NIBIB - Developmental Resource for Biophysical Imaging Opto-Electronics

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Extremely bright, water-soluble quantum dots can be used to image capillaries within the dermis with cellular resolution. In this image, collagen is imaged by second harmonic generation (blue) and the quantum dots are imaged by two photon fluorescence excitation (yellow). Red blood cells exclude the quantum dots, forming shadows within the capillaries, which can be monitored over time (yellow trace).

Image by Dan Larson. For additional information, see Larson, Zipfel, Williams, Clark, Bruchez, Wise, and Webb, Science 2003; 300(5624):1434-1436.

Developmental Resource for Biophysical Imaging Opto-Electronics

Contact Information
Grant Number
Research Emphasis
Current Research
Resource Capabilities
References

Contact Information

Cornell University
School of Applied and Engineering Physics
223 Clark Hall
Ithaca, NY 14853-2501

http://www.drbio.cornell.edu

Principal Investigator/Contact

Watt W. Webb, Sc.D.

Principal Investigator/Director
Phone: 607-255-3331
Fax: 607-255-7658
www2@cornell.edu

Associate Director
Warren R. Zipfel, Ph.D.

Phone: 607-255-0663
Fax: 607-255-7658
wrz2@cornell.edu

Grant Number

Grant No. EB001976

Research Emphasis

The resource focuses on creation and critical applications of quantitative optical instrumentation for biophysical and biomedical research, including multiphoton excitation fluorescence microscopy capable of diffraction-limited 3-D imaging of dynamic processes deep in living cells, tissues and organisms, photochemical micropharmacology by photoactivation of caged reagents, and dynamic ultrasensitive measurements at the single-molecule level. Simultaneous molecular absorption of multiple (2 or more) infrared photons permits quantitative fluorimetric measurements involving standard biological probes and intrinsic cellular fluorophores, with reduced photodamage. We carry out research in photophysical properties of fluorescent molecules undergoing multiphoton excitation to provide a quantitative basis for biological applications. Other research areas include structure sensitive imaging of cell membrane heterogeneity, imaging in brain of energy metabolism dynamics of neurons and astrocytes, tracking of individual macromolecules on cell surfaces with 5 nm spatial sensitivity, fluorescence correlation spectroscopy (FCS) measurements of the dynamics of diffusion, chemical kinetics, and protein folding. FCS is capable of providing single-molecule sensitivity in solution, and when combined with multiphoton microscopy is applicable in living cells.

Current Research

We are developing instrumentation to extend imaging depth in thick tissue preparations and to optimize sensitivity for in vivo imaging in living specimens, such as transgenic mice and for exploratory areas such as analytic imaging of intrinsic tissue fluorescence and second harmonic generation imaging of tissue structure for biomedical pathology and research. In situ photobleaching, phototoxicity, and induced fluorescence measurements to discern the mechanistic aspects of photodamage. Application of multiphoton microscopy of intrinsic tissue fluorescence for rapid detection of pathology of disease in biopsy or in vivo is advancing rapidly, and development of multiphoton microscopy for endoscopic applications is on the agenda.

Resource Capabilities

Multiphoton photophysics and photochemistry, 3-D resolved microscopic imaging of fluorescence at single-molecule sensitivity in and on cells and in vivo. Molecular dynamics and mobility by fluorescence correlation spectroscopy in cells and dilute solutions.

Instruments

Four laser scanning microscopes equipped with mode-locked lasers provide 100 fs pulses at 80 MHz with wavelengths tunable from 690 nm to 1,000 nm for multiphoton imaging of UV and visible absorbing fluorophores
Inverted (2) and upright (2) microscopes equipped for electrophysiology
Instrumentation for microscopic fluorescence emission spectroscopy; molecular diffusion measurements in 2-D or 3-D by fluorescence photobleaching recovery and FCS; chemical kinetics by FCS with one- and two-photon excitation and time domain fluorescence lifetime measurements

References

Kasischke KA, Vishwasrao HD, Fisher PJ, Zipfel WR, Webb WW. Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis. Science 2004;305(5680):99-103.
Baumgart T, Hess ST, Webb WW. Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension. Nature 2003;425:821-824.
Zipfel WR, Williams RM, Christie RH, Nikitin AY, Hyman BT, Webb WW. Live tissue intrinsic emission microscopy using multiphoton excited intrinsic fluorescence and second harmonic generation. PNAS 2003;100(12):7075-7080.
Larson DT, Zipfel WR, Williams RM, Clark S, Bruchez M, Wise F, Webb WW. Water-Soluble Quantum Dots with Large Two-Photon Cross-sections for Multiphoton Fluorescence Imaging in vivo. Science 2003;300(5624):1434-1436.
Dombeck DA, Kasischke KA, Vishwasrao HD, Ingelsson M, Hyman BT, Webb WW. Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy. PNAS 2003;100(12):7081-7086.

Last reviewed on: 12/21/2006

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