University of Maryland School of Medicine
725 West Lombard Street
Baltimore, MD 21201
Grant No. P41 RR008119
Principal Investigator and Contact
Joseph R. Lakowicz, Ph.D.
410-706-8409; Fax: 410-706-8408
The goal of the Center for Fluorescence Spectroscopy (CFS) is to advance the field of fluorescence spectroscopy
and its applications to the biological, medical and life sciences. The center develops the use of time-resolved
fluorescence in clinical and analytical chemistry as well as new functional probes. Current studies are the effects
of nobel metallic colloids, islands, and particles on the intrinsic properties of fluorophores, a new phenomenon
in fluorescence that is likely to find enormous impact in sensing, microscopy, resonance energy transfer, probe
chemistry, and multiphoton excitation.
Fluorophores are being placed in close proximity to metallic particles and surfaces by a variety of fabrication
techniques, using protein and Langmuir Blodgett fatty-acid spacers, to produce the desirable metal-enhanced
fluorescence effects, such as increased quantum yields and increased photostability. The center is currently
investigating the possibility of resonance energy transfer over much larger than traditional distances (> 100 Å)
using biologically relevant donors and acceptor labeled proteins, as well as the reduced self-quenching of highly
labeled species. Other projects involve the use of metallic colloids and particles to significantly improve signal
levels in sensing, imaging, single-molecule detection, and in vivo optical imaging.
Both time-domain and frequency-domain instruments are available. FD measurements are provided from 100 KHz to
10 GHz with time resolution from a microsecond to a picosecond time scale. The high-frequency measurements are made
using CFS custom-designed circuits for the use of high-speed microchannel plate detectors in FD measurements.
Both the TD and FD instruments are equipped with PMTs and MCPs, allowing their use in study of time-resolved
fluorescence in the cuvette, scattered media, and tissue through the skin. The CFS has also recently acquired
a single-photon timing Streak-Camera with outstanding time-resolution and gated emission capabilities.
Software for data analysis is available on site or remotely over the Internet and runs on Silicon Graphics workstations or personal computers. Copies of individual programs can be obtained from the Internet or by
contacting CFS staff.
Once a year, CFS provides a short course (one week) on Principles and Applications of
Time-Resolved Fluorescence Spectroscopy. The CFS also is home for the Journal of Fluorescence, the
Society of Fluorescence, and the Who抯 Who in Fluorescence annual volume.
- Lakowicz, J. R., Shen, Y., D扐uria, S., Malicka, J., Fang, J., Gryczynski, Z., and Gryczynski, I.,
Radiative decay engineering 2. Effects of silver island films on fluorescence intensity lifetimes and resonance
energy transfer. Analytical Biochemistry 301:261�7, 2002.
- Lakowicz, J. R., Radiative decay engineering: Biophysical and biomedical applications. Analytical
Biochemistry 298:1�, 2001.
University of Illinois at Urbana-Champaign
Department of Physics
1110 West Green Street
Urbana, IL 61801-3080
Grant No. P41 RR003155
Enrico Gratton, Ph.D.
Director and Contact
Theodore L. Hazlett, Ph.D.
217-244-5620; Fax: 217-244-7187
Biological fluorescence spectroscopy has been undergoing a transition from studies of extracted biomolecules梥uch
as proteins, nucleic acid polymers, lipid assemblies, and other more complex systems in large-volume cuvettes梩o
microscopic samples with an ultimate resolution at the single-molecule level. In this environment, the research
emphasis of the Laboratory for Fluorescence Dynamics (LFD) is geared toward the development of technologies that
facilitate the transition to microscope-based systems. The confluence of multiphoton laser excitation techniques
and fluorescence microscopy applications has led to rapid advances in imaging techniques, as well as related methods
such as fluorescence correlation spectroscopy (FCS). FCS offers high spatial resolution and observation of motional
dynamics in single molecules identified from correlation functions or photon-counting histogram analysis methods.
Other research areas include fluorescence in turbid media (such as tissue), global analysis software that allows tests
of models (such as quenching, lifetime heterogeneity, energy transfer, etc.), and interferometry.
New methods and instrumentation to study cellular structure and function; imaging of tissue, particularly skin;
and macromolecular dynamics and interactions. Examples of cellular studies include GFP-conjugates, receptor
(HDL) function, ion distribution, and photosynthesis in algae. Other studies relate to membrane fluidity and
domains (in large unilamellar vesicles), membrane-associated enzymes, protein-lipid interactions, protein folding,
DNA-protein interactions, and vascular injury. Monitoring of physiological function of tissue based on variations
in spectroscopic properties (such as fluorescence lifetime-based detection in medical endoscopy).
Time-resolved and steady-state fluorescence instrumentation: The frequency domain (phase/modulation; 300 MHz)
instrumentation with laser excitation (titanium sapphire, argon ion, or mode-locked Nd/YAG pumped dye laser)
covers the ultraviolet to the near-infrared regions. The photon-counting scanning fluorometers record
emission/excitation and polarization spectra, as well as kinetics. The sample compartments of the
lifetime and spectral instruments accommodate high pressure, gas quenching, and other types of vessels
in addition to thermostatting conventional cuvettes.
Light/fluorescence microscopy: For the study of cells and other microscopic structures, several laser-based
multiphoton excitation systems (Ti:sapphire; 700�00 nm) are coupled to light microscopes with fluorescence
capabilities. These systems allow for imaging (in plane and 3-D), particle tracking, and a variety of
image-capture approaches based on fluorescence spectra, intensity, polarization, generalized polarization,
and lifetime. In addition, these systems are compatible with measurements of fluorescence correlation
spectroscopy. Software has been developed to process the images and the FCS data (auto- and cross-correlation
and photon-counting histograms).
The laboratory houses a data analysis center, spectroscopy laboratories, microscopy laboratories, a wet
chemistry laboratory, and a tissue culture facility.
- Chen, Y., M黮ler, J. D., Ruan, Q. Q., and Gratton, E., Molecular brightness characterization of EGFP
in vivo by fluorescence fluctuation spectroscopy. Biophysical Journal 82:133�4, 2002.
- Sukhishvili, S. A., Chen, Y., et al., Surface diffusion of poly(ethylene glycol). Macromolecules
- Sanchez, S. A., Chen, Y., Mueller, J. D., et al., Solution and interface aggregation states of C.
atrox venom phospholipase A2 by 2-photon excitation fluorescence correlation spectroscopy. Biochemistry
Massachusetts Institute of Technology
George R. Harrison Spectroscopy Laboratory
Building 6, Room 014
77 Massachusetts Avenue
Cambridge, MA 02139
Grant No. P41 RR002594
Michael S. Feld, Ph.D.
617-253-7700; Fax: 617-253-4513
Ramachandra R. Dasari
The Laser Biomedical Research Center: develops methods of spectral diagnosis for in vivo analysis of
diseased biological tissue; studies light propagation in turbid media by means of photon migration;
develops spectroscopic techniques for imaging functional change in biological tissue; uses low-coherence
interferometry to measure nanometer length changes and small-scale dynamic processes in living cells and
tissues; develops near-infrared Raman spectroscopy for accurate concentration measurements of blood analytes,
diagnosis of breast cancer, and in vivo studies of atherosclerosis; and studies intrinsic noise in gene
Includes development of point contact and imaging instruments for use in a clinical setting. These systems are
used to study reflectance, fluorescence, elastic light scattering, and Raman spectra from human tissues. Tri-model
spectroscopy梐 clinical technique that combines intrinsic fluorescence spectroscopy, diffuse reflectance spectroscopy,
and light-scattering spectroscopy for spectral diagnosis梚s being developed and tested in the esophagus, colon, bladder,
cervix, and oral cavity. Near-infrared Raman spectroscopy is being developed for transcutaneous measurement of blood
analyte concentrations. Raman histochemistry of breast cancer and atherosclerosis is being studied. Optical fiber
probes for in vivo Raman spectroscopy are being developed, with clinical studies under way in femoral and carotid
arteries. Low-coherence interferometry is being developed to measure nanometer-scale dynamic processes
in biological systems. Instrumentation for ultra-sensitive fluorescence microscopy to explore stochastic gene
expression by a single gene is also being developed.
Instruments and Experimental Set-Ups
The center houses state-of-the-art facilities for Raman spectroscopy in the near infrared, visible, and
ultraviolet, with micro-Raman capability and optical fiber probes for remote measurements; a ps/fs laser
laboratory with a mode-locked Ti:sapphire laser with second, third, and fourth harmonic generation, and an
OPA to generate tunable radiation in the visible; a streak camera capable of 2 ps resolution; a fast-gated
CCD (up to 100 MHz); instrumentation for fluorescence microspectroscopy; spectral endoscopes for fluorescence
imaging of disease; excitation-emission matrix (EEM) spectrometers with multi-wavelength excitation that collect
an entire EEM in less than one second.
- Georgakoudi, I., Sheets, E. E., et al., Tri-modal spectroscopy for the detection and
characterization of cervical pre-cancers in vivo. American Journal of Obstetrics and Gynecology 186:374�2, 2002.
- Wax, A., Yang, C., et al., Cellular organization and substructure measured using angle-resolved
low-coherence interferometry. Biophysical Journal 82:2256�64, 2002.
- Georgakoudi, I., Jacobson, B. C., Muller, M. G., et al., NAD(P)H and collagen as quantitative
fluorescent biomarkers of epithelial pre-cancerous changes. Cancer Research 62:682�7, 2002.
- Georgakoudi, I., Jacobson, B., et al., Fluorescence, reflectance and light-scattering spectroscopy for
evaluating dysplasia in patients with Barrett抯 esophagus. Gastroenterology 120:1620�29, 2001.
University of California, Irvine
Beckman Laser Institute
1002 Health Sciences Road East
Irvine, CA 92612
Grant No. P41 RR001192
Bruce J. Tromberg, Ph.D.
949-824-8705; Fax: 949-824-6969
Tatiana B. Krasieva, Ph.D.
Albert Cerussi, Ph.D.
The program is designed to develop optical instrumentation and biophysical models for
understanding mechanisms of light-tissue interaction.
Mechanisms of coherent image formation; models for understanding the origin and degradation
of coherent light interactions in biological tissues; combined two-photon excited
fluorescence and second harmonic generation imaging in vivo.
Applications: Monitoring/imaging hemodynamics before, during, and after
interventional procedures; imaging cell-extracellular matrix interactions.
Tissue absorption and scattering measurements in complex, heterogeneous, biological systems;
relationships between tissue optical properties and physiology/cellular structure; tissue
phantoms, preclinical animal models, and human subjects. Applications: Tumor diagnostics
in breast, brain, and cervix; structural origins of tissue optical properties.
Confocal Ablation Trapping System: Integrates independently controlled, tunable trapping and
ablation beams into a Zeiss laser scanning confocal microscope. Detectors include a cooled color
CCD camera and three photomultiplier tubes.
Functional Optical Coherence Tomography (OCT): Optical fiber-based OCT systems utilizing superluminescent
diodes, photonic crystals, and ultrafast lasers. Capable of multi-modal imaging in vivo blood flow
and tissue structure simultaneously to depths of 2-4 mm. Fiber probe and microscope platforms
available with video scanning.
Multi-Photon Microscopy: Two-photon fluorescence (TPF) and second harmonic generation (SHG)
are detected simultaneously from tissue using 2 photomultiplier tube channels and a dedicated
spectrometer. Intrinsic SHG signals are sensitive to collagen in the extracellular matrix, while
TPF reports cellular and tissue autofluorescence. Typical scans are 256 times 256 pixels in 1�seconds.
Diffuse Optical Spectroscopy: Multi-wavelength, high-bandwidth (1 GHz) portable frequency-domain
photon migration systems for quantitative, noninvasive measurements of tissue optical and physiological
properties. 300 KHz�GHz photon density waves (PDWs) are produced using up to 10 intensity-modulated
diode lasers (674�0 nm) combined with a broadband continuous light source. The frequency-dependence
of PDW phase and amplitude is used to calculate absorption, µa, and reduced scattering, µs', spectra
with 3 nm resolution. The wavelength-dependence of absorption is used to determine tissue hemoglobin
concentration (total, oxy- and deoxy-forms) and tissue oxygen saturation, lipid, and water concentration.
- Zoumi, A., Yeh, A., Tromberg, B. J., Imaging cells and extracellular matrix in vivo using
second-harmonic generation and two-photon excited fluorescence. Proceedings of the National
Academy of Sciences USA 99:11014�019, 2002.
- Ren, H., et al., Imaging transverse flow velocity using the Doppler bandwidth in phase-resolved
F-OCT. Optics Letters 27:409�1, 2002.
- Shah, N., et al., Non-invasive functional optical spectroscopy of human breast tissue.
Proceedings of the National Academy of Sciences USA 98:4420�25, 2001.
- Hayakawa, C. K., et al., Use of perturbation Monte Carlo methods to solve inverse photon migration
problems in heterogeneous tissues. Optics Letters 26:1335�37, 2001.
Los Alamos National Laboratory
Bioscience Division, Mail Stop M-888
Los Alamos, NM 87545
|Principal Investigator and Contact|
John P. Nolan, Ph.D.
505-667-1623; Fax: 505-665-3024
The National Flow Cytometry Resource advances flow cytometric analyses through innovative research,
development, and collaborations. Flow cytometry is a technique for high-speed analysis of individual
particles ranging in size from single molecules and macromolecular complexes to subcellular organelles,
cells, and cellular aggregates. Particles pass rapidly through one or more focused laser beams where probe
molecules bound to specific components, such as DNA in cells, are excited and the emitted fluorescence photons
are detected. Measurement of fluorescence emissions and scattered excitation light provides quantitative
information about the particles. For cells, measurements are made of DNA, RNA, and protein content; surface
molecules; and physiological parameters. Since individual particles are analyzed, distributions of these and
other measured parameters are obtained at analysis rates of thousands of events per second. Based on the
measurements, particles in selected subpopulations can be physically separated by sorting. Unique flow
cytometric capabilities include high-resolution chromosome analysis and sorting; fluorescence lifetime
measurement; rapid-mix analyses with subsecond time resolution; phase-sensitive fluorescence detection;
DNA fragment size quantification; ultrasensitive fluorescence detection; multiplexed analysis; and
multivariate data display and analysis. Expert advice and assistance are available to collaborators
preparing cellular and chromosome samples for analysis; multiplexed genetic and protein analysis, rapid
kinetic analyses, macromolecular assembly dynamics, multivariate data acquisition and analysis; sorting
procedures; and other areas.
Microsphere-based analysis of molecular interactions; kinetic analyses of signal transduction processes in
cells; DNA fragment-size distribution analyses; high-throughput screening of molecular interactions.
Associated research: Cell cycle analysis and control; applications of chromosome analysis and sorting
in neoplastic transformation; chromosome sorting for recombinant DNA library construction; bacterial
and viral detection and identification; and medical applications of flow cytometry.
Four multiwavelength sorting systems with two or three lasers for sequential excitation
(high- and low-power Argon, Krypton lasers), phase-sensitive/lifetime flow cytometer,
rapid mix flow cytometer, DNA fragment-size analysis cytometer; optical chromosome selection
cytometer; BD FACSCaliber five-parameter cytometer; fluorescence microscope with cooled CCD
camera; and static and time-resolved spectrophotometers.
Data acquisition capabilities for up to 512 parameters per event; axial light loss measurements;
Coulter volume; multiwavelength excitation/emission measurements; rapid-mix/sample delivery with
subsecond capabilities; bulk chromosome sorting; offline data analysis with local workstations or
central computing facility.
- Lauer, S., Goldstein, B., Nolan, R., and Nolan, J. P., Analysis of cholera toxin-ganglioside
interactions by flow cytometry. Biochemistry 41:1742�51, 2002.
- Lauer, S. and Nolan, J. P., Development and characterization of Ni-NTA microspheres.
Cytometry 48:138�5, 2002.
- Graves, S. W., Nolan, J. P., Jett, J. H., et al., Effects of nozzle design on rapid delivery in
flow cytometry. Cytometry 47:127�7, 2002.
- Nolan, J. P. and Sklar, L. A., Suspension array technology: Evolution of the flat
array paradigm. Trends in Biotechnology 20:9�, 2002.
University of Pennsylvania
231 South 34th Street
Philadelphia, PA 19104-6323
Robin M. Hochstrasser, Ph.D.
215-898-8410; Fax: 215-898-0590
Thomas Troxler, Ph.D.
Erwen Mei, Ph.D.
The laboratory develops methods for the investigation of rapid structural changes and ultrafast processes in
proteins, enzymes, and nucleic acids. Examples include protein and peptide conformational changes,
electron and energy transfer in light-harvesting protein, heme protein dynamics, photoisomerization in
light-sensitive proteins, photophysics, and folding of single biomolecular assemblies.
Ultrafast (femtosecond, picosecond, and nanosecond) methodologies are developed for these investigations:
Phase-controlled infrared (IR) pulses are developed for IR analogues of NMR, structure determination,
and probing nuclear motions. Time-correlated single-photon counting, transient spectroscopy
(UV/Vis/vibrational IR/terahertz), photon echoes, two-photon absorption, other 4-wave optical
and IR methods, laser-induced temperature jumps, femtosecond infrared methods, and time-resolved
Two-dimensional IR spectroscopy (IR analogues of NMR) to study the dynamics of structures occurring
in proteins and peptides; coherent IR methods to examine structural fluctuations through vibrational
correlation functions; IR pump-probe methods with vibrational mode selectivity to study vibrational
dynamics, mode coupling, and energy transfer; transient IR probing of protein folding and conformational
dynamics through the application of T-jump, stopped-flow, and isotope-editing techniques; time- and
frequency-resolved spectroscopy of single proteins and biological assemblies, including the application
of new lifetime imaging techniques; ultrafast optical, infrared, and terahertz methods to study excited
state dynamics, molecular motion, energy transfer, and photophysics in biomolecules.
Phase-controlled IR femtosecond pulses and tunable IR pulses; femtosecond to nanosecond fluorescence
spectrometers; femtosecond to millisecond transient absorption spectrometers using Ti:Sapphire lasers;
inverted confocal microscope with time-correlated single-photon counting, array detector, and femtosecond
pulse excitation capabilities; rapid recording of fluorescence in the range of 100 fs to many ns;
time-correlated photon counting and fluorescence upconversion methods; facilities for pump-probe
experiments using all optical wavelengths available from OPAs.
The laboratory is developing phase-controlled tunable IR pulses for multidimensional spectroscopy of peptides
and small proteins. Precision transient absorption spectra can be acquired in the fs to ms regime.
Instrumentation has also been developed to perform transient IR and terahertz spectroscopy. Atomic force
and confocal microscopes have been constructed that are investigating single molecules, molecular assemblies,
and protein folding and unfolding. Regional Laser and Biomedical Technology Laboratories can accommodate
essentially any laser-based experiment with emphasis on short pulsed methods. The staff is skilled in
creating novel experimental configurations based on lasers needed for both short-term and long-term projects.
- Rubtsov, I. and Hochstrasser, R. M., Vibrational dynamics, mode coupling, and structural constraints
for acetylproline-NH2. Journal of Physical Chemistry B 106:9165�71, 2002.
- Ge, N. H., Zanni, M. T., and Hochstrasser, R. M., Effects of vibrational frequency correlations on
two-dimensional infrared spectra. Journal of Physical Chemistry A 106:962972, 2002.
- Huang, C. Y., Getahun, Z., Zhu, Y. J., et al., Helix formation via conformation diffusion search. Proceedings of the National Academy of Sciences USA 99:2788�93, 2002.