Research and Development

The Laboratory for Fluorescence Dynamics (LFD) designs, tests, and implements advances in the technology of hardware, software, and biomedical applications. Technical developments of the five core projects are strongly interrelated and driven by collaborative research.

As of 2008.

Core Projects

New Hardware and Software for the Microscope Environment

This core project enhances the overall techniques and methods developed at the LFD. The subprojects are not mere updates or maintenance of instrumentation but are novel on their own.

Current developments:

• New concepts in fluorescence microscopy based on integration of scanning and multiple modes of data acquisition.
• Data acquisition hardware for ultra high bandwidth data collection for fluorescence lifetime-resolved imaging microscopy: the FLIMbox.
• A total internal reflection fluorescence (TIRF) microscope with a fast-frame camera for detecting dynamics in combination with spatial correlations.
• Rapidly changing the z-focus for 3-D particle tracking.
• Detection of ultra low concentration of particles and detection via scanning fluorescence correlation spectroscopy.
• Polarized fluorescence correlation spectroscopy.

Previous developments:

• New hardware for increased sensitivity and discrimination between fluorescent species based on photon arrival time.
• Methods for fast, multiple point correlations.
• Giant unilamellar vesicles, GUVs, as a means to study fluctuations in membranes, lipid-lipid interactions and protein-membrane interactions.
• A hydrostatic pressure cell for the microscope with single-molecule sensitivity.
• Novel mechanical solutions for high precision optical microscopy: design of the high performance optical microscope (HPM).

Spatio-Temporal Fluctuation Correlation Spectroscopy

This core project expands the analysis and visualization of fluctuation correlation spectroscopy towards multiple channels and images.

Current developments:

• Methods based on circular and raster scanning to obtain information about dynamic processes in cells.
• Methods for the analysis of fast kinetic processes in cells: binding of EGFP complexes to immobile structures.
• A method to analyze data from fluctuation experiments based on intensity distributions to recover the underlying populations of molecular species: the Photon Counting Histogram (PCH).

Previous developments:

• Methods for multidimensional PCH analysis and cross-correlation analysis.
• Derivation of molecular populations from single trajectory analysis.

3-D Particle Tracking and Distance Measurements

This core project is filling the gap between distance measurement methods based on energy transfer and direct optical resolution.

Current developments:

• A method to measure distances between two fluorescent particles (molecules) in the 10-200 nm range.
• Particle tracking in three dimensions.
• Methods of reconstructing the position of identical fluorescent particles below the diffraction limit.

Fluorescence Lifetime-resolved Imaging (FLIM)

This core project enables access to the complex fluorescence lifetime information in images without the need to become an expert spectroscopist.

Current developments:

• Identification of molecular species in fluorescence lifetime images based on the phasor approach.
• Using higher harmonic phasor plots to better determine the localization and physical rates for Förster Resonance Energy Transfer (FRET).
• Exploiting natural differences in expression levels of fluorescent proteins among cells in culture to separate the contribution of the background fluorescence from the proteins and to quantify FRET efficiencies.

Previous developments:

• Lifetime resolved medical endoscopy.
• Full field fluorescence lifetime-resolved imaging at video-rate.
• Data visualization for lifetime resolved imaging.

Data Analysis Software

The development of the Globals software envelops all activities of the LFD and serves as the bridge between the technical development and the dissemination and training of data analysis methods.

Current developments:

• Image fluctuation analysis: RICS, STICS
• Analysis of fluorescence lifetime images using the phasor approach: FLIM, FRET
• Documentation for Web-training and workshops.

Previous developments:

• Fluctuation analysis: line and circular scanning FCS.
• Porting Globals Unlimited to the Windows platform: Globals for Spectroscopy.
• Data visualization.

Previous Core Projects

Several previous developments by the LFD have been patented and are now available commercially.

• Parallel multiharmonic frequency-domain fluorometer.
• Using optical modulation amplitude (OMA) for lifetime measurements over the emission spectrum.
• Lifetime stopped-flow instrument.
• Theory and software for distribution analysis.
• Lifetime-resolved camera.
• Pump-probe fluorescence/absorption instrument.
• Photon migration in tissues using frequency-domain near infrared spectroscopy (Fd-NIRS).
• Two-photon microscope.
• Two-photon FCS.
• Stimulated emission imaging.
• Fluorescence in turbid media.

Collaborative Research Projects

Collaborative research projects are externally funded projects that provide challenging problems to determine the applicability, validity, and scope of the new technologies developed in the LFD. They are used as showcase examples or test cases for core development, and sometimes develop into core projects.

• Paxillin Dynamics at Focal Adhesions.
  Collaborators: Alan R. Horwitz, and Claire M. Brown (University of Virginia)
  
• Rapid Motion of Chromatin In Interphase Cells Revealed by Fast Particle Tracking.
  Collaborator: Andrew S. Belmont (University of Illinois)
  
• Real-Time 3-D Tracking of Viral Particles in Live Cells: Rhinovirus entry into cells and Dynamics of Bacteriophage HK97 Capsid Maturation.
  Collaborators: Jack Johnson and Kelly Lee (Scripps Research Institute), Andre Gomes and Andrea Oliveira (Universidade Federal Rio De Janeiro)
  
• Oligomeric Alzheimer's b-Peptide in Biological Fluids.
  Collaborator: Harry LeVine (University of Kentucky)
  
• Regulation of Renal Phosphate Transport by Lipids.
  Collaborator: Moshe Levi (University of Colorado Health Science Center)
  
• Lipid Rafts Reconstituted in Model Membranes.
  Collaborators: Kenneth Jacobson (University of North Carolina)
  
• Organelle Transport along Microtubules in Xenopus Melanophores: Evidence for Cooperation between Multiple Motors.
  Collaborators: Vladimir Gelfand and Anna S. Serpinskaya (University of Illinois)
• Dual-color Photon Counting Histogram in Cells.
  Collaborator: Joachim Muller and Yan Chen (University of Minnesota)
  
• B2R Dynamics in HEK293 Cells Measured by Fluorescence Fluctuation Spectroscopy.
  Collaborator: Suzanne Scarlata (SUNY, Stony Brook)
  
• Rotational Fluctuation Correlation Spectroscopy.
  Collaborator: George Barisas (Colorado State University)
  
• A Laser Microbeam Biotechnology Resource: Simultaneous OCT and 2-Photon Microscopy.
  Collaborators: Zhongping Chen, Shuo Tang, Tatiana B. Krasieva, and Bruce J. Tromberg (Beckman Laser Institute and University of California)
  
• Dynamics of Urokinase Plasminogen Activator Receptor (uPAR) Interactions in Cell Migration and Adhesion.
  Collaborator: Valeria R. Caiolfa, Fondazione Centro San Raffaele, Milano, Italy.