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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.

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.

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.

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).

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.