Imaging diffusion in living cells using time-correlated single-photon counting

Current efforts to monitor the diffusion of proteins in living cells are based on either fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching, or image correlation spectroscopy. However, these methods cannot generate a map of diffusion times. Here, we introduce a new method termed diffusion imaging microscopy that combines scanning confocal microscopy, time-correlated single-photon counting, and FCS and thus allows us to measure spatially resolved diffusion times. In our approach, we record scan images with time-resolved photon streams within each individual pixel. By extending the pixel dwell time to 25-100 ms, a software correlation of individual photons within each pixel yields the average diffusion time. Additionally, information on fluorescence intensity (number of photons) and fluorescence lifetime is available and can be used to sort fluorescence photons and to discriminate from autofluorescence. We evaluated our method by measuring diffusion times of dT20-TMR in solutions of different viscosity. We further demonstrate the applicability of the method to living cells and recorded a diffusion map of a living 3T3 mouse fibroblast incubated with dT20-ATTO488.     

Optical design and evaluation of a three-dimensional imaging and ranging system based on time-correlated single-photon counting

The design and operation of a noncontact surface profilometry system based on the time-correlated single-photon-counting technique are described. This system has a robust optomechanical design and uses an eye-safe laser that makes it particularly suitable for operation in an uncontrolled industrial environment. The sensitivity of the photon-counting technique permits its use on a variety of target materials, and its mode of operation does not require the continual presence of an operator. The system described has been optimized for a 1-25-m standoff, has a distance repeatability of <30 microm, and has a transverse spatial resolution of approximately 60 microm at a 2-m standoff and approximately 400 microm at a 13-m standoff.     

Four-dimensional microscopy of defects in integrated circuits

We demonstrate four-dimensional microscopy of defects in integrated circuits by a technique that combines laser-scanning confocal reflectance microscopy with one-photon optical-beam-induced current (1P-OBIC) imaging. Accurate information is obtained about the three-dimensional structure of the defect and the kind of material (metal, semiconductor, or dielectric) that is damaged by the defect. The same focused probe beam simultaneously produces the 1P-OBIC and reflectance signals from the illuminated sample spot. The hardware development cost is minimal for a laser-scanning confocal microscope, and the image reconstruction procedure is computationally efficient. Imaging is demonstrated on defects that are caused by electrical overstress and unwanted generation centers. Exclusive three-dimensional distributions of the semiconductor and metal sites in the integrated circuit reveal defect features that are difficult to recognize with confocal or 1P-OBIC imaging alone.     

Laser spectral characterization in multiphoton microscopy

Spectral and temporal characterization is a fundamental task when a tunable Ti:sapphire ultrafast laser system is operated for multiphoton microscopy applications. In the present paper simple procedures are reported that perform laser-peak-emission wavelength and bandwidth measurements without the need of any further instrumentation but a simple and inexpensive diffraction grating, by taking advantage of the confocal microscope imaging capabilities.     

Optical manipulation in combination with multiphoton microscopy for single-cell studies

We demonstrate how optical tweezers can be incorporated into a multiphoton microscope to achieve three-dimensional imaging of trapped cells. The optical tweezers, formed by a cw 1064 nm Nd:YVO4 laser, were used to trap live yeast cells in suspension while the 4',6-diamidino-2-phenylindole-stained nucleus was imaged in three dimensions by use of a pulsed femtosecond laser. The trapped cell was moved in the axial direction by changing the position of an external lens, which was used to control the divergence of the trapping laser beam. This gives us a simple method to use optical tweezers in the laser scanning of confocal and multiphoton microscopes. It is further shown that the same femtosecond laser as used for the multiphoton imaging could also be used as laser scissors, allowing us to drill holes in the membrane of trapped spermatozoa.     

Two-dimensional imaging without scanning by multifocal multiphoton microscopy

We describe multifocal multiphoton microscopy giving images without laser scanning. A multitude of 8 x 8 laser beams is focused into a sample yielding two-photon excitation in a plane. The focal spots are arranged in a rectangular array with close spacing between individual points (approximately 0.5 microm). The fluorescence emission from the sample is recorded with a CCD camera, but, owing to the close distance between the beams, they can no longer be regarded as individual points but rather as an illumination of the plane that is covered by the array of focal points. The axial sectioning capability is comparable with an ordinary single-beam two-photon microscope. Interference between the beams that could compromise the axial sectioning capability does not occur in our setup owing to small temporal delays between the individual beams. The axial sectioning capability of the setup is discussed in detail by means of the step response in which the foci are scanned axially into a uniformly fluorescent medium.     

