Lecture Abstracts
The 3rd
Symposium on Biological Imaging
Multidimensional Biological Imaging:
Approaches and
Innovations
September 9th, 2005
BioPharmaceutical Technology Center
Madison, Wisconsin
Sponsored
by:
W.M. Keck Laboratory
for Biological Imaging (UW-Madison)
Laboratory for Optical and
Computational Instrumentation (UW-Madison)
Promega Corporation
BioPharmaceutical Technology Center
Institute
Imaging
Exocytosis and Endocytosis
by Total Internal Reflection
(TIRF)
Wolfhard Almers
The Vollum Institute
Oregon Health & Science University
Total internal reflection generates a 100 nm thin layer of light (the evanescent field, EF) where water borders on glass. Where cells adhere to glass, the EF illuminates only the plasma membrane and the adjacent 100 nm of cytoplasm. All other regions remain in the dark. We have found the method to be broadly useful to study the mechanisms of exocytosis and endocytosis. In particular, it is useful to image single secretory granules, synaptic vesicles and various types of endocytic vesicles as they approach the plasma membrane and undergo exocytosis, or draw inwards from the plasma membrane during endocytosis. It may be combined with FRET to observe Ca-triggered conformational changes in SNAREs, the proteins that mediate exocytic membrane fusion, and with multi-color imaging to record the recruitment of cytosolic proteins to single sites of endocytosis. In particular, we describe a type of endocytosis that is not mediated by clathrin. Instead, it seems specialized to internalize the membrane of granules that have recently undergone exocytosis but have not yet flattened into the plasma membrane. It may be derived from phagocytosis, an ancestral mechanism by which the simplest cells ingest particles.
Multidimensional
Image
Informatics and
Visualization
Kevin Eliceiri
Laboratory for
Optical and
Computational
Instrumentation
University of
Wisconsin-Madison
Of
Mice and Men:
Molecular Imaging in Living Subjects
Sanjiv Sam Gambhir
Molecular Imaging Program at
Stanford
Stanford University
Technologies for small animal imaging are rapidly evolving and include micro positron emission tomography (microPET), micro computed tomography (microCAT), and charge coupled device (CCD) based optical cameras for imaging very low levels of light. We have developed and validated reporter genes that can be imaged using PET and also married these approaches to optical reporter genes (e.g., firefly luciferase). The Herpes Simplex Type 1 Virus Thymidine Kinase (HSV1-tk) and Dopamine Type 2 Receptor (D2R) reporter genes have been extensively studied. These reporter genes when expressed allow for trapping of positron labeled tracers. Cells that do not express the reporter gene do not significantly trap these tracers. Methods to improve the sensitivity of these approaches including transcriptional amplification strategies have been developed. These approaches allow repetitive and quantitative study of basic cellular events in living subjects while utilizing multiple imaging modalities to provide synergistic information. The approaches are very generalizable because the reporter gene can be activated based on many different cellular events. This is accomplished through the use of any promoter/regulatory region of choice driving expression of the reporter gene. The developed reporter genes are being incorporated with various in vivo gene delivery approaches, cell trafficking models, and transgenic models to study specific biological processes in vivo. In addition, the use of split reporter strategies have been validated for imaging protein-protein interactions in living subjects. Applications of the developed approaches for optimizing gene therapy & studying cancer biology are now under active investigation. The PET reporter gene approaches have also been translated into clinical studies of cancer gene therapy and demonstrate the ability to monitor gene therapy in patients.
Fluorescence
Nanoscopy: Breaking the
Diffraction Barrier by the RESOLFT Concept
Stefan Hell
Department of NanoBiophotonics
Max Planck Institute for Biophysical Chemistry
Since its
discovery by Abbe in 1873, the microscopy diffraction barrier
has received a lot of attention. However, the concepts to
improve the spatial resolution of a focusing (far-field) light
microscope of the mid 20th century were either too vague or
subject to unrealistic physical conditions. Consequently,
far-field fluorescence microscopes remained diffraction-limited
in theory and practice. In this lecture, we discuss the
principle of fundamentally breaking the diffraction barrier
through reversible saturable optical (fluorescence) transitions
(RESOLFT). This principle was first put forward in the
form of Stimulated Emission Depletion (STED) and Ground State
Depletion (GSD) microscopy. In these concepts, the
diffraction barrier is broken by a saturated optical transition
(depletion) between two states of a marker, whereby the
transition is effected with an intensity distribution featuring
one or more intensity minima (zero). The saturation level
defines the size of the ultrasharp focal spot and/or the
concomitantly enlarged bandwidth of the optical transfer
function (OTF). We show that in a RESOLFT concept the
resolution can be approximated by Δx = λ/(πn
√I/Isat),
whereby Isat is the characteristic intensity required for
saturating the transition, and I denotes the intensity applied. If the minima are produced
by focusing optics with a numerical aperture nsin α, the
minimal distance at which two identical objects can be discerned
is Δx = λ / (2n sin α√1+I/Isat
We give evidence of STED-microscopy
displaying PSF of 10-20 nm FWHM, corresponding to a 15-fold
enlargement of the OTF over Abbe’s barrier. The reduction
in fluorescence spot size provided by STED also allows
fluorescence fluctutation (correlation) spectroscopy with
subdiffraction probing volumes. The success of STED stems
from the fact that the saturation of the single-photon
transition of stimulated emission provides strong nonlinearities
at comparatively low intensities. The reason for
that is simple but critical: Unlike in multiphoton events,
saturation is not effected by the joint action of
multiple photons, but stems from the population of the
fluorophore states.
