Showing papers in "SPIE milestone series in 2003"

Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy

Abstract: We propose a new type of scanning fluorescence microscope capable of resolving 35 nm in the far field. We overcome the diffraction resolution limit by employing stimulated emission to inhibit the fluorescence process in the outer regions of the excitation point-spread function. In contrast to near-field scanning optical microscopy, this method can produce three-dimensional images of translucent specimens.

Two-photon polymerization initiators for three-dimensional optical data storage and microfabrication

Abstract: Two-photon excitation provides a means of activating chemical or physical processes with high spatial resolution in three dimensions and has made possible the development of three-dimensional fluorescence imaging, optical data storage, and lithographic microfabrication. These applications take advantage of the fact that the two-photon absorption probability depends quadratically on intensity, so under tight-focusing conditions, the absorption is confined at the focus to a volume of order λ3 (where λ is the laser wavelength). Any subsequent process, such as fluorescence or a photoinduced chemical reaction, is also localized in this small volume. Although three-dimensional data storage and microfabrication have been illustrated using two-photon-initiated polymerization of resins incorporating conventional ultraviolet-absorbing initiators, such photopolymer systems exhibit low photosensitivity as the initiators have small two-photon absorption cross-sections (δ). Consequently, this approach requires high laser power, and its widespread use remains impractical. Here we report on a class of π;-conjugated compounds that exhibit large δ (as high as 1, 250 × 10−50 cm4 s per photon) and enhanced two-photon sensitivity relative to ultraviolet initiators. Two-photon excitable resins based on these new initiators have been developed and used to demonstrate a scheme for three-dimensional data storage which permits fluorescent and refractive read-out, and the fabrication of three-dimensional micro-optical and micromechanical structures, including photonic-bandgap-type structures.

Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex

Abstract: Cortical blood flow at the level of individual capillaries and the coupling of neuronal activity to flow in capillaries are fundamental aspects of homeostasis in the normal and the diseased brain. To probe the dynamics of blood flow at this level, we used two-photon laser scanning microscopy to image the motion of red blood cells (RBCs) in individual capillaries that lie as far as 600 μm below the pia mater of primary somatosensory cortex in rat; this depth encompassed the cortical layers with the highest density of neurons and capillaries. We observed that the flow was quite variable and exhibited temporal fluctuations around 0.1 Hz, as well as prolonged stalls and occasional reversals of direction. On average, the speed and flux (cells per unit time) of RBCs covaried linearly at low values of flux, with a linear density of ≈70 cells per mm, followed by a tendency for the speed to plateau at high values of flux. Thus, both the average velocity and density of RBCs are greater at high values of flux than at low values. Time-locked changes in flow, localized to the appropriate anatomical region of somatosensory cortex, were observed in response to stimulation of either multiple vibrissae or the hindlimb. Although we were able to detect stimulus-induced changes in the flux and speed of RBCs in some single trials, the amplitude of the stimulus-evoked changes in flow were largely masked by basal fluctuations. On average, the flux and the speed of RBCs increased transiently on stimulation, although the linear density of RBCs decreased slightly. These findings are consistent with a stimulus-induced decrease in capillary resistance to flow.

CMOS image sensors: Electronic camera-on-a-chip

Abstract: CMOS active pixel sensors (APS) have performance competitive with charge-coupled device (CCD) technology, and offer advantages in on-chip functionality, system power reduction, cost, and miniaturization. This paper discusses the requirements for CMOS image sensors and their historical development, CMOS devices and circuits for pixels, analog signal chain, and on-chip analog-to-digital conversion are reviewed and discussed.

Near-field fluorescence microscopy based on two-photon excitation with metal tips

Abstract: We present a new scheme for near-field fluorescence imaging using a metal tip illuminated with femtosecond laser pulses of proper polarization. The strongly enhanced electric field at the metal tip ( $\ensuremath{\approx}15\mathrm{nm}$ end diameter) results in a localized excitation source for molecular fluorescence. Excitation of the sample via two-photon absorption provides good image contrast due to the quadratic intensity dependence. The spatial resolution is shown to be better than that of the conventional aperture technique. We used the technique to image fragments of photosynthetic membranes, as well as $J$-aggregates with spatial resolutions on the order of 20 nm.

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability

Abstract: A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H2O2 may account, in part, for this inhibition. Thus, two-photon microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos.

