescence Research in Biomedical Sciences. Interactive video image acquisition and analysis, and supermini computers for data acquisition and reduction are also part of the imaging facility. Four separate laboratories are networked to allow multiple user applications. A wide variety of projects in cell biology, cell physiology, developmental biology, regulatory biology, and biological chemistry will be supported by in the instrumentation. Among the projects to be supported are analyses of the growth factor stimulation of quiescent cells and macrophage chemotaxis. Ratio imaging will be used as a spectroscopic method for determining the spatial and temporal changes in (cellular) physiological parameters such as pH and pCa. Multiple parameter analysis of two or more separable fluorescent probes will also be used to correlate spatial and temporal dynamics of a variety of cellular funcitons. Three dimensional reconstruction will be carried out on actin networks in living cells. A method of two wavelength total internal reflection image analysis will be used to quantitate the distance from the cell substrate to selected molecules. Methods for mapping the spatial and temporal variations in fluorescence lifetime and fluorescence anisotropy of suitably labeled proteins in living cells will be developed. The first target for mapping with be calmodulin. Overall, the equipment, coupled with other resources in the Center, will permit state-of-the-art measurements greatly extending our understanding of cellular function.

9217217 Lanni The goal of this project is the construction of a fluorescence microscope for high-speed, high-resolution, three-dimensional imaging of biological specimens. The approach to be taken is a direct extension of Standing-Wave Fluorescence Microscopy, demonstrated in this laboratory previously. The proposed instrument will incorporate a laser-based optical system for excitation field sYnthesis, in which standing wave fields are time-multiplexed in the specimen to create an effective intensity field that is peaked at the in-focus object plane. Fluorescence emission will therefore be excited maximally from only in-focus object features. Serial focal plane images will then be recorded in the usual way, by use of an electronic (CCD) camera. The optical transfer function for this field-synthesis fluorescence microscope (FSFM) covers a band of axial spatial frequencies not accessible through classical optical sectioning microscopy. In principle, this instrument has the speed and transverse resolution of an imaging system, and axial resolution comparable to or better than a scanning confocal microscope. The significance of this project in the biological sciences can be stated concisely. Microscopes are most severely limited in axial, as opposed to transverse, resolution. This problem is rectified by confocal scanning, but with a loss of the fast, efficient information transfer that occurs in direct image formation. In many biological problems, particularly in studies of live cells, speed and photometric efficiency are primary constraints. The field-synthesis fluorescence microscope is designed specifically to give improved axial resolution while still retaining the high-performance characteristics of an imaging system.

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.