Therefore, transitions that are easy to saturate (i.e. low Isat), allow huge ς at low intensities. Examples include the saturation of the marker’s triplet state, which reduces Isat by ~ 103 as compared to STED. Of similar interest is the ‘switching’ between conformational fluorophore states, which gives a factor of >106. Suitable candidates for saturable switches are encountered in photochromic compounds and photoswitchable GFP-like proteins, which should ultimately give nanoscale resolution at intensities provided by a lamp.
Insights
Into Cell Compartmentalization and
Protein Trafficking Using GFP Technology
Jennifer Lippincott-Schwartz
Cell Biology and Metabolism Branch
National Institutes of Health
The development of fluorescent proteins as molecular tags over the past decade has spurred a revolution by allowing complex biochemical processes to be correlated with the functioning of proteins in living cells. Fluorescent proteins such as green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its variants can be fused to virtually any protein of interest to analyze protein geography, movement and chemistry in living cells. As such, they have provided an important new tool for understanding protein function, filling an urgent need now that the genome sequence of many organisms is complete. The modified GFPs have been used as markers to track and quantify individual or multiple protein species, as probes to monitor protein-protein interactions, and as photochemically modulatable proteins to highlight and follow the fate of specific protein populations within a cell. Here, I will discuss the kinetic microscopy methods of photobleaching and photoactivation that are being used to monitor the appearance, location, movement and degradation of GFP fusion proteins in living cells. Results from these applications are providing profound new insights into protein function and cellular processes in the complex environment of the cell.
Quantitative
Multi-Dimensional Image Analysis:
The FARSIGHT Approach
Badrinath Roysam
Center for Subsurface Sensing and Imaging Systems
Rensselaer Polytechnic Institute
Optical microscopy
and associated technologies have matured into a powerful tool
for diverse biological investigations at the sub-cellular,
cellular, and tissue levels. Three-dimensional (x,y,z) imaging
is now well established. Increasingly, the spatial dimension is
important in combination with other dimensions, for example,
changes in the spatial biochemical composition of the cell's
environment, dynamic transport phenomena, signaling events, and
dynamic changes in anatomy and chemical composition can be
recorded. From a systems biology perspective, modern microscopy
is valuable for its ability to record structures and functional
markers in intact tissue avoiding the need to fragment cells.
Spatial distributions and interactions among multiple structural
and functional markers can be recorded in a linked manner unlike
biochemical assays, gene arrays and flow cytometry, in which
spatial information is disrupted.
The end result of these
developments is the growing availability of sophisticated, and
voluminous, imagery data containing a wealth of information that
must be analyzed and interpreted. There is a need for automated
technologies for translating this massive data into quantitative
database representations that can be queried together with
genomic and proteomic databases and ontologies to generate
systematic insight.
This talk will focus on the FARSIGHT method that greatly simplifies and systematizes the above multi-dimensional image analysis problem.
Imaging
Dynamics at the Kinetochore Microtubule Interface
Ted Salmon
Department of Biology
University of North Carolina at Chapel Hill
My lab is interested in the spindle and cell cycle mechanisms that achieve accurate chromosome segregation. We have specialized in the development of imaging methods for analysis of protein function at kinetochores, spindle fibers and poles in living cells and re-constituted preparations. We are using fluorescent speckle microscopy methods (FSM) and spinning-disk confocal microscopy (SDCM) in combination with fluorescent photo-activation techniques to sort out the molecular mechanisms that pull kinetochores poleward either by forces coupled to microtubule depolymerization at kinetochores (Pac-Man Mechanism) or by microtubule poleward flux (Traction fiber or Pole Reeling-In Mechanism). Our studies are currently focused on the Ndc80 protein complex. It functions at the kinetochore outer plate to dynamically link kinetochores to the plus ends of spindle microtubules, to signal the spindle checkpoint through Mad2, and to prevent errors in microtubule attachment, like merotelic attachment of kinetochores to microtubules from opposite poles. We have also shown that merotelic kinetochore orientation is a major mechanism of aneuploidy in mammalian tissue cells that is not detected by the spindle assembly checkpoint. Live cell imaging studies show that error correction mechanisms function before anaphase to reduce kinetochore microtubules from the wrong pole, while anaphase spindle mechanics prevents the mis-segregation of most but not all chromosomes with merotelic kinetochores.
The
Role of Optics in Breast Cancer Detection
Bruce Tromberg
Department of Biomedical
Engineering
University of California, Irvine
Diffuse optical spectroscopy and imaging are non-invasive diagnostic techniques that employ near-infrared (NIR) light to quantitatively characterize the optical properties of thick, multiple-scattering tissues. Although NIR was first applied to breast diaphanography more than 70 years ago, quantitative optical methods employing time- or frequency-domain photon migration technologies have only recently been used for breast imaging. In this talk I will review principles of photon migration (i.e. diffuse optics) and describe the development of broadband methods for quantitatively measuring the bulk absorption and scattering properties of thick tissues. Clinical study results will be shown highlighting the sensitivity of optical methods to breast tissue metabolic changes associated with aging, hormonal stimulation, tumor growth, and chemotherapy. These finding will be placed in the context of conventional breast imaging in order to assess the role of optics in breast cancer research and clinical management.