Rapid progress in digital electronics allows digitisation of monitor signals at a very early stage of the signal processing chain, providing optimum performance and maximum flexibility for today’s accelerator instrumentation. While the analog front-ends of such systems are usually specific for each monitor type, the subsequent digital part of the processing chain can be unified for many different measurement tasks. The “generic VME PMC Carrier board” (VPC) [1] was developed to achieve this unification for the PSI electron and proton accelerator diagnostics and fast data acquisition and feedback systems. The core of the VME64x board consists of two Virtex2Pro FPGAs with two PowerPCs each, a floating point DSP and RAM. The FPGAs can acquire and process measurement data from the VMEbus P0/P2 connectors or from two applicationdependent PMC mezzanine modules. Two 2 GBaud fibre optics transceivers may also be used to acquire or distribute measurement data. Envisaged applications include digital beam position (DBPM) and current monitors for proton beams, data processing for a muon decay experiment, and general beam diagnostics as well as global feedbacks at SLS accelerators and beamlines.

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.

Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 microm. 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 one-photon 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.

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 similar to 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 multicolor 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.

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.

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).

Optical single molecule detection is an emerging field which currently impacts various scientific disciplines In particular, laser induced fluorescence detection and spectroscopy of individual fluorescent molecules open new avenues for the development of fluorescent based assays in biophysical research but also in biotechnology and pharmaceutical industries The ability to detect and identify either tagged or autofluorescent single molecules allows for the separation of sub-populations from heterogeneous ensembles which therefore renders single molecule detection (SMD) an ideal tool for selecting, trapping, sorting, tracking, picking, and even manipulating biological (macro)molecules Moreover, since the physical observables in SMD experiments are unmasked by ensemble averaging, time-dependent pathways of chemical reactions can be resolved without the need to synchronize all the molecules of the ensemble Currently, optical SMD has been successfully applied in separation science [1–7] near field [8–10] and far field microscopy [11–14], and total internal reflection (TIR) microscopy on quartz-liquid interfaces [15–17].

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 excited-state 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-mum-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.

We report the application of pump-probe fluorescence microscopy in time-resolved polarization imaging. We derived the equations governing the pump-probe stimulated emission process and characterized the pump and probe laser power levels for signal saturation. Our emphasis is to use this novel methodology to image polarization properties of fluorophores across entire cells. As a feasibility study, we imaged a 15-μm orange latex sphere and found that there is depolarization that is possibly due to energy transfer among fluorescent molecules inside the sphere. We also imaged a mouse fibroblast labeled with CellTracker Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethyl-rhodamine). We observed that Orange CMTMR complexed with gluthathione rotates fast, indicating the relatively low fluid-phase viscosity of the cytoplasmic microenvironment as seen by Orange CMTMR. The measured rotational correlation time ranged from ∼30 to ∼150 ps. This work demonstrates the effectiveness of stimulated emission measurements in acquiring high-resolution, time-resolved polarization information across the entire cell.

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.

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.

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.

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 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.