Sensitive measurement of optical nonlinearities using a single beam

Abstract: A sensitive single-beam technique for measuring both the nonlinear refractive index and nonlinear absorption coefficient for a wide variety of materials is reported. The authors describe the experimental details and present a comprehensive theoretical analysis including cases where nonlinear refraction is accompanied by nonlinear absorption. In these experiments, the transmittance of a sample is measured through a finite aperture in the far field as the sample is moved along the propagation path (z) of a focused Gaussian beam. The sign and magnitude of the nonlinear refraction are easily deduced from such a transmittance curve (Z-scan). Employing this technique, a sensitivity of better than lambda /300 wavefront distortion is achieved in n/sub 2/ measurements of BaF/sub 2/ using picosecond frequency-doubled Nd:YAG laser pulses. >

Brominated 7-hydroxycoumarin-4-ylmethyls: Photolabile protecting groups with biologically useful cross-sections for two photon photolysis

Abstract: Photochemical release (uncaging) of bioactive messengers with three-dimensional spatial resolution in light-scattering media would be greatly facilitated if the photolysis could be powered by pairs of IR photons rather than the customary single UV photons. The quadratic dependence on light intensity would confine the photolysis to the focus point of the laser, and the longer wavelengths would be much less affected by scattering. However, previous caged messengers have had very small cross sections for two-photon excitation in the IR region. We now show that brominated 7-hydroxycoumarin-4-ylmethyl esters and carbamates efficiently release carboxylates and amines on photolysis, with one- and two-photon cross sections up to one or two orders of magnitude better than previously available. These advantages are demonstrated on neurons in brain slices from rat cortex and hippocampus excited by glutamate uncaged from N-(6-bromo-7-hydroxycoumarin-4-ylmethoxycarbonyl)-L-glutamate (Bhc-glu). Conventional UV photolysis of Bhc-glu requires less than one-fifth the intensities needed by one of the best previous caged glutamates, gamma-(alpha-carboxy-2-nitrobenzyl)-L-glutamate (CNB-glu). Two-photon photolysis with raster-scanned femtosecond IR pulses gives the first three-dimensionally resolved maps of the glutamate sensitivity of neurons in intact slices. Bhc-glu and analogs should allow more efficient and three-dimensionally localized uncaging and photocleavage, not only in cell biology and neurobiology but also in many technological applications.

High-resolution nonlinear optical imaging of live cells by second harmonic generation

Abstract: By adapting a laser scanning microscope with a titanium sapphire femtosecond pulsed laser and transmission optics, we are able to produce live cell images based on the nonlinear optical phenomenon of second harmonic generation (SHG). Second harmonic imaging (SHIM) is an ideal method for probing membranes of living cells because it offers the high resolution of nonlinear optical microscopy with the potential for near-total avoidance of photobleaching and phototoxicity. The technique has been implemented on three cell lines labeled with membrane-staining dyes that have large nonlinear optical coefficients. The images can be obtained within physiologically relevant time scales. Both achiral and chiral dyes were used to compare image formation for the case of single- and double-leaflet staining, and it was found that chirality plays a significant role in the mechanism of contrast generation. It is also shown that SHIM is highly sensitive to membrane potential, with a depolarization of 25 mV resulting in an approximately twofold loss of signal intensity.

Multifocal multiphoton microscopy

Abstract: We present a real-time, direct-view multiphoton excitation fluorescence microscope that provides three-dimensional imaging at high resolution. Using a rotating microlens disk, we split the near-infrared light of a mode-locked titanium:sapphire laser into an array of beams that are transformed into an array of high-aperture foci at the object. We typically scan at 225 frames per second and image the fluorescence with a camera that reads out the images at video rate. For 1.4 aperture oil and 1.2 water immersion lenses at 780-nm excitation we obtained axial resolutions of 0.84 and 1.4 µm, respectively, which are similar to that of a single-beam two-photon microscope. Compared with the latter setup, our system represents a 40–100-fold increase in efficiency, or imaging speed. Moreover, it permits the observation with the eye of high-resolution two-photon images of (live) samples.

Measuring serotonin distribution in live cells with three-photon excitation

Abstract: Tryptophan and serotonin were imaged with infrared illumination by three-photon excitation (3PE) of their native ultraviolet (UV) fluorescence. This technique, established by 3PE cross section measurements of tryptophan and the monoamines serotonin and dopamine, circumvents the limitations imposed by photodamage, scattering, and indiscriminate background encountered in other UV microscopies. Three-dimensionally resolved images are presented along with measurements of the serotonin concentration ( approximately 50 mM) and content (up to approximately 5 x 10(8) molecules) of individual secretory granules.

