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Imaging Technology
Center for Light Microscope Imaging & Biotechnology
Pittsburgh, PA
USA

Year: 1996
Status: Award Recipient
Category: Science
Nominating Company: Eastman Kodak Company

Automated light microscopes acquire, process, analyze, display and archive 4-dimensional image data on the chemical dynamics that are responsible for the functioning of living cells.
Background

The cell is the basic unit of life. Hundreds of chemical reactions are
performed in beautifully orchestrated processes within the living cell
to produce the functions of life. Basic cell functions include the
duplication of cells, locomotion, production of energy and communication
within and between cells. The myriad of chemical reactions are performed
with complex temporal and spatial relationships that encode the
necessary information to carry-out cellular functions. Normal cells
orchestrate the communication of information properly, while disease
states such as cancer occur as a result of faulty "information" at the
level of the archival information stored in the DNA and/or the
alteration of normal chemical reactions.

Light microscopes have been used by biologists and clinicians for
hundreds of years to view the structures of cells and tissues and to
detect some of the chemicals involved in cell functions. Until recently,
the light microscope has been primarily a tool to see patterns of
structures and chemicals in cells and tissues that are chemically
stabilized (fixed). Although a wealth of information has been obtained
over the years by studying these dead cells, the complex interplay of
chemical reactions that are responsible for life have not been defined.
A major step forward was made by combining electronic cameras, digital
imaging technologies and robotic controls of microscope components to
create a quantitative, machine vision tool to replace the qualitative
human observer. Different modes of optical contrast that yield distinct
structural and chemical information can be applied to the same cells to
assemble detailed dynamic maps of structure and chemistry. The digital
information technologies have enabled scientists to extract quantitative
and dynamic information from living cells in ways not possible before.

The creation of novel, fluorescent chemicals used as "sensors" of the
functions of living cells has been the final component of the
technologies required to fulfill the goal of investigating the dynamics
of life at the cellular level. Fluorescent molecules are usually small
organic compounds that have the property of absorbing light of one color
and giving off another color. The properties of the fluorescent light
that is emitted from these molecules, called fluorescent dyes, can vary
depending on the immediate chemical environment of the dyes. It has been
possible to construct fluorescent protein "biosensors" by combining the
optimal fluorescent dye with specific sites on proteins. These
"biosensors" can be incorporated into living cells where they report the
temporal and spatial dynamics of the chemical reactions that they were
designed to "sense". The automated microscopes are then used to record,
analyze, display and archive the 4-dimensional information. Several
colored dyes can be used in the same cell so that multiple chemical
events can be correlated in time and space. A new class of "dye" has
recently been defined that allows specific components to be labeled
using advances in genetic engineering. This will make it even easier to
construct "sensors".

Objectives

Our objective has been to develop and integrate the necessary
technologies to create an approach (and corresponding tool) that can
detect and measure the dynamic chemical events that produce cell
functions that are responsible for life. A tool for investigating living
cells was necessary to decipher the highly orchestrated and dynamic
chemistry of life. Information technologies have been at the heart of
the developments, since digital imaging through a light microscope is
the key component of this methodology.

Description of the Application

The application is the automated light microscope used in conjunction
with fluorescent "biosensors" that have been incorporated into living
cells. The application has been discussed in several reviews [1, 2, 3,
4, 5]. A standard light microscope has been modified so that all of the
moving parts are robotic and under computer control. Electronic cameras
are used to acquire image data sets in 4 or more dimensions (3-D space,
time and different modes of microscope contrast). Digital imaging
technologies are used to process, analyze, display and archive the
complex data sets. Specific "biosensors" are incorporated into living
cells that are then investigated with the automated microscope. Our
application can be considered as a functional imaging tool for chemical
reactions in living cells. This application is to living cells what
x-ray and nuclear magnetic resonance imaging tomography is to whole
humans. Since the cell is the unit of life, we are exploring the very
mechanisms responsible for life.

