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Conceptually, on-bead screening is one of the most efficient high-throughput screening (HTS) methods. One of its inherent advantages is that the solid support has a dual function: it serves as a synthesis platform and as a screening compartment. Compound purification, cleavage and storage and extensive liquid handling are not necessary in bead-based HTS. Since the establishment of one-bead one-compound library synthesis, the properties of polymer beads in chemical reactions have been thoroughly investigated. However, the characterization of the kinetics and thermodynamics of protein-ligand interactions on the beads used for screening has received much less attention. Consequently, the majority of reported on-bead screens are based on empirically derived procedures, independent of measured equilibrium constants and rate constants of protein binding to ligands on beads. More often than not, on-bead screens reveal apparent high affinity binders through strong protein complexation on the matrix of the solid support. After decoding, resynthesis, and solution testing the primary hits turn out to be unexpectedly weak binders, or may even fall out of the detection limit of the solution assay. Only a quantitative comparison of on-bead binding and solution binding events will allow systematically investigating affinity differences as function of protein and small molecule properties. This will open up routes for optimized bead materials, blocking conditions and other improved assay procedures. By making use of the unique features of our previously introduced confocal nanoscanning (CONA) method, we investigated the kinetic and thermodynamic properties of protein-ligand interactions on TentaGel beads, a popular solid support for on-bead screening. The data obtained from these experiments allowed us to determine dissociation constants for the interaction of bead-immobilized ligands with soluble proteins. Our results therefore provide, for the first time, a comparison of on-bead versus solution binding thermodynamics. Our data indicate that affinity ranges found in on-bead screening are indeed narrower compared to equivalent interactions in homogeneous solution. A thorough physico-chemical understanding of the molecular recognition between proteins and surface bound ligands will further strengthen the role of on-bead screening as an ultimately cost-effective method in hit and lead finding.

Solid phase combinatorial chemistry provides fast and cost-effective access to large bead based libraries with compound numbers easily exceeding tens of thousands of compounds. Incubating one-bead one-compound library beads with fluorescently labeled target proteins and identifying and isolating the beads which contain a bound target protein, potentially represents one of the most powerful generic primary high throughput screening formats. On-bead screening (OBS) based on this detection principle can be carried out with limited automation. Often hit bead detection, i.e. recognizing beads with a fluorescently labeled protein bound to the compound on the bead, relies on eye-inspection under a wide-field microscope. Using low resolution detection techniques, the identification of hit beads and their ranking is limited by a low fluorescence signal intensity and varying levels of the library beads' autofluorescence. To exploit the full potential of an OBS process, reliable methods for both automated quantitative detection of hit beads and their subsequent isolation are needed. In a joint collaborative effort with Evotec Technologies (now Perkin-Elmer Cellular Technologies Germany GmbH), we have built two confocal bead scanner and picker platforms PS02 and a high-speed variant PS04 dedicated to automated high resolution OBS. The PS0X instruments combine fully automated confocal large area scanning of a bead monolayer at the bottom of standard MTP plates with semiautomated isolation of individual hit beads via hydraulic-driven picker capillaries. The quantification of fluorescence intensities with high spatial resolution in the equatorial plane of each bead allows for a reliable discrimination between entirely bright autofluorescent beads and real hit beads which exhibit an increased fluorescence signal at the outer few micrometers of the bead. The achieved screening speed of up to 200,000 bead assayed in less than 7 h and the picking time of approximately 1 bead/min allow exploitation of one-bead one-compound libraries with high sensitivity, accuracy, and speed.

Careful regulation of mRNA half-lives is a fundamental mechanism allowing cells to quickly respond to changing environmental conditions. The mRNA-binding Hu proteins are important for stabilization of short-lived mRNAs. Here we describe the identification and mechanistic characterization of the first low-molecular-weight inhibitors for Hu protein R (HuR) from microbial broths (Actinomyces sp.): dehydromutactin (1), MS-444 (2) and okicenone (3). These compounds interfere with HuR RNA binding, HuR trafficking, cytokine expression and T-cell activation. A mathematical and experimental analysis of the compounds' mode of action suggests that HuR homodimerizes before RNA binding and that the compounds interfere with the formation of HuR dimers. Our results demonstrate the chemical drugability of HuR; to our knowledge HuR is the first example of a drugable protein within the Hu family. MS-444, dehydromutactin and okicenone may become valuable tools for studying HuR function. An assessment of HuR inhibition as a central node in malignant processes might open up new conceptual routes toward combatting cancer.