Polarization coherent anti-Stokes Raman scattering microscopy

Abstract: We report polarization coherent anti-Stokes Raman scattering (P-CARS) microscopy that allows vibrational imaging with high sensitivity and spectral selectivity. The nonresonant background signals from both Raman scatterers and the solvent are efficiently suppressed in P-CARS microscopy. We demonstrate P-CARS imaging of unstained cells based on the contrast of the protein amide I band.

Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy

Abstract: Three‐dimensional maps of cellular metabolic oxidation/reduction states of rabbit cornea in situ were obtained by imaging the fluorescence of the naturally occurring reduced pyridine nucleotides (both reduced nicotinamide‐adenine dinucleotide, NADH, and reduced nicotinamide‐adenine dinucleotide phosphate, NADPH, denoted here as NAD(P)H). Autofluorescence images with submicrometre lateral resolution were obtained throughout the entire 400 μm thickness of the cornea. Two‐photon excitation scanning laser microscopy with near‐infrared excitation provided high fluorescence collection efficiency, reduced photodamage, and eliminated ultraviolet chromatic aberration, all of which have previously degraded the visualization of pyridine nucleotide fluorescence. Sharp autofluorescence images of the basal epithelium (40 μm within the cornea) show substantial subcellular detail, providing the ability to monitor autofluorescence intensity changes over time, which reflect changes in oxidative metabolism and cellular dynamics necessary for maintenance of the ocular surface. The autofluorescence was confirmed to be mostly of NAD(P)H origin by cyanide exposure, which increased the fluorescence from all cell types in the cornea by about a factor of two. Autofluorescence images of individual keratocytes in the stroma were observed only after cyanide treatment, while in the predominant extracellular collagen (> 90% of the stromal volume), fluorescence was not distinguished from the background. Observation of keratocyte metabolism demonstrates the sensitivity made available by two‐photon microscopy for future redox fluorescence imaging of cellular metabolic states.

Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes

Abstract: The influence of the pulse length, ?, of ultrashort laser pulses at 780 and 920??nm on cell vitality and cellular reproduction has been studied. A total of 2400 nonlabeled cells were exposed to a highly focused scanning beam from a mode-locked 80-MHz Ti:sapphire laser with 60??s pixel dwell time. For the same pulse energy, destructive effects were more pronounced for shorter pulses. The damage behavior was found to follow approximately a P2/? dependence (P, mean power), indicating that cell destruction is likely based on a two-photon excitation process rather than a one- or a three-photon event. Therefore, femtosecond as well as picosecond pulses provide approximately the same relative optical window for safe two-photon fluorescence microscopy.

Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis

Abstract: Cell fusions produce multinucleate syncytia that are crucial to the structure of essential tissues in many organisms [1-5]. In humans the entire musculature, much of the placenta, and key cells in bones and blood are derived from cell fusion. Yet the developmental fusion of cell membranes has never been directly observed and is poorly understood. Similarity between viral fusion proteins and recently discovered cellular proteins implies that both cell-cell and virus-cell fusion may occur by a similar mechanism [6-8]. Paradoxically, however, fusion of enveloped viruses with cells involves an opening originating as a single pore [9-11], whereas electron microscopy studies of cell-cell fusion describe simultaneous breakdown of large areas of membrane [12, 13]. Here, we have shown that developmental cell fusion is indeed consistent with initiation by a virus-like, pore-forming mechanism. We examined live cell fusions in the epithelia of Caenorhabditis elegans embryos by a new method that integrates multiphoton, confocal, and electron microscopy. The fusion aperture always originated at a single point restricted to the apical adherens junction and widened slowly as a radial wavefront. The fusing membranes dispersed by vesiculation, rather than simple unfolding of the conjoined double bilayer. Thus, in these cells fusion appears to require two specialized sequential processes: formation of a unique primary pore and expansion of the opening by radial internalization of the interacting cell membranes.