Time multiplexing and parallelization in multifocal multiphoton microscopy

We investigate the imaging properties of high-aperture multifocal multiphoton microscopy on the basis of diffraction theory. Particular emphasis is placed on the relationship between the sectioning property and the distance between individual foci. Our results establish a relationship between the degree of parallelization and the axial resolution for both two- and three-photon excitation. In addition, we show quantitatively that if a matrix of temporal delays is inserted between the individual foci, it is, for the first time to our knowledge, possible to solve the classical conflict between the light budget and the sectioning property in three-dimensional microscopy and to provide a virtually unlimited density of foci at best axial resolution.     

Standardless atom counting in scanning transmission electron microscopy

We demonstrate that high-angle annular dark-field imaging in scanning transmission electron microscopy allows for quantification of the number and location of all atoms in a three-dimensional, crystalline, arbitrarily shaped specimen without the need for a calibration standard. We show that the method also provides for an approach to directly measure the finite effective source size of a scanning transmission electron microscope.     

Performance and design of InGaAs /InP photodiodes for single-photon counting at 1.55 microm

The performance of selected, commercially available InGaAs/InP avalanche photodiodes operating in a photon-counting mode at an incident wavelength of 1.55 mum is described. A discussion on the optimum operating conditions and their relationship to the electric field distribution within the device is presented.     

Cryogenic thermoelectric (QVD) detectors: Emerging technique for fast single-photon counting and non-dispersive energy characterization

Abstract

”QVD”detectors are based on thermoelectric heat-to-voltage (Q V) conversion and digital (V D) readout. We have devised and analyzed the performance of QVD detectors with several different sensor designs that enable use of high thermoelectric figure of merit samples, be they of thin film, bulk crystal, or whisker form. Our first QVD devices had the well-studied material Au-Fe as thin film sensors. More recently, we have confirmed the literature reports of substantially higher Seebeck coefficient at cryogenic temperatures in lanthanum (cerium) hexaborides. We have also investigated the kinetic properties of La(Ce)B₆ crystals with different La – Ce ratios. Currently we are exploring prototype devices based on bulk single-crystalline sensors. These include a successfully tested candidate with a sharpend hexaboride sensor and small-size bismuth absorber – a whisker prototype. In theory, QVD sensors are competitive with superconducting tunnel junction (STJ) and transition edge sensor (TES) devices in energy resolution ability. However, QVD sensors ought to be able to respond at very much faster rates than these competitors; the lanthanum-cerium hexaboride sensors are expected to reach rates of 100 MHz counting rates for UV/optical photons. In addition to traditional astrophysical applications, these detectors can be applied to the tasks of quantum computing and communication.     

Quantum cryptography with perfect multiphoton entanglement

Multiphoton entanglement in the same polarization has been shown theoretically to be obtainable by type-I spontaneous parametric downconversion (SPDC), which can generate bright pulses more easily than type-II SPDC. A new quantum cryptographic protocol utilizing polarization pairs with the detected type-I entangled multiphotons is proposed as quantum key distribution. We calculate the information capacity versus photon number corresponding to polarization after considering the transmission loss inside the optical fiber, the detector efficiency, and intercept-resend attacks at the level of channel error. The result compares favorably with all other schemes employing entanglement.     

Three-dimensional time-resolved fluorescence imaging by multifocal multiphoton microscopy for a photosensitizer study in living cells

Two-photon fluorescence microscopy is widely applied to biology and medicine to study both the structure and dynamic processes in living cells. The main issue is the slow acquisition rate due to the point scanning approach limiting the multimodal detection (x, y, z, t). To extend the performances of this powerful technique, we present a time-resolved multifocal multiphoton microscope (MMM) based on laser amplitude splitting. An array of 8 x 8 foci is created on the sample that gives a direct insight of the fluorescence localization. Four-dimensional (4D) imaging is obtained by combining simultaneous foci scanning, time-gated detection, and z displacement. We illustrate time-resolved MMM capabilities for 4D imaging of a photosensitizer inside living colon cancer cells. The aim of this study is to have a better understanding of the photophysical processes implied in the photosensitizer reactivity.     

Errors in counting identical objects with a counting balance

Measurement Techniques -     

Origin and effect of high-order dispersion in ultrashort pulse multiphoton microscopy in the 10 fs regime

Short pulses can induce high nonlinear excitation, and thus they should be favorable for use in multiphoton microscopy. However, the large spectral dispersion can easily destroy the advantages of the ultrashort pulse if there is no compensation. The group delay dispersion (GDD), third-order dispersion, and their effects on the intensity and bandwidth of second-harmonic generation (SHG) signal were analyzed. We found that the prism pair used for compensating the GDD of the two-photon microscope actually introduces significant negative high-order dispersion (HOD), which dramatically narrowed down the two-photon absorption probability for ultrashort pulses. We also investigated the SHG signal after GDD and HOD compensation for different pulse durations. Without HOD compensation, the SHG efficiency dropped significantly for a pulse duration below 20 fs. We experimentally compared the SHG and two-photon excited fluorescence (TPEF) signal intensity for 11 fs versus 50 fs pulses, a pulse duration close to that commonly used in conventional multiphoton microscopy. The result suggested that after adaptive phase compensation, the 11fs pulse can yield a 3.2- to 6.0-fold TPEF intensity and a 5.1-fold SHG intensity, compared to 50 fs pulses.     