We developed a versatile set of chemical labeling reagents which allow dye ligation to the C-terminus of a protein or a single internal cysteine and target purification in a simple two-step process. This simple process results in a fully 1:1 labeled conjugate suitable for all quantitative fluorescence spectroscopy and imaging experiments. We refer to a "generic labeling toolbox" because of the flexibility to choose one of many available dyes, spacers of different lengths and compositions which increase the target solubility, a variety of affinity purification tags, and different cleavage chemistries to release the 1:1 labeled proteins. Studying protein function in vitro or in the context of live cells and organisms is of vital importance in biological research. Although label free detection technologies gain increasing interest in molecular recognition science, fluorescence spectroscopy is still the most often used detection technique for assays and screens both in academic as well as in industrial groups. For generations, fluorescence spectroscopists have labeled their proteins of interest with small fluorescent dyes by random chemical linking on the proteins' exposed lysines and cysteines. Chemical reactions with a certain excess of activated esters or maleimides of longer wavelength dyes hardly ever result in quantitative labeling of the target protein. Most of the time, more than one exposed amino acid side chain reacts. This results in a mixture of dye-protein complexes of different labeling stoichiometries and labeling sites. Only mass spectrometry allows resolving the precise chemical composition of the conjugates. In "classical" ensemble averaging fluorescent experiments, these labeled proteins are still useful, and quantification of, e.g., ligand binding experiments, is achieved via knowledge of the overall protein concentration and a fluorescent signal change which is proportional to the amount of complex formed. With the development of fluorescence fluctuation analysis techniques working at single molecule resolution, like fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), fluorescence intensity diffusion analysis (FIDA), etc., it became important to work with homogeneously labeled target proteins. Each molecule participating in a binding equilibrium should be detectable when it freely fluctuates through the confocal focus of a microscope. The measured photon burst for each transition contains information about the size and the stoichiometry of a protein complex. Therefore, it is important to work with reagents that contain an exact number of tracers per protein at identical positions. The ideal fluorescent tracer-protein complex stoichiometry is 1:1. While genetic tags such as fluorescent proteins (FPs) are widely used to detect proteins, FPs have several limitations compared to chemical tags. For example, FPs cannot easily compete with organic dyes in the flexibility of modification and spectral range; moreover, FPs have disadvantages in brightness and photostability and are therefore not ideal for most biochemical single molecule studies. We present the synthesis of a series of exemplaric toolbox reagents and labeling results on three target proteins which were needed for high throughput screening experiments using fluorescence fluctuation analysis at single molecule resolution. On one target, Hu-antigen R (HuR), we demonstrated the activity of the 1:1 labeled protein in ribonucleic acid (RNA) binding, and the ease of resolving the stoichiometry of an RNA-HuR complex using the same dye on protein and RNA by Fluorescence Intensity Multiple Distribution Analysis (FIMDA) detection.

The goal of this article is to provide a review of deep tissue studies based on two-photon microscopy. We will further explore three new developments that have significantly enhanced the power of two-photon imaging: (1) simultaneous two-photon fluorescence and confocal reflected-light imaging, (2) two-photon video-rate imaging, and (3) fluorescent spectroscopic measurements in deep tissue.

We present the basic concept and the realization of our fully configurable data-acquisition hardware for the neutron scattering instruments at SINQ. This system allows collection of the different data entities and event-related signals generated by the various detector units. It offers a variety of synchronization options, including a time-measuring mode for time-of-flight determinations. Based on configurable logic (FPGA, CPLD), event rates up to the MHz range can be processed and transmitted to a programmable online data-reduction system (Histogram Memory). It is implemented on a commercially available VME Power PC module running a real-time operating system (VxWorks).