Figure Captions (for the five slides submitted)

The Multimode Microscope is our fourth generation automated microscope.
It is capable of acquiring multiple modes of microscopy on live cells,
including Reflection Interference Microscopy, 3-D Fluorescence
Microscopy, Differential Interference Contrast-Video Enhanced Contrast
Microscopy (VEC-DIC), Fluorescence Anisotropy, Fluorescence Recovery
After Photobleaching, and Multispectral Fluorescence. The user in SLIDE
#1 is running the microscope through the computer interface.

The Multimode Microscope, together with a variety of fluorescent probes,
enabled the first application of five spectrally distinct biological
probes to be analyzed in living cells. SLIDE #2 demonstrates this
technology by showing five distinct parameters numbered 1:nucleus,
2:actin analog, 3:endosomes, 4:mitochondria, 5:cytoplasmic volume, and a
composite overlay of probes 1-4. Four parameter fluorescence imaging
alone with DIC-VEC imaging, was used to correlate the spatial changes of
four parameters in live fibroblasts migrating into a "wound". The arrows
on the right panels indicate the direction of cell migration. Stress
fibers containing actin are absent during late migration (bottom right
image) and the mitochondria and endosomes maintained in the perinuclear
region during early cell migration (top right image) become distributed
throughout the cell after polarized locomotion was initiated.

The method of ratio imaging developed by us, has been used to detect
patterns of relative concentration of proteins in living cells. SLIDE #3
shows pseudocolored, ratio images of myosin II (a molecular motor)
concentration during the dynamic events of cell division (red - regions
of high myosin II concentration; blue - regions of low myosin II
concentration). Recent results from this study revealed new mechanisms
of recruitment and transport of myosin II in forming the cleavage
furrow, the structure that generates force for cell division.

SLIDE #4 demonstrates one of the new biosensors that has been developed
and used to detect the activation of myosin II by attaching a phosphate
to a specific site on this motor protein within living cells. This
biosensor uses fluorescence resonance energy transfer to detect the
level of myosin II phosphorylation. Ratio imaging of donor:acceptor
wavelengths during the process of early cell migration detected an
increasing gradient of myosin II phosphorylation (activation) (panel A)
from the leading edge of the cell to the rear of the cell containing the
nucleus (N). The Multimode Microscope, using a rapid acquisition
program, also collects a third image in the same live cell at the same
time point that can be used to create a second ratio image of the
relative concentration of myosin II. The arrow indicates the direction
of migration and blue = low levels of myosin II phosphorylation
activation (Panel A) or low concentrations of myosin II (Panel B), red =
higher levels of myosin II phosphorylation activation (Panel A) or high
concentrations of myosin II (Panel B). The results demonstrated that
cell locomotion involves the assembly of the myosin II motor at the
leading edge, and transport of the contractile machinery into the rear
of the cell where the myosin II motor is turned on and force-generating
contractions occur.

SLIDE #5 depicts the user interface for the fifth generation, automated
microscope called the Automated Interactive Microscope (AIM). This is
the main AIM window on a Silicon Graphics, Onyx computer. The right side
of this window has user-friendly controls for defining the experimental
parameters including optical configurations, and all optical-mechanical
motion controls. On the left side of this window are two sub-windows for
image display. The upper live window depicts a migrating cell whose
boundaries are tracked by the "snake" algorithm. The bottom window
displays multi-color, 3-D fluorescence images. In the present case, a
labeled sea urchin in early development is being investigated. These
image display windows can be used with our 5-D viewer tool to observe
cellular dynamics.

Publications

1) Taylor, D. L., M. Nederlof, F. Lanni, and A. Waggoner. 1992. The new
vision of light microscopy. American Scientist 80:322-335.

2) Farkas, D.L., G. Baxter, R. DeBiasio, A. Gough, M. Nederlof, D. Pane,
J. Pane, D. Patek, K. Ryan, and D. L. Taylor. 1993. Multimode light
microscopy and the dynamics of molecules, cells and tissues. Annu. Rev.
Physiol. 55: 785-817.

3) Taylor, D. L., R. DeBiasio, G. LaRocca, D. Pane, P. Post, J. Kolega,
K. Giuliano, K. Burton, B.Gough, A. Dow, J. Yu, A. Waggoner, and D.L.
Farkas.1994. Potential of machine-vision light microscopy in toxicologic
pathology. Toxicologic Pathology 22: 145-159.

4) Giuliano, K. and D.L. Taylor. 1995. Light optical-based reagents for
the measurement and manipulation of ions, metabolites, and
macromolecules in living cells. Methods in Neuroscience 27: 1-15.

5) Giuliano, K, P. Post, K. Hahn, and D. L. Taylor. 1995. Fluorescent
protein biosensors: measurement of molecular dynamics in living cells.
Annu. Rev. Biophys. Biomol. Struct. 24: 405-434.
The application has helped basic and clinical scientists begin to define
the mechanisms responsible for normal cell functions, such as how cells
control duplication (division), how immune cells attack "foreign cells",
and how cells locomote to "fill in" wounded regions of tissue or track
down and engulf bacteria and viruses. The application has also
stimulated the development of new, high-speed methods for screening the
effects of drugs on specific properties of living cells, as well as
methods for determining the properties of cancerous cells that make them
different from normal cells.

How has it affected them? What are its most important benefits?

The application has put a completely new tool in the hands of scientists
and clinicians. Before this technology was developed, these
investigators used chemically fixed, dead, cells to try to understand
cellular chemistry. They then attempted to extrapolate the results to
explain how cells worked. The new technology allows specific chemical
reactions to be studied in living cells, while the cell is functioning.
Cells can now be treated as "living test tubes". It has produced a
renaissance and revolution in the use of the light microscope in biology
and medicine.

What impact will it have on society?

The information stored in living cells is the most sophisticated
information "technology" available to mankind. Cells are remarkable,
"living computers". The modern revolution in the biological sciences is
based on the the fact that biological information can be deciphered and
manipulated at ever higher rates. Biological information falls into
three general categories that represent increasing levels of complexity:
1) one-dimensional information in DNA, the digital archive of life, with
a four-letter language of nucleotides; 2) the three-dimensional
information of proteins, the cellular machines of life, with a
twenty-letter language of amino acids; and 3) the multi-dimensional
information in living cells, the orchestration of chemical reactions in
time and space, with as yet only partially defined language. This last
category encodes the information that will unlock the mystery of life
and how we can use it. Our application is a critical tool for defining
the "language" of cell functions and thus an important gateway to
understanding and harnessing living cells. The application uses digital
imaging information technology to access the multi-dimensional
information of living cells. This knowledge will lead to improvements in
human health and create new biotechnology industries.

The impact has recently extended into the field of education. Together
with the Carnegie Science Center, in Pittsburgh, PA, we developed a
planetarium show entitled "Journey into the Living Cell". Our
multi-dimensional imaging tools were used to create a group immersive
visualization environment (G.I.V.E.) to take students and the general
public on an interactive journey to explore the functions of cells. The
dynamic chemistry of cells will be presented to more than a million
people over the next year.
The central component of this application has been the development of
digital imaging tools to control the automated acquisition of image data
sets and then process, analyze, archive, and visualize the chemical
reactions that the "biosensors" report. We modified and extended digital
imaging technologies for the specific application of studying living
cells. New algorithms were created to process, analyze and display
4-dimensional data sets. A fully integrated system of automated control,
image acquisition, processing, analysis, display and archiving was
developed specifically for the application. In addition, algortihms used
in other fields were adapted for use on dynamic image sets of cells. For
example, we extended a "snake" boundary detection algorithm, originally
developed for another field, to identify and track the boundaries of
moving cells. We also developed a ratio imaging approach to map spectral
and polarization changes of fluorescence-based sensors in living cells.
This latter method converted the microscope into a dynamic spectroscopic
tool for measuring changes in cell chemistry. Finally, we developed a
"5-D" viewer for analyzing 3-D data sets, in time and at several colors
of fluorescence. This tool allows the investigator to view the dynamic
interplay of the chemical changes while measurements are made. The
combination of using some existing imaging tools borrowed from other
fields and developing novel algorithms created the heart of the
application.
Our application is unique and original. We were the first to integrate
the development of robotic light microscopes, electronic cameras and
imaging sciences with the development of very specific "biosensors" of
protein functions based on fluorescence to explore the chemical and
molecular dynamics of living cells. Our work has stimulated the use of
this technology by an ever increasing number of scientists around the
world. We continue to develop the application by pushing the performance
boundaries of the computational tools, electronic imaging devices, light
optical methods and fluorescence-based "biosensors". We are now building
the fifth generation automated microscope using high performance
computing and communication. Our present goal is to acquire, process,
analyze, display and archive n-dimensional image data sets in real-time.
This will open a new avenue for automated, interactive exploration of
living cells by scientists. We will then be in a position to manipulate
the chemical reactions that we measure, while the reactions are going
on. We continue to lead in the extension of the original application.