Critical flocculation concentrations, binding isotherms, and ligand exchange properties of peptide-modified gold nanoparticles studied by UV-visible, fluorescence, and time-correlated single photon counting spectroscopies

Protocols for modifying gold nanoparticles with peptide-bovine serum albumin (BSA) conjugates are described within. The resulting constructs were characterized using a number of techniques including static fluorescence spectroscopy and time-correlated single photon counting spectroscopy (TCSPC) in order to quantify peptide-BSA binding isotherms, exchange rates, critical flocculation concentrations, and the composition of mixed peptide-BSA monolayers on gold nanoparticles. TCSPC has proven to be a powerful technique for observing the microenvironment of protein-gold nanoparticle conjugates because it can distinguish between surface-bound and solution-phase species without the need for separation steps. Full characterization of the composition and stability of peptide-modified metal nanoparticles is an important step in their use as intracellular delivery vectors and imaging agents.     

Infrared multiphoton dissociation with a hollow fiber waveguide

A novel scheme for performing infrared multiphoton dissociation (IRMPD) is presented in which a hollow fiber waveguide (HFWG) is used to transmit IR radiation into the ion storage region of a mass spectrometer. Efficient dissociation of oligonucleotide and protein ions is demonstrated on an ESI-FTICR instrument in which IRMPD is performed in the external ion reservoir and on a quadrupole ion trap. Using a simple optical scheme consisting of a single focusing lens and an x, y translator, the 10.6-microm IR laser beam, initially 3.5 mm in diameter, is focused into the vacuum-sealed HFWG. The small internal diameter and the high transfer efficiency of the waveguide allow IR radiation of high power density to be employed for IRMPD. In studies performed on a quadrupole ion trap, a 500-microm-i.d. waveguide was used as a medium to transmit IR radiation directly through a 700-microm orifice in the ring electrode. Efficient IRMPD of both a 12-mer oligonucleotide and the protein melittin were performed at laser powers of 0.5 and 3.2 W, respectively.     

Superconducting NbN single-photon detector integrated with quarter-wave resonator

The spectral dependence of the quantum efficiency of superconducting NbN single-photon detectors integrated with quarter-wave resonators based on Si₃N₄, SiO₂, and SiO layers has been studied.     

Quantitative counting of single fluorescent molecules by combined electrochemical adsorption accumulation and total internal reflection fluorescence microscopy

We developed an ultrasensitive quantitative single-molecule imaging method for fluorescent molecules using a combination of electrochemical adsorption accumulation and total internal reflection fluorescence microscopy (TIRFM). We chose rhodamine 6G (R6G, fluorescence dye) or goat anti-rat IgG(H+L) (IgG(H+L)-488), a protein labeled by Alexa Fluor 488 or DNA labeled by 6- CR6G (DNA-R6G) as the model molecules. The fluorescent molecules were accumulated on a light transparent indium tin oxide (ITO) conductive microscope coverslip using electrochemical adsorption in a stirred solution. Then, images of the fluorescent molecules accumulated on the ITO coverslip sized 40 x 40 microm were acquired using an objective-type TIRFM instrument coupled with a high-sensitivity electron multiplying charge-coupled device. One hundred images of the fluorescent molecules accumulated on the coverslip were taken consecutively, one by one, by moving the coverslip with the aid of a three-dimensional positioner. Finally, we counted the number of fluorescent spots corresponding to single fluorescent molecules on the images. The linear relationships between the number of fluorescent molecules and the concentration were obtained in the range of 5 x 10(-15) to 5 x 10(-12) mol/L for R6G, 3 x 10(-15) to 2 x 10(-12) mol/L for IgG(H+L)-488, and 3 x 10(-15) to 2 x 10(-12) mol/L for DNA-R6G.     

Ultra-low dead time free-running InGaAsP single-photon detector with active quenching

High afterpulse probability and the consequent long dead time of free-running InGaAs(P) single-photon detectors have long been the limiting factors to their practical applications. Here we present a free-running InGaAsP single-photon detector for 1.06?μm wavelength with ultra-low dead time and afterpulse probability. With the optimized active-quenching circuit and packaging, the avalanche pulse discrimination level is down to 2.4?mV, and the full-width at half-maximum of the avalanche pulse is as short as 500?ps, which greatly lessens afterpulsing effects. As a result, the dead time of the detector is as low as 35?ns, comparable to free-running silicon detectors, with a low afterpulse probability of 11.6% at 10% detection efficiency, 242?K. The proposed detector provides a high-performance, small-sized, low-power approach to single-photon detection for practical applications such as light detection and ranging and free-space quantum key distribution systems.