SummaryScreening of one-bead one-compound libraries by incubating beads with fluorescently labeled target protein requires isolation and structure elucidation of a large number of primary hit beads. However, the potency of the identified ligands is only revealed after time consuming and expensive larger scale resynthesis and testing in solution. Often, many of the resynthesized compounds turn out to be weak target binders in solution due to large differences between surface and solution binding affinities. For an industry style high-throughput screening (HTS) process a high false positive rate is detrimental. We have therefore combined single bead and single molecule/single cell techniques into an integrated HTS process in which the picomole amount of substance contained on one isolated hit bead is sufficient for quality control, structure determination, and precise affinity determination to the target protein in solution.

Multifocal multiphoton microscopy (MMM) enhances imaging speed by parallelization. It is not well understood why the imaging depth of MMM is significantly shorter than conventional single-focus multiphoton microscopy (SMM). In this report, we show that the need for spatially resolved detectors in MMM results in a system that is more sensitive to the scattering of emission photons with reduced imaging depth. For imaging depths down to twice the scattering mean free path length of emission photons (2xl (s) (em)), the emission point spread function (PSF(em)) is found to consist of a narrow, diffraction limited distribution from ballistic emission photons and a broad, relatively low amplitude distribution from scattered photons. Since the scattered photon distribution is approximately 100 times wider than that of the unscattered photons at 2xl (s) (em), image contrast and depth are degraded without compromising resolution. To overcome the imaging depth limitation of MMM, we present a new design that replaces CCD cameras with multi-anode photomultiplier tubes (MAPMTs) allowing more efficient collection of scattered emission photons. We demonstrate that MAPMT-based MMM has imaging depth comparable to SMM with equivalent sensitivity by imaging tissue phantoms, ex vivo human skin specimens based on endogenous fluorophores, and green fluorescent protein (GFP) expressing neurons in mouse brain slices.

We present the implementation of intensity-modulated laser diodes for applications in frequency-domain pump-probe fluorescence microscopy. Our technique, which is based on the stimulated-emission approach, uses two sinusoidally modulated laser diodes. One laser (635 nm) excites the chromophores under study, and the other laser (680 nm) is responsible for inducing stimulated emission from excitedstate molecules. Both light sources are modulated in the 80-MHz range but with an offset of 5 kHz between them. The result of the interaction of the pump and the probe beams is that a cross-correlation fluorescence signal at 5 kHz is generated primarily at the focal volume. Microscope imaging at the cross-correlation signal results in images with high contrast, and time-resolved high-frequency information can be acquired without high-speed detection. A detailed experimental arrangement of our methodology is presented along with images acquired from a 4.0-mm-diameter fluorescent sphere and TOTO-3-labeled mouse STO cells. (TOTO-3 is a nucleic acid stain.) Our results demonstrate the feasibility of using sinusoidally modulated laser diodes for pump-probe imaging, creating the exciting possibility of high-contrast time-resolved imaging with low-cost laser-diode systems. © 2001 Optical Society of America.

The majority of proteins contain intrinsic fluorophores as natural sensors of molecular structures, dynamics, and interactions. The intrinsic protein fluorescence signal allows for the label-free and, hence, undisturbed and rapid study of protein-ligand interactions. Ultraviolet-based drug screening is hampered by the background, photobleaching, light scattering, inner filter effects, and interfering assay compounds. Such problems can be overcome by means of molecular three-photon excitation (3PE) with infrared femtosecond light pulses since longer excitation wavelengths result in less Raleigh scattering, and the subfemtoliter (confocal-like) 3PE volume minimizes out-of-focus photobleaching, background generation, and inner filter effects. We demonstrate the general feasibility of 3PE for protein spectroscopy and illustrate the technique's excellent potential for high-throughput screening. By using the intrinsic fluorescence intensity of a protein-substrate, we were able to discriminate between ligands of different affinities in binding assays.

Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 mm. The major damage mechanism during two-photon skin imaging is associated with the formation of cavitation at the epidermal-dermal junction, which results in thermal mechanical damage of the tissue. In this report, we verify that this damage mechanism is of thermal origin and is associated with onephoton absorption of infrared excitation light by melanin granules present in the epidermal-dermal junction. The thermal mechanical damage threshold for selected Caucasian skin specimens from a skin bank as a function of laser pulse energy and repetition rate has been determined. The experimentally established thermal mechanical damage threshold is consistent with a simple heat diffusion model for skin under femtosecond pulse laser illumination. Minimizing thermal mechanical damage is vital for the potential use of two-photon imaging in noninvasive optical biopsy of human skin in vivo. We describe a technique to mitigate specimen thermal mechanical damage based on the use of a laser pulse picker that reduces the laser repetition rate by selecting a fraction of pulses from a laser pulse train. Since the laser pulse picker decreases laser average power while maintaining laser pulse peak power, thermal mechanical damage can be minimized while two-photon fluorescence excitation efficiency is maximized. © 2004 Society of Photo-Optical Instrumentation Engineers.

We present a multicolor multiphoton fluorescence microscope with single-photon counting sensitivity. The system integrates a standard multiphoton fluorescence microscope, an optical grating spectrograph operating in the UV-Vis wavelength region, and a 16-anode photomultiplier tube (PMT). The major technical innovation is in the development of a multichannel photon counting card (mC-PhCC) for direct signal collection from multi-anode PMTs. The electronic design of the mC-PhCC employs a high-throughput, fully-parallel, single-photon counting scheme along with a high-speed electrical or fiber-optical link interface to the data acquisition computer. There is no electronic crosstalk among the detection channels of the mC-PhCC. The collected signal remains linear up to an incident photon rate of 10(8) counts per second. The high-speed data interface offers ample bandwidth for real-time readout: 2 MByte lambda-stacks composed of 16 spectral channels, 256 x 256 pixel image with 12-bit dynamic range can be transferred at 30 frames per second. The modular design of the mC-PhCC can be readily extended to accommodate PMTs of more anodes. Data acquisition from a 64-anode PMT has been verified. As a demonstration of system performance, spectrally resolved images of fluorescent latex spheres and ex-vivo human skin are reported. The multicolor multiphoton microscope is suitable for highly sensitive, real-time, spectrally-resolved three-dimensional imaging in biomedical applications.

High-speed two-photon imaging based on multi-foci excitation requires the use of spatially resolved detectors, such as charge coupled device (CCD) cameras, instead of single channel photomultiplier tube (PMT). The performance of systems based on both a PMT and a CCD in turbid medium was evaluated by measuring the image point spread function (PSF) and the image contrast as a function of depth and scattering coefficient with single point scanning. We found no significant change in the full-width at half maximum of the point spread function (PSF) for depth up to 100 μm. However, the CCD lost contrast significantly faster as a function of depth and increase scattering. This discrepancy is resolved by measuring a low amplitude but broad tail in the PSF distribution. The tail of the PSF distribution can be up to 200 μm in diameter. We further evaluate scattering effects in the imaging of GFP neurons in a mouse brain slice.

Summary form only given. We developed multi-photon fluorescence spectroscopy based on a 16/spl times/1 multianode photo multiplier tube and we continue to develop video rate microscopy based on an 8/spl times/8 multianode PMT. The spectroscopy consists of grating, 16/spl times/1 multianode PMT and a 16 channel photon counting card. It can take spectrum of pixels in addition to acquiring images. Multiple color fluorescence spheres, cells stained with multiple color fluorescence dyes and tissues were examined with the system. Video rate multi-photon microscopy is constructed based on an 8/spl times/8 micro lenslet array, an 8/spl times/8 multianode PMT and 64 photon counting card for deep tissue imaging which is difficult due to scattering of tissue samples.

Tissue physiology and pathology can be better understood using two-photon microscopy that allows tissue structure and biochemistry to be assayed on the subcellular level. Histological analysis provides high-resolution tissue characterization and is the only imaging method that can be applied to tissues of arbitrary thicknesses. Scanning confocal microscopy was invented in 1962 by Minsky demonstrating that three-dimensional (3-D) resolved images can be obtained in translucent specimens without physical sectioning. An alternative 3-D imaging technology is two-photon excitation microscopy, which was introduced by Denk, Webb, and coworkers in 1990. The electronic transition of a fluorophore can be induced by the simultaneous absorption of two photons. Depth discrimination is the most important feature of two-photon microscopy. The design of a typical two-photon fluorescence microscope design is based on three basic components: a femtosecond laser system, high-throughput microscope optics, and high-sensitivity detection optoelectronics. An important advantage of two-photon imaging is its suitability for in vivo imaging.

CHAPTER 21 Fluorescence-Lifetime Imaging Techniques for Microscopy Chen Y. Dong, 1 Todd French, 2 Peter T. C. So, 1 C. Buehler, 1 Keith M. Berland, 3 and Enrico Gratton 1 Laboratory for Fluorescence Dynamics Department of Physics University of Illinois at Urbana-Champaign Urbana, Illinois 61801 Molecular Devices Corporation Sunnyvale, California 94089 Physics Department Emory University Atlanta, Georgia 30322 I. Introduction II. Time-Resolved Fluorescence Methods A. Time-Domain and Frequency-Domain Measurements B. Simultaneous Multiple-Lifetime-Component Measurement C. Photobleaching Effects III. Fluorescence-Lifetime-Resolved Camera A. Instrumentation B. Camera-Based Microscope Examples IV. Two-Photon Fluorescence Lifetime Microscopy A. Instrumentation B. Two-Photon Microscopy Examples V. Pump-Probe Microscopy A. Instrumentation B. Pump-Probe Microscopy Examples VI. Conclusion References METHODS IN CELL BIOLOGY, VOL. 72 Copyright 1998, Elsevier Inc. All rights reserved. 0091-679X/03 $35.00

Two-photon scanning microscopy has been successfully applied in studying tissue structures and biochemistry with subcellular spatial resolution. We developed a 16-channel two-photon scanning microscope with high-performance single photon counting readout electronics. The apparatus incorporates a mode-locked Ti:Sapphire laser, a scanning microscope, a spectrograph with multi-anode PMT (16 channels with ~ 7 to 10 nm spectral resolution each), and a 16-channel photon counting card (PhCC) with integrated high-speed data link (payload 1.2 Gbps). Each PhCC detection channel features single-photon sensitivity and 100 MHz photon counting bandwidth. The multi-color single-photon counting detection scheme allows ultra-sensitive measurements in a broad spectrum of biomedical applications. We present spectroscopic studies of ex vivo tissue autofluorescence.

In biological applications of optical microscopy, technical developments often lead to novel imaging modalities with significant applications. For example, the development of confocal microscopy led to microscopic imaging with enhanced contrast (see Chapter 5), and the more recent development of two-photon fluorescence microscopy revolutionized fluorescence microscopy by providing an imaging modality capable of high image contrast, reduced photodamage, and exciting possibilities in controlling localized photochemical reactions in three dimensions (see Chapters 9-13).

Multiphoton microscopy is one of the fastest growing areas in biomedical imaging. The potential of multiphoton excitation was first theorized by Maria GoppertMayer in 1931. Generating three-dimensionally resolved microscopic images based on nonlinear optical excitation was postulated in the 1970s (Gannaway and Sheppard, 1978; Wilson and Sheppard, 1984). The definitive experiment was done by Denk, Webb, and co-workers (1990), who accomplished two-photon, three-dimensional (3D) imaging of biological specimens. Furthermore, they demonstrated 3D localized uncaging and photobleaching using two-photon excitation to trigger a photochemical reaction in a subfemtoliter volume.

