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Vibrational excitations of low frequency collective modes are essential for functionally important conformational transitions in proteins. Here we report the first direct measurement on the lifetime of vibrational excitations of the collective modes at 87 pm (115 cm-1) in bacteriorhodopsin, a transmembrane protein. The data show that these modes have extremely long lifetime of vibrational excitations, over 500 picoseconds, accommodating 1500vibrations. We suggest that there is a connection between this relativelyslow anharmonic relaxation rate of approximately 10 g sec-1 and thesimilar observed rate of conformational transitions in proteins, which require require multi-level vibrational excitations and energy exchanges with othervibrational modes and collisional motions of solvent molecules.

The extremely slow α-helix/β-sheet transition of proteins is a crucial step in amylogenic diseases and represents an internal rearrangement of local contacts in an already folded protein. These internal structural rearrangements within an already folded protein are a critical aspect of biological action and are a product of conformational flow along unknown metastable local minima of the energy landscape of the compact protein. We use a diffusional IR mixer with time-resolved Fourier transform IR spectroscopy capable of 400-μs time resolution to show that the trifluoroethanol driven β-sheet to α-helix transition of β-lactoglobulin proceeds via a compact β-sheet intermediate with a lifetime of 7 ms, small compared with the overall folding time of β-lactoglobulin.

We have fabricated using electron beam nanolithography a fixed slit near-field optical scanning device which uses near-field fluorimetry to achieve 200 nm spatial resolution of objects moving over the slits. We explore the basic physics of operating narrow slits in the waveguide cutoff mode and present data from the passage of extended double-stranded DNA molecules passing over the slits as a first example of how this device can be used to do ultrahigh spatial resolution mapping of long polymers.

A versatile free electron laser (FEL) user facility has recently come on line at the Thomas Jefferson National Accelerator Facility (Jefferson Lab) providing high average (kilowatt-level) power laser light in the infrared. A planned upgrade of the FEL in this facility will extend the wavelength range through the visible to the deep UV and provide the photobiology community with a unique light source for a variety of studies. Planned and potential applications of this FEL include: IR studies of energy flow in biomolecules, IR and visible imaging of biomedical systems, IR and visible studies of photodynamic effects and UV and near visible studies of DNA photodamage.

We show that myoglobin, which is almost entirely α helix in secondary structure, has an unusually long-lived 12 ps vibrational excited state lifetime generated by optically pumping at the blue side 5.85 microns of the amide I band, indicating the generation of a long-lived trapped soliton- antisoliton breather mode.

A separation technique employing a microfabricated sieve has been demonstrated by observing the motion of DNA molecules of different size. The sieve consists of a two-dimensional lattice of obstacles whose asymmetric disposition rectifies the Brownian motion of molecules driven through the device, causing them to follow paths that depend on their diffusion coefficient. A nominal 6% resolution by length of DNA molecules in the size range 15–30 kbp may be achieved in a 4-inch (10-cm) silicon wafer. The advantage of this method is that samples can be loaded and sorted continuously, in contrast to the batch mode commonly used in gel electrophoresis.

Novel microfabricated devices promise to accomplish fractionation of chromosomal size DNA more quickly, more accurately, at lower cost, and by using smaller sample amounts. Chromosomes are released by lysing cells directly in the device. The chromosomal DNA is further concentrated on a platinum wire in a 10 μm wide band using the phenomenon of dielectric trapping in AC fields. The DNA is then electrophoretically driven into a microfabricated array of posts arranged in a hexagonal lattice. Under electric fields whose direction periodically changes by 120°, the longer DNA molecules move at lower speeds than the shorter ones, and separation according to size is achieved. This technique allows application of electric fields as large as 1000 V/cm and, thus, promises to reduce considerably separation times compared to the presently used technique of pulsed-field gel electrophoresis.

Biology is deeply influenced by the structure of biomolecules, the dynamics of biomolecules, the way that biomolecules interact with each other, and how the cellular architecture is influenced by the structure and dynamics of the molecular components. At its most fundamental level, the length scale of these structures and dynamics of primary biological components is on the 1–100 nm length scale. Although there are ingenious optical techniques that reach into that length domain, they involve highly specific and robust optical probes and averaging techniques to find the standard deviation of the mean, greatly limiting our ability to observe the real-time dynamics of cell components at a fundamental level. It is now clear that unraveling the response of cells to ionizing radiation is critical for understanding many fundamental problems in biology, from aging to apoptosis (programmed cell death) to the origins of cancer. Since a cell is spatially highly heterogeneous, simply irradiating cells results in the activation of many different biological pathways. The high brightness of a coherent X-ray/VUV light source would allow irradiating cell components with 100 nm or better spatial resolution, which would be a major step forward in unraveling the response of cells to genomic and component damage.

