Laser Applications

Research Emphasis

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.

Current Research

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.

Resource Capabilities

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.

Special Features

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’s Who in Fluorescence annual volume.

1. Lakowicz, J. R., Shen, Y., D’Auria, 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 301:261–277, 2002.
2. Lakowicz, J. R., Radiative decay engineering: Biophysical and biomedical applications. Analytical Biochemistry 298:1–24, 2001.

Research Emphasis

Biological fluorescence spectroscopy has been undergoing a transition from studies of extracted biomolecules—such as proteins, nucleic acid polymers, lipid assemblies, and other more complex systems in large-volume cuvettes—to 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.

Current Research

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).

Resource Capabilities

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; 700–1000 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.

1. Chen, Y., Müller, J. D., Ruan, Q. Q., and Gratton, E., Molecular brightness characterization of EGFP in vivo by fluorescence fluctuation spectroscopy. Biophysical Journal 82:133–144, 2002.
2. Sukhishvili, S. A., Chen, Y., et al., Surface diffusion of poly(ethylene glycol). Macromolecules 35:1776–1784, 2002.
3. 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 40:6903–6911, 2001.

Research Emphasis

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 regulatory networks.

Current Research

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—a clinical technique that combines intrinsic fluorescence spectroscopy, diffuse reflectance spectroscopy, and light-scattering spectroscopy for spectral diagnosis—is 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.

Resource Capabilities

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.

1. 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 186:374–382, 2002.
2. Wax, A., Yang, C., et al., Cellular organization and substructure measured using angle-resolved low-coherence interferometry. Biophysical Journal 82:2256–2264, 2002.
3. 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 62:682–687, 2002.
4. Georgakoudi, I., Jacobson, B., et al., Fluorescence, reflectance and light-scattering spectroscopy for evaluating dysplasia in patients with Barrett’s esophagus. Gastroenterology 120:1620–1629, 2001.

Research Emphasis

The program is designed to develop optical instrumentation and biophysical models for understanding mechanisms of light-tissue interaction.

Current Research

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.

Resource Capabilities

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 1–2 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–1 GHz photon density waves (PDWs) are produced using up to 10 intensity-modulated diode lasers (674–980 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.

1. 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 99:11014–11019, 2002.
2. Ren, H., et al., Imaging transverse flow velocity using the Doppler bandwidth in phase-resolved F-OCT. Optics Letters 27:409–411, 2002.
3. Shah, N., et al., Non-invasive functional optical spectroscopy of human breast tissue. Proceedings of the National Academy of Sciences USA 98:4420–4425, 2001.
4. Hayakawa, C. K., et al., Use of perturbation Monte Carlo methods to solve inverse photon migration problems in heterogeneous tissues. Optics Letters 26:1335–1337, 2001.

Research Emphasis

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.

Current Research

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.

Resource Capabilities

Instruments

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.

Special Features

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.

1. Lauer, S., Goldstein, B., Nolan, R., and Nolan, J. P., Analysis of cholera toxin-ganglioside interactions by flow cytometry. Biochemistry 41:1742–1751, 2002.
2. Lauer, S. and Nolan, J. P., Development and characterization of Ni-NTA microspheres. Cytometry 48:138–145, 2002.
3. Graves, S. W., Nolan, J. P., Jett, J. H., et al., Effects of nozzle design on rapid delivery in flow cytometry. Cytometry 47:127–137, 2002.
4. Nolan, J. P. and Sklar, L. A., Suspension array technology: Evolution of the flat array paradigm. Trends in Biotechnology 20:9–12, 2002.

Research Emphasis

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 confocal microscopy.

Current Research

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.

Resource Capabilities

Instruments

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.

Special Features

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.

1. Rubtsov, I. and Hochstrasser, R. M., Vibrational dynamics, mode coupling, and structural constraints for acetylproline-NH2. Journal of Physical Chemistry B 106:9165–9171, 2002.
2. 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 106:962–972, 2002.
3. Huang, C. Y., Getahun, Z., Zhu, Y. J., et al., Helix formation via conformation diffusion search. Proceedings of the National Academy of Sciences USA 99:2788–2793, 2002.