Single-molecule detection by two-photon-excited fluorescence

Abstract: We present a technique for observing single fluorophore molecules in solution. A mode-locked laser beam is focused into the solution, thereby defining a two-photon excitation volume localized in three dimensions. Molecules diffusing into and out of this volume produce fluorescence bursts, which are detected with a high signal-to-background ratio. The theoretical foundations for the technique are laid out, including the attendant fluorescence rate distribution, and agree with experimental results obtained for Rhodamine B molecules in water.

Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery

Abstract: Multiphoton fluorescence photobleaching recovery (MP-FPR) is a technique for measuring the three-dimensional (3D) mobility of fluorescent molecules with 3D spatial resolution of a few microns. A brief, intense flash of mode-locked laser light pulses excites fluorescent molecules via multiphoton excitation in an ellipsoidal focal volume and photobleaches a fraction. Because multiphoton excitation of fluorophores is intrinsically confined to the high-intensity focal volume of the illuminating beam, the bleached region is restricted to a known, three-dimensionally defined volume. Fluorescence in this focal volume is measured with multiphoton excitation, using the attenuated laser beam to measure fluorescence recovery as fresh unbleached dye diffuses in. The time course of the fluorescence recovery signal after photobleaching can be analyzed to determine the diffusion coefficient of the fluorescent species. The mathematical formulas used to fit MP-FPR recovery curves and the techniques needed to properly utilize them to acquire the diffusion coefficients of fluorescently labeled molecules within cells are presented here. MP-FPR is demonstrated on calcein in RBL-2H3 cells, using an anomalous subdiffusion model, as well as in aqueous solutions of wild-type green fluorescent protein, yielding a diffusion coefficient of 8.7 x 10(-7) cm(2)s(-1) in excellent agreement with the results of other techniques.

Influence of optical properties on two-photon fluorescence imaging in turbid samples

Abstract: A numerical model was developed to simulate the effects of tissue optical properties, objective numerical aperture (N.A.), and instrument performance on two-photon-excited fluorescence imaging of turbid samples. Model data are compared with measurements of fluorescent microspheres in a tissuelike scattering phantom. Our results show that the measured two-photon-excited signal decays exponentially with increasing focal depth. The overall decay constant is a function of absorption and scattering parameters at both excitation and emission wavelengths. The generation of two-photon fluorescence is shown to be independent of the scattering anisotropy, g, except for g > 0.95. The N.A. for which the maximum signal is collected varies with depth, although this effect is not seen until the focal plane is greater than two scattering mean free paths into the sample. Overall, measurements and model results indicate that resolution in two-photon microscopy is dependent solely on the ability to deliver sufficient ballistic photon density to the focal volume. As a result we show that lateral resolution in two-photon microscopy is largely unaffected by tissue optical properties in the range typically encountered in soft tissues, although the maximum imaging depth is strongly dependent on absorption and scattering coefficients, scattering anisotropy, and objective N.A..

Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer

Abstract: The biochemical and biophysical properties of a red fluorescent protein from a Discosoma species (DsRed) were investigated. The recombinant DsRed expressed in E. coli showed a complex absorption spectrum that peaked at 277, 335, 487, 530, and 558 nm. Excitation at each of the absorption peaks produced a main emission peak at 583 nm, whereas a subsidiary emission peak at 500 nm appeared with excitation only at 277 or 487 nm. Incubation of E. coli or the protein at 37 degrees C facilitated the maturation of DsRed, resulting in the loss of the 500-nm peak and the enhancement of the 583-nm peak. In contrast, the 500-nm peak predominated in a mutant DsRed containing two amino acid substitutions (Y120H/K168R). Light-scattering analysis revealed that DsRed proteins expressed in E. coli and HeLa cells form a stable tetramer complex. DsRed in HeLa cells grown at 37 degrees C emitted predominantly at 583 nm. The red fluorescence was imaged using a two-photon laser (Nd:YLF, 1047 nm) as well as a one-photon laser (He:Ne, 543.5 nm). When fused to calmodulin, the red fluorescence produced an aggregation pattern only in the cytosol, which does not reflect the distribution of calmodulin. Despite the above spectral and structural complexity, fluorescence resonance energy transfer (FRET) between Aequorea green fluorescent protein (GFP) variants and DsRed was achieved. Dynamic changes in cytosolic free Ca2+ concentrations were observed with red cameleons containing yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), or Sapphire as the donor and RFP as the acceptor, using conventional microscopy and one- or two-photon excitation laser scanning microscopy. Particularly, the use of the Sapphire-DsRed pair rendered the red cameleon tolerant of acidosis occurring in hippocampal neurons, because both Sapphire and DsRed are extremely pH-resistant.