Symmetry is usually prized in nature, but the deliberate skewing of symmetry in nanofluidic devices can lead to elegant new ways of sorting biomolecules.

We will discuss two recent directions of our work: (1) The influence of submicon length scales on polymer dynamics, (2) Ultra-rapid mixing via sub-micron hydrodynamic focusing. (1) Polymer dynamics at sub-micron length scales. We have explored the changes in the dynamics of long polymers as the thickness of the quasi-2 dimensional space is varied from 0.09 microns to 10 microns. We will show how the thickness of this space, scaled with the persistence length of the polymer, changes the dynamics of the polymer. The consequences of this qualitative change in polymer dynamics is quite important, since it controls the elongation of the polymer at a given force field and hence the ability of the array to fractionate the polymer. (2) Mixing at the sub-micron length scale cannot be tubulent but only diffusive in nature. We will show how it is possible using hydrodynamics to produce liquid jets of width under 20 nanometers which can mix fluids in under 1 microsecond times.

We describe a method for wetting micro- and nanofluidic devices with water or any other pure liquid. The process is performed by enclosing the fluidic device in a liquid-filled cell, heating the cell to a temperature above the critical point of the liquid, and subsequent cooling of the cell to room temperature. Because the process liquid is essentially a gas during wetting, arbitrary shapes can be wetted. We demonstrate wetting of micro- and nanostructures in a fused-silica device with only a single inlet. The process is low-cost, fast, safe, and very reliable.

We demonstrate a novel hydrodynamic shear activation of leucocyte adhesion, using physiological flow conditions and a microfabricated array of channels with length scales similar to those of human capillaries. Vital chromosome stains and cell specific fluorochrome labeled antibodies reveal that the eventual adhesion of the leukocytes to the silicon array displays a strong dependence on cell type and nuclear morphology, with granulocytes activating more rapidly with distance and penetrating a smaller distance than lymphocytes. Further, the granulocytes interact with the lymphocytes in a self-exclusionary manner under shearing flow with the eventual separation of the two cell types in the array. Such arrays of microfabricated obstacles thus have an interesting potential for sorting white blood cells by type from a 10 microliter drop of whole blood.© (1997) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

1 Importance of DNA Sorting in Biology 2 Physical Description of DNA 3 Reptation of DNA in Sequencing Electrophoresis 4 Obstacle Courses on Microchips 5 Complex Dynamics of Long DNA Molecules 6 Pulsed-Field Gel Electrophoresis: Separation of Restriction Fragments 7 Efficient Pulsed-Field Fractionation in Silicon Arrays 8 Summary Acknowledgment References

The feasibility of using nanofabricated arrays as electrophoretic chambers for DNA sequencing is investigated. A specific array design, consisting of rows of closely spaced posts, separated by longer open spaces, is proposed. Molecules driven through the array by an electric field get hooked over obstacles at successive rows and their progress through the device is delayed as a consequences. The dependence of the delay time on molecular size is derived. Numerical evaluation indicates that a device of modest dimensions, operating at high fields, can rapidly resolve oligonucleotides containing several hundred bases.

The Compact Infrared Free Electron Laser (CIRFEL) was built as part of a joint collaboration between Northrop Grumman and Princeton University to develop FEL's for use by researchers in the materials, medical and physical sciences. The CIRFEL was designed to laser in the Mid-IR and Far-IR regimes with picosecond pulses, megawatt level peak powers and an average power of a few watts. The CIRFEL utilizes an RF photocathode gun to produce high-brightness time synchronized electron bunches. The micropulse separation is 7 nsec which allows a number of relaxation phenomena to be observed. In addition, the photocathode illumination laser can be used in combination with the FEL IR light for pump- probe experiments. The CIRFEL is presently being commissioned and working towards lasing. The present status of the machine is presented.

Publisher Summary This chapter provides some practical advice on the way to do low-temperature spectroscopy. The chapter discusses the internal structure of visible absorption spectra and their temperature dependence. It also discusses the utilization of low-temperature spectroscopy to probe protein dynamics. The chapter goes beyond the traditional use of low temperatures to trap intermediate chemical states and to speculate the insight that low-temperature spectroscopy provides the role of protein dynamics in controlling chemical reactions. Probably the main reason for the neglect of low-temperature spectroscopy in biophysics is the unstated opinion of many biophysicists that these kinds of studies are irrelevant to the biological aspects of the reaction. Another reason, connected to the first claim of irrelevance, is the fact that any such study of low-temperature dynamics necessarily involves construction of theoretical models, and in complex molecules this is a notoriously difficult and speculative thing to do. Thus, the chapter provides a comprehensive review of all low-temperature studies on biological systems.

Biomolecules are notorious for their unpredictable flexibility. Some of the smallest nanopores ever created are being used to manipulate individual DNA molecules, with far-from simple results.

This is a theoretical paper which examines at a game theoretical perspective the dynamics of cooperators and cheater cells under metabolic stress conditions and high spatial heterogeneity. Although the ultimate aim of this work is to understand the dynamics of cancer tumor evolution under stress, we use a simple bacterial model to gain fundamental insights into the progression of resistance to drugs under high competition and stress conditions.

Publisher Summary This chapter describes one particular experimental approach to obtaining substantive information regarding the protein dynamics relevant to biological function. This chapter discusses both the promise and limitations of transient infrared (IR) spectroscopy of proteins for advancing the microscopic understanding of mechanistic biochemistry. The vast majority of work has concentrated on heme proteins as model systems. Picosecond transient IR absorption work in proteins is still in its infancy. Because laser technology is costly and technically difficult, the biological insight gained from these experiments is small. Although a fair amount is known about myoglobin structural relaxation, the basic steps involved in hemoglobin (Hb) structural relaxation are not well known. Time-resolved ultraviolet (UV) Raman work seems to have been the most productive, in spite of the promise of IR spectroscopy. However, it is hoped that this chapter might entice scientists interested in the conformational dynamics of protein function to work with laser technologists to address this issue in a meaningful manner.

Free-electron lasers and high-energy physics accelerators have increased the demand for very high-brightness beam sources. This paper describes the design of an accelerator which has produce beams of 2.1 /spl pi/ mm-mrad at 1 nC and emittances of 3.7 and 6.5 /spl pi/ mm-mrad for 2 and 3 nC, respectively. The accelerator has been operated between 10 and 18 MeV. The beam emittance growth in the accelerator is minimized by using a photoinjector electron source integrated into the design of the linac, a focusing solenoid to correct the emittance growth caused by space charge, and a special design of the coupling slots between accelerator cavities to minimize quadrupole effects. >

Using synthetic 45 bp long DNAs of known sequences, we have studied (i) the unusual structure that the phased A-tracts have at temperatures below 37 degrees C, (ii) the thermodynamics of the loss of that aberrant structure well below the duplex melting temperature, and (iii) the conformational changes that occur with temperature. Using temperature-dependent circular dichroism, we detect a low-temperature structural transition in a 45-mer duplex with four segments of phased (dA)5 tracks separated by five segments of five randomized G-C pairs, but none in the corresponding isomeric random sequence 45-mer duplex. Differential scanning calorimetry measurements reveal the enthalpy of this preglobal melting transition to be 3.5 kcal/mol.AT pair or 4.4 kcal/mol.AA step. The integrated enthalpy change for this helix-to-helix intramolecular event only is about 16% of the global duplex-to-single-strands melting enthalpy and is relatively broad compared to the global melting event (about 30 vs 15 degrees C for the full width at half maxima). Electric birefringence decay measurements show that the phased 45 bp duplex has a rotational time constant of 100 ns at 5 degrees C which increases to 220 ns above 40 degrees C. Simple modeling of the dynamics within the junction model for bending yields that the bend per A-tract is 42 degrees at 5 degrees C, decreasing to 0 degrees above 40 degrees C. We suggest that this sequence-dependent structure which "melts" at physiological temperatures may be relevant to DNA topology and function.

There has been a significant financial effort poured into the technology of the Free Electron Laser (FEL) over the last 15 years or so. Much of that money was spent in the hopes that the FEL would be a key element in the Strategic Defense Initiative, but a small fraction of money was allocated for the Medical FEL program. The Medical FELs program was aimed at exploring how the unique capabilities of the FEL could be utilized in medical applications. Part of the Medical FEl effort has been in clinical applications, but some of the effort has also been put into exploring applications of the FEL for fundamental biological physics. It is the purpose of this brief text to outline some of the fundamental biophysics I have done, and some plans we have for the future. Since the FEL is (still) considered to be an avant garde device, the reader should not be surprised to find that much of the work proposed here is also rather radical and avant garde.

In this paper, we address the hydraulic flow of fluids on and within nanofabricated hydrophilic (as opposed to hydrophobic) surfaces, and the influence of an extended precursor film on the wetting dynamics of water penetrating a nano-textured array of posts that has no sealing surface (open-top). The width of the precursor film in front of the wetting edge δ is extraordinarily large, approximately 2 mm for our device, and it drives the wetting penetration film in the open-top array. We discuss the way this thin precursor film controls the movement of the macroscopic wetting front Lw and its dynamics in the nanofabricated array of posts. Rapid and controllable transport of nanoparticles in such an open top array of posts, as controlled by the precursor film, is demonstrated using a bump array textured morphology.

The effects of the nanocrystal size on the emission spectra and decay rates of upconverting hexagonal NaYF(4):Yb,Er nanocrystals are investigated. The influence of nanocrystal size is represented in terms of the surface area/volume ratio (SA/Vol). Our results show that a small nanocrystal size, or large SA/Vol ratio increases the decay rate, in particular, the green luminescence decay rate varies linearly with the SA/Vol ratio.

Bacterial systems offer excellent tests of how well the general theoretical predictions of ecology dynamics do or do not in fact conform to reality. We believe that the basic rules that govern the cohabitation of competing species for limited resources are the same from bacteria to man, we just don't know the rules, and that fundamental studies of the games bacteria play will give fundamental insight into the vastly more complex systems we hope to attack later. In this tutorial review we discuss how simplified micro-ecologies constructed using tools of micro and nanofabrication techniques offer some idea of how physical principles and analysis can address the issue of complex ecology dynamics.

We have used optical microlithography to fabricate capped quasi-two-dimensional obstacle courses in SiO2. We report here observations using epifluorescence microscopy of the electrophoresis and length fractionation of large DNA molecules confined in arrays. Simple reptation theory, based on the work of deGennes, predicts that at low electric fields the electrophoretic mobility of a polymer of length L much greater than the persistence length p scales inversely with L (ref. 2). But elongation of the coil in the matrix at sufficiently strong electric fields results in a length-independent electrophoretic mobility. The application of suitably timed pulsed electric fields restores the fractionating power of gels for long molecules but the protocols of pulsed-field electrophoresis are semi-empirical because the complex and ill-understood gel matrix plays a critical role in fractionation. Microlithographically constructed obstacle arrays, with their low dimensionality, small volume and extremely reproducible topography, will make it possible to understand the motion and fractionation of large polymer molecules in complex but well characterized topologies.

Experiments on the field-free transient electric birefringence decay of dilutesolutions of monodisperse DNA fragments whose length L is much larger than the persistence length L, are reported. They reveal that the highly nonexponential decay is independent of L. We present a simple theoretical model which includes the internal rigidity of the polymer, explains the qualitative results of the measurements, and describes quantitatively the dynamics of long semiflexible polymers such as DNA, providing an accurate determination of the persistence length

Upconverting nanoparticles (UCNPs) when excited in the near-infrared (NIR) region display anti-Stokes emission whereby the emitted photon is higher in energy than the excitation energy. The material system achieves that by converting two or more infrared photons into visible photons. The use of the infrared confers benefits to bioimaging because of its deeper penetrating power in biological tissues and the lack of autofluorescence. We demonstrate here sub-10 nm, upconverting rare earth oxide UCNPs synthesized by a combustion method that can be stably suspended in water when amine modified. The amine modified UCNPs show specific surface immobilization onto patterned gold surfaces. Finally, the low toxicity of the UCNPs is verified by testing on the multi-cellular C. elegans nematode.

In this work we demonstrate a new microfluidic method for the rapid assessment of platelet size and morphology in whole blood. The device continuously fractionates particles according to size by displacing them perpendicularly to the fluid flow direction in a micro-fabricated post array. Whole blood, labeled with the fluorescent, platelet specific, antibody PE-anti-CD41, was run through the device and the positions of fluorescent objects noted as they exited the array. From this, histograms of platelet size were created which show marked increases in size after exposure to thrombin or a temperature of 4 degrees C. We infer that the well known morphological changes that occur during activation are causing the observed increase in size.