How did your application evolve? What is its background?

The application evolved out of the need for biologists to detect and
measure the dynamics of living cells in order to define the functions.
It became clear that biochemistry, molecular biology and static, fixed
cell structural approaches would not solve the problem. Therefore, the
necessary technologies were integrated to build the automated light
microscope. No existing tool was available to solve the problem. An
interdisciplinary team of biologists, chemists, physicists, engineers
and computer scientists was assembled to design, build and use the
technology to first approach fundamental questions about the mechanisms
of cell locomotion. Subsequently, the application has been directed
toward questions in developmental biology, neuroscience and applied
biotechnology, including drug screening and multicolor
fluorescence-based clinical diagnostics.
The application has exceeded its goals. The original goal was to measure
the chemical dynamics of living cells in order to define the mechansim
of cell locomotion. However, each of the integrated technologies have
all undergone major developments that have extended the power of the
application over the last five years. For example, solid state cameras
replaced night vision cameras, so the quantitation of the light signals
became much simpler and better. High performance computers are making it
possible to perform the measurements of chemical events in real time.
Finally, our development of the standing wave fluorescence microscope
has improved the axial resolution of the fluorescence microscope by a
factor of five to ten. Therefore, the quality and type of measurements
have been dramatically better than we envisioned five years ago. In
addition, the application is now being embraced by the fields of cell
biology, developmental biology, neurosciences, immunology, toxicology
and pathology, to name a few.

Our fourth generation automated microscope is fully operational and is
being used daily in biomedical investigations. In parallel, we are
building the fifth generation system. It is difficult to define how many
people benefit, since the numbers increase yearly. The existing
generation technologies have been transferred to industry and various
companies make systems and components available to the biomedical
community. The plans for the future are to collaborate with a major
microscope manufacturer to design the optimal robotic microscope and
develop the high performance computing tools to extend the power of the
application. In addition, further advances in the construction of
biosensors will accelerate the impact of the application. In addition,
we are beginning to collaborate with pharmaceutical companies in the
design of a system specifically for high throughput drug discovery. We
have also initiated studies, in collaboration with cancer biologists, to
define differences between normal and cancerous cells, by imaging from
the molecular to the full organism (in vivo) level.
The most important challenge was the integration of the distinct
technologies to create a user-friendly tool for biologists. In addition,
the first and second generation automated microscopes were based on less
than optimal electronic cameras (night vision cameras) and computers
that were state of the art for the day, but under-powered for the task.
Therefore, the application evolved with the improvement in electronic
cameras (cooled charge-coupled devices) and high performance computing.
Finally, we have been able to harness the rapid developments in the
field of molecular biology to create "biosensors" based on genetic
engineering.

The earliest stages of the application were limited by financial
resources, but sizeable grants, particularly from the National Science
Foundation, accelerated our developments. The intellectual resources,
including outstanding undergraduate and graduate students, have always
been present. The interdisciplinary nature of the project attracted
excellent faculty from the university, as well as corporate involvement.
In fact, corporate scientists and engineers have played important roles
in helping to solve some technical questions.

The creation of the Center for Light Microscope Imaging and
Biotechnology formed the organizational structure needed for an
interdisciplinary program. The early stages of the program required the
scientists and engineers from different fields to learn the "languages"
of the other areas. This was successfully accomplished through the
extensive involvement of undergraduate and graduate students who formed
the heart of the effort. The students "trained" their faculty mentors
and actually stimulated the involvement of faculty from different
departments to serve on thesis committees. A two-day retreat one year
ago cemented the communication between the fields.