A CCD-based optical CT scanner for high-resolution 3D imaging of radiation dose distributions: equipment specifications, optical simulations and preliminary results

Abstract: Methods based on magnetic resonance imaging for the measurement of threedimensional distributions of radiation dose are highly developed. However, relatively little work has been done on optical computed tomography (OCT). This paper describes a new OCT scanner based on a broad beam light source and a two-dimensional charge-coupled device (CCD) detector. A number of key design features are discussed including the light source; the scanning tank, turntable and stepper motor control; the diffuser screen onto which images are projected and the detector. It is shown that the non-uniform pixel sensitivity of the low-cost CCD detector used and the granularity of the diffuser screen lead to a serious ring artefact in the reconstructed images. Methods are described for eliminating this. The problems arising from reflection and refraction at the walls of the gel container are explained. Optical ray-tracing simulations are presented for cylindrical containers with a variety of radii and verified experimentally. Small changes in the model parameters lead to large variations in the signal intensity observed in the projection data. The effect of imperfect containers on data quality is discussed and a method based on a ‘correction scan’ is shown to be successful in correcting many of the related image artefacts. The results of two tomography experiments are presented. In the first experiment, a radiochromic Fricke gel sample was exposed four times in different positions to a 100 kVp x-ray beam perpendicular to the plane of imaging. Images of absorbed dose with slice thickness of 140 µm were acquired, with ‘true’ in-plane resolution of 560 × 560 µm 2 at the edge of the 72 mm field of view and correspondingly higher resolution at the centre. The nominal doses measured correlated well with the known exposure times. The second experiment demonstrated the well known phenomenon of diffusion in the dosemeter gels and yielded a value of (0.12 ± 0.02) mm 2 s −1 for the

Photonic force microscope based on optical tweezers and two-photon excitation for biological applications

Abstract: A new scanning probe microscope, the photonic force microscope (PFM), based on optical tweezers and two-photon absorption processes for biological applications is described. Optical tweezers are used to trap a fluorescent latex bead with a diameter of 200 nm in an aqueous solution in all three dimensions. The fluorescent dye is chosen to fulfill the two-photon absorption criterion for the 1064-nm line of a Nd:YVO4 laser. The intensity of the fluorescence emission is utilized as a very sensitive position sensor along the optical axis. Two-dimensional images are formed by laterally scanning the trapped latex bead across biological samples while recording the two-photon-induced fluorescences intensity. A scanning probe image of the outer surface of a small neurite from a cultured rat hippocampal neuron is shown, which is hardly visible under differential interference contrast microscopy. The lateral resolution is given by the bead diameter; the axial resolution is 40 nm. Under the experimental conditions the maximal imaging force applied by the probe is below 5 pN.

Cell damage in near-infrared multimode optical traps as a result of multiphoton absorption

Abstract: We report on cell damage of single cells confined in continuous-wave (cw), near-infrared (NIR) multimode optical traps as a result of multiphoton absorption phenomena. Trapping beams at NIR wavelengths less than 800 nm are capable of damaging cells through a two-photon absorption process. Cell damage is more pronounced in multimode cw traps compared with single-frequency true cw NIR traps because of transient power enhancement by longitudinal mode beating. Partial mode locking in tunable cw Ti:sapphire lasers used as trapping beam sources can produce unstable subnanosecond pulses at certain wavelengths that amplify multiphoton absorption effects significantly. We recommend the use of single-frequency long-wavelength NIR trapping beams for optical micromanipulation of vital cells.

High-speed, two-photon scanning microscope

Abstract: 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 µs/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.

A first-order model for computation of laser-induced breakdown thresholds in ocular and aqueous media: Part II-Comparison to experiment

Abstract: For pt. I see ibid., vol.31, no.12, p.2241-9 (1995). An analytic, first-order model has been developed to calculate irradiance thresholds for laser-induced breakdown (LIB) in condensed media, including ocular and aqueous media. A complete derivation and description of the model was given in a previous paper (Part I). The model has been incorporated into a computer code and code results have been compared to experimentally measured irradiance thresholds for breakdown of ocular media, saline, and water by nanosecond, picosecond, and femtosecond laser pulses in the visible and near-infrared. The comparison included both breakdown data from the literature and from our own measurements. Theoretical values match experiment to within a factor of 2 or better, over a range of pulsewidths spanning five orders of magnitude.

Scanning two-photon fluctuation correlation spectroscopy: Particle counting measurements for detection of molecular aggregation

Abstract: Scanning fluctuation correlation spectroscopy (FCS) is an experimental technique capable of measuring particle number concentrations by monitoring spontaneous equilibrium fluctuations in the local concentration of a fluorescent species in a small (femtoliter) subvolume of a sample. The method can be used to detect molecular aggregation for dilute, submicromolar samples by directly "counting particles". We introduce the application of two-photon excitation to scanning FCS and discuss its important advantages for this technique. We demonstrate the capability of measuring particle number concentrations in solution, first with dilute samples of monodisperse 7-nm and 15-nm radius latex spheres, and then with B phycoerythrin. The detection of multiple species in a single sample is shown, using mixtures containing both sphere sizes. The method is then applied to study protein aggregation in solution. We monitor the concentration-dependent association/ dissociation equilibrium for glycogen phosphorylase A and malate dehydrogenase. The measured dissociation constants, 430 nM and 144 nM respectively, are in good agreement with previously published values. In addition, oligomer dissociation induced by pH titration from pH 8 to pH 5.0 is detectable for the enyme phosphofructokinase. The possibility of measuring dissociation kinetics by scanning two-photon FCS is also demonstrated using phosphofructokinase.

Two-photon image correlation spectroscopy and image cross-correlation spectroscopy

Abstract: We introduce two-photon image correlation spectroscopy (ICS) using a video rate capable multiphoton microscope. We demonstrate how video rate two-photon microscopic imaging and image correlation analysis may be combined to measure molecular transport properties over ranges typical of biomolecules in membrane environments. Using two-photon ICS, we measured diffusion coefficients as large as 10(-8) cm2 s(-1) that matched theoretical predictions for samples of fluorescent microspheres suspended in aqueous sucrose solutions. We also show the sensitivity of the method for measuring microscopic flow using analogous test samples. We demonstrate explicitly the advantages of the image correlation approach for measurement of correlation functions with high signal-to-noise in relatively short time periods and discuss situations when these methods represent improvements over non-imaging fluorescence correlation spectroscopy. We present the first demonstration of two-photon image cross-correlation spectroscopy where we simultaneously excite (via two-photon absorption) non-identical fluorophores with a single pulsed laser. We also demonstrate cellular application of two-photon ICS for measurements of slow diffusion of green fluorescent protein/adhesion receptor constructs within the basal membrane of live CHO fibroblast cells.

Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope

Abstract: The combination of pulsed-mode excitation multifocal multiphoton microscopy with a high-repetition, time-gated intensified CCD camera enables efficient three-dimensional (3D) fluorescence lifetime imaging. With a 200-ps gate opening at 76 MHz repetition rate, fluorescence decay can be traced in a sequence of images with varying delays between pulse and gate. Fluorophore lifetimes are measured with a precision of a few picoseconds. As an application we show that, upon two-photon excitation at 800 nm, certain pollen samples feature a multiexponential fluorescence relaxation. Our results indicate that efficient four-dimensional microscopy with hundreds of nanometer spatial and tens of picoseconds temporal resolution is within reach.

Free charge transfer in charge-coupled devices

Abstract: The free charge-transfer characteristics of charge-coupled devices (CCD's) are analyzed in terms of the charge motion due to thermal diffusion, self-induced drift, and fringing field drift. The charge-coupled structures considered have separations between the gates equal to the thickness of the channel oxide. The effect of each of the above mechanisms on charge transfer is first considered separately, and a new method is presented for the calculation of the self-induced field. Then the results of a computer simulation of the charge-transfer process that simultaneously considers all three charge-motion mechanisms is presented for three-phase CCD's with gate lengths of 4 and 10 µ. The analysis shows that while the majority of the charge is transferred by means of the self-induced drift that follows a hyperbolic time dependence, the last few percent of the charge decays exponentially under the influence of the fringing field drift or thermal diffusion, depending on the design of the structure. The analysis shows that in CCD's made on relatively high resistivity substrates, the transfer by fringing-field drift can be very fast, such that transfer efficiencies of 99.99 percent are expected at 5- to 10- MHz bit rates for 10-µ gate lengths and at up to 100 MHz for 4-µ gate lengths.