We provide a brief summary of current problems in DNA-protein interactions and argue that measurement of the elastic properties of DNA is critical to understanding these problems We discuss the ing1et depletion technique and various improvements we have been able to Achieve. Previous results using this technique are discussed, and we will present our plans for future experiments.

Separation of blood cells by native susceptibility and by the selective attachment of magnetic beads has recently been demonstrated on microfluidic devices. We discuss the basic principles of how forces are generated via the magnetic susceptibility of an object and how microfluidics can be combined with micron-scale magnetic field gradients to greatly enhance in principle the fractionating power of magnetic fields. We discuss our efforts and those of others to build practical microfluidic devices for the magnetic separation of blood cells. We also discuss our attempts to integrate magnetic separation with other microfluidic features for developing handheld medical diagnostic tools.

The fractionation of small particles in a liquid based on their size in a micropost array by deterministic lateral displacement was recently demonstrated with unprecedented resolution (L. R. Huang, E. C. Cox, R. H. Austin and J. C. Sturm, Science, 2004, 304, 987-990, ). In this paper, we present a model of how the critical particle size for fractionation depends on the micropost geometry, depending specifically on the gap between posts, the offset of posts in one row with respect to another, and whether the fluid is driven by hydrodynamics or by electroosmosis. In general the critical particle diameter is much smaller than the gap, which prevents clogging. The model is supported by data with particles from 2.3 to 22 microm.

The near-field radiation pattern of a long thin slit (with a width much smaller than the excitation wavelength) in a uniform aluminum surface was measured and modeled by numerical computation. In particular, the interplay between the incident light polarization and the slit width is found to play an essential role in the near-field profile on the back side of the nanoslits. Two-dimensional finite-difference time-domain computer simulations were performed to calculate the near-field intensity profile for different slit widths and metal thicknesses. This method will allow the optimization of three-dimensional near-field radiation patterns for a variety of near-field molecular scanning schemes.

We have used diffusive mixing and dielectrophoretic trapping to lyse Escherichia coli cells in a microfabricated environment and trap the E. coli chromosome. We characterize the conditions needed for efficient lysis of the cells, and conditions needed for the dielectrophoretic trapping of the chromatin without the trapping of cytoplasmic proteins.

The fluorescence decay of the rare earth terbium when bound to the protein calmodulin changes from a simple exponential decay to a complex nonexponential decay as the temperature is lowered below 200 K. We have fit the observed decay curves by assuming that the terbium emission is a forced electric dipole transition and proteins have a distribution of continuous conformational states. Quantitative fits to the data indicate that the root-mean-square configurational deviation of the atoms surrounding the terbium ion is 0.2 A, in good agreement with other measurements. We further point out that because the protein seems to undergo a glass transition yet retains configurational order at room temperature, the proper name for the physical state of a protein at room temperature is the rubber-like state.

Protoheme rebinding of carbon monoxide after photodissociation has been observed at temperatures from 5 to 340 K for times from 2 μs to 1 ks. Below 80 K, binding is nonexponential in time and CO‐concentration independent, above 230 K exponential and the rate is CO‐concentration proportional. A model is proposed in which the carbon monoxide, moving from the solvent to the binding site at the ferrous heme iron, encounters two successive barriers. The outer is formed by the solvent, the inner is a property of the heme and probably connected to the motion of the iron from the spin‐2 deoxy to the spin‐0 carbon monoxide state. The temperature dependence of the two processes yields all activation enthalpies and entropies for the two barriers. The nonexponential rebinding observed at low temperatures implies that the inner barrier possesses distributed activation enthalpy and entropy. The enthalpy spectrum and the entropy spread are determined. The spectrum demonstrates that heme exists in many different conformational states. At low temperatures, these states are frozen; above about 230 K, rapid conformational relaxation renders rebinding exponential. Below 15 K, quantum‐mechanical molecular tunneling dominates. The tunneling rate yields the width of the innermost barrier. Earlier experiments on carbon monoxide binding to myoglobin had provided evidence for four barriers. The present results imply that the innermost barrier in myoglobin is caused by the heme, the outermost by the solvent, and the two intermediate ones by the globin.