Non-invasive optical diagnosis of cellular and extracellular structure and biochemistry in thick tissue is becoming a reality with the maturation of the two-photon imaging. Today, the slow imaging speed of typical two-photon microscopes is a major hurdle in realizing their clinical potential. We have developed a high-speed two-photon microscope optimized for acquiring 3-D tissue images in real time. The scanning speed improvement of this system is obtained by the use of an air bearing polygonal mirror. The maximum achievable scanning rate is 40 microseconds per line, which is about 100 times faster than conventional scanning microscopes. High-resolution fluorescence images were recorded in real-time by an intensified CCD camera. Using this instrument, we have monitored the movements of protozoas and mapped the collagen/elastin fiber structures in excised human skin.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

We report the implementation of intensity modulated diode lasers in frequency-domain pump-probe studies, diode lasers are compact, stable, and economical units that require little maintenance. In our study, a 365 nm diode laser is used as the excitation source and the output of a 680 nm unit induces stimulated emission from excited state fluorophores. By modulating the intensities of the two diode lasers at slightly different frequencies, and detecting the fluorescence signal at the cross-correlation frequency, both time-resolved and high spatial resolution imaging can be achieved. The laser diodes are modulated in the 100 MHz cross-correlation signal has been used for time-resolved imaging of fluorescent microspheres and mouse fibroblasts labeled with nucleic acid stains TOTO-3. These results demonstrate and feasibility of using intensity modulated diode lasers for frequency-domain, pump-probe studies.

Publisher Summary The chapter discusses multiphoton excitation microscopy based on NAD(P)H functional imaging. Progress is also being made in synthesizing molecules with very large two-photon absorption cross sections. For multiphoton excitation processes, the number of photon pairs absorbed for each laser pulse is related inversely to the pulse width. The passage of ultrashort pulses from the mode-locked laser through a dielectric medium results in pulse broadening because of group velocity dispersion. This pulse broadening, called “pulse dispersion,” is a serious problem in multiphoton excitation microscopy because it results in a reduction in the probability of multiphoton excitation. Multiphoton excitation microscopy has the following important advantages: (1) reduced phototoxicity, (2) reduced photobleaching, (3) increased penetration depth, (4) ability to perform uncaging or photobleaching in a diffraction limited volume, (5) ability to excite fluorophores in the ultraviolet without a ultraviolet laser, (6) the excitation and the fuorescence wavelengths are well separated, and (7) no spatial filter is required. The chapter discusses functional metabolic imaging of cellular metabolism based on NAD(P)H fluorescence, ultraviolet confocal fluorescence microscopy compared with multiphoton excitation microscopy, laser sources for multiphoton excitation microscopy, and pulse compensation techniques.

A new versatile system for the measurement of time-resolved fluorescence emission spectra of biomolecules is presented. Frequency doubling and tripling of a Ti:Sapphire laser allows excitation overa wide wavelength range. The influence of increasing the spectral resolution on the time resolution hasbeen investigated. System performance can be optimized for best resolution in the spectral or timedomain, respectively. The currently achieved temporal resolution is 6 psec, and the best spectralresolution is 3 nm. Long fluorescence decays can be resolved with optimal time resolution by way oftaking into account the flyback of the streak camera. With the system described, the core complex (a)3APCLc8.9 of the phycobilisome from the photosynthetic cyanobacteria Mastigocladus laminosus has been analysed. Lifetime analysis clearlydemonstrated the influence of the linker polypeptide on the phycobiliprotein complex and the identityof native and reconstituted complex. Introduction A large variety of instruments capable of acquiring fluorescence spectra and lifetimes have beenpresented. Most of them scan through the spectrum or acquire spectra and decay using two separatechannels [1]. The acquisition of fluorescence parameters is therefore time consuming. A short excitation

We have developed a high-speed two-photon microscope with submicrometer resolution in real time. The imaging speed improvement of this system is obtained by the use of a high-speed polygonal mirror scanner. The maximum achievable scanning rate is 40 micros/line, which is approximately 100 times faster than conventional scanning microscopes. High-resolution fluorescence images were recorded in real time by an intensified CCD camera. Using this instrument, we have resolved cellular architecture in three dimensions and have monitored the movements of protozoas. More important, photodamage to biological specimens during video-rate imaging can be minimized with two-photon excitation as compared with other one-photon modalities.