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.     

Low-cost, scalable laser scanning module for real-time reflectance and fluorescence confocal microscopy

We present a low-cost, high-speed, retrofitted laser scanning module for microscopy. The cage-mounted system, with various available fiber-coupled sources, offers a real-time imaging alternative to costly commercial systems with capabilities for conventional or confocal reflectance and fluorescence applications as well as advanced laser scanning microscopy implementations. Reflectance images of a resolution target and confocal images of fluorescent polystyrene beads are presented for system characterization. Confocal fluorescence image stacks of T84 epithelial cancer cells are presented to demonstrate application to biological studies. This laser scanning module is a flexible, scalable, high-speed alternative to commercial laser scanning systems suitable for applications requiring a simple imaging tool and for teaching laboratories.     

Two-photon fluorescence microscopy with a diode-pumped Cr:LiSAF laser

We demonstrate the acquisition of high-quality two-photon fluorescence microscopy images using an all-solid-state self-mode-locked Cr:LiSAF laser. We contrast the performance of the two-photon technique with single-photon confocal fluorescence microscopy images taken with an argon-ion laser. Examples of improved depth penetration and reduced dye bleaching are presented.     

Direct observation of minority carrier leakage in operating laser diodes by Kelvin probe force microscopy

A new approach to investigating the leakage of nonequilibrium holes and electrons from the active region of a semiconductor laser diode is proposed. According to this, the scanning Kelvin probe force microscopy is used to measure averaged local changes in the contact potential difference on the surface of laser mirrors of a device operating at a pulsed bias voltage. It is shown that the measured signal level is determined by the degree of charge exchange between the slow surface states and nonequilibrium minority carriers, the concentration of which is directly related to the leakage current.     

Quantum noise of laser diodes

Abstract

Single-mode laser theory for semiconductor lasers predicts sub-Poissonian light generation for a laser quietly driven far above threshold. Experiments have shown however that only few laser diodes exhibits such reduced intensity noise. We present a review of different mechanisms that have been proposed to explain the excess noise observed in semiconductor lasers, including imperfect anticorrelations in multimode lasers, and Petermann excess noise factor in single-mode lasers.     

Determination of elastic constants by time-resolved line-focusacoustic microscopy

The determination of elastic constants by using time-resolved line-focus acoustic microscopy (TRL-FAM) is discussed. Waveforms have been measured with a time-resolved line-focus acoustic microscopy system. A measurement model which simulates the measurements is also introduced for parametric studies of the waveforms. The determination of elastic constants is achieved by systematically comparing the relative wave-mode time-delays obtained from the measurement model and the experiments. The method has been applied to determine elastic constants of both isotropic and anisotropic materials     

Applicability of blue/UV laser diodes for the measurement of vaporized fuel fluorescence around stoichiometric concentrations

A fluorescence-based fiber-optic probe is implemented for use with a blue/UV laser diode to form a rugged, nonintrusive fuel sensor. Measurements are obtained from a test vessel controlled to replicate the temperature and pressure environment in a typical combustion engine. A fuel containing a specific polycyclic aromatic hydrocarbon (perylene, C20H12) is used to obtain wavelength resolved information and concentration variation characteristics. Emission intensity is examined in atmospheres with and without oxygen at pressures from 1 to 10 bar. The signals from a retail gasoline fuel present a greater challenge as they result from a complex mixture of hydrocarbon components. Detection of gasoline vapor concentrations in the range of 4% to 125% of stoichiometry is demonstrated.     

A spectroscopically resolved photo- and electroluminescence microscopy technique for the study of high-power and high-brightness laser diodes

A novel and advanced characterization technique is described for performing optical studies of the luminescence properties of materials. It was developed for the investigation of semiconductor materials, including semiconductor laser diodes and photonic integrated circuits. A quantitatively calibrated, spatially and spectrally resolved imaging technique is described, which is based upon the technologies of photoluminescence microscopy and photoluminescence spectroscopy. The principles of the spectroscopically resolved photo- and electroluminescence microscopy techniques are outlined in this paper, and the developed instrument is described in detail. Design calculations used to select and set up the experimental apparatus are presented, and results are found to compare well with this analysis. Various experimental measurements are used to demonstrate the performance of the new instrument. The study of strain and defects in high-power laser diodes is presented as one of the more challenging applications of the new technique. The results presented demonstrate the ability of this technique to image photoluminescence shifts occurring in the substrate of packaged laser bars, enabling the investigation of strain, defects and their evolution with aging. Other applications of the technique include the spectroscopic measurement of near- and far-field patterns and virtual sources of laser diodes, investigations of spectral hole burning and optical scattering processes in lasers and photonic integrated circuits, and studies of organic LEDs. In the future, applications are also envisaged in medicine and the biological sciences.     

Defects and laser action in GaAs diodes

Zn-diffused, Te-doped GaAs injection lasers were examined by means of infrared microscopy, etching, transmission electron microscopy and scanning electron probe microanalysis.Bands of high infrared absorption, i.e., striations, were present in many diodes and the lines of intersection of the striations with the p-n junction were minimum emission filaments. Misfit dislocation networks were present in the p-n junction region but did not influence the emission pattern. Precipitates and diffusion-induced dislocations were found in the zinc-diffused region. All the properties of the material that enter into the expression for the threshold current for laser action were considered. It was concluded that the striations affect laser emission patterns mainly through the variation of the infra-red absorption coefficient.     

Peculiarities of using laser diodes in linear measurements

The position of the emitting region of a laser diode depends on the working current. This phenomenon, as well as the polarization of radiation, may influence the accuracy of linear measurements using such lasers.     

Mechanical properties of single cells by high-frequency time-resolved acoustic microscopy

In this paper, we describe a new, high-frequency, time-resolved scanning acoustic microscope developed for studying dynamical processes in biological cells. The new acoustic microscope operates in a time-resolved mode. The center frequency is 0.86 GHz, and the pulse duration is 5 ns. With such a short pulse, layers thicker than 3 microm can be resolved. For a cell thicker than 3 microm, the front echo and the echo from the substrate can be distinguished in the signal. Positions of the first and second pulses are used to determine the local impedance of the cell modeled as a thin liquid layer that has spatial variations in its elastic properties. The low signal-to-noise ratio in the acoustical images is increased for image generation by averaging the detected radio frequency signal over 10 measurements at each scanning point. In conducting quantitative measurements of the acoustic parameters of cells, the signal can be averaged over 2000 measurements. This approach enables us to measure acoustical properties of a single HeLa cell in vivo and to derive elastic parameters of subcellular structures. The value of the sound velocity inside the cell (1534.5 +/- 33.6 m/s) appears to be only slightly higher than that of the cell medium (1501 m/s).     

Implementation of time-resolved step-scan fourier transform infrared (FT-IR) spectroscopy using a kHz repetition rate pump laser

Time-resolved step-scan Fourier transform infrared (FT-IR) spectroscopy has been shown to be invaluable for studying excited-state structures and dynamics in both biological and inorganic systems. Despite the established utility of this method, technical challenges continue to limit the data quality and more wide ranging applications. A critical problem has been the low laser repetition rate and interferometer stepping rate (both are typically 10 Hz) used for data acquisition. Here we demonstrate significant improvement in the quality of time-resolved spectra through the use of a kHz repetition rate laser to achieve kHz excitation and data collection rates while stepping the spectrometer at 200 Hz. We have studied the metal-to-ligand charge transfer excited state of Ru(bipyridine)(3)Cl(2) in deuterated acetonitrile to test and optimize high repetition rate data collection. Comparison of different interferometer stepping rates reveals an optimum rate of 200 Hz due to minimization of long-term baseline drift. With the improved collection efficiency and signal-to-noise ratio, better assignments of the MLCT excited-state bands can be made. Using optimized parameters, carbonmonoxy myoglobin in deuterated buffer is also studied by observing the infrared signatures of carbon monoxide photolysis upon excitation of the heme. We conclude from these studies that a substantial increase in performance of ss-FT-IR instrumentation is achieved by coupling commercial infrared benches with kHz repetition rate lasers.     

Mathematical model for time-resolved and frequency-domain fluorescence spectroscopy in biological tissues

In general it is not possible to write an analytic expression for the fluorescence signal generated by a fluorophore distributed in a scattering medium such as tissue. However, by assuming that the scattering properties of the tissue are the same at the excitation and emission wavelengths, we have derived a simple relation between the fluorescence and the scatter signals. Along with diffusion theory, this was used to write expressions for the fluorescence signal detected at the tissue surface in both the time and the frequency domains. Experiments using the fluorophore aluminum chlorosulfonated phthalocyanine in tissue-simulating materials confirmed the accuracy of the model. Applications to in vivo spectroscopy are discussed.     

Characterization of time-resolved fluorescence response measurements for distributed optical-fiber sensing

A distributed optical-fiber sensing system based on pulsed excitation and time-gated photon counting has been used to locate a fluorescent region along the fiber. The complex Alq3 and the infrared dye IR-125 were examined with 405 and 780 nm excitation, respectively. A model to characterize the response of the distributed fluorescence sensor to a Gaussian input pulse was developed and tested. Analysis of the Alq3 fluorescent response confirmed the validity of the model and enabled the fluorescence lifetime to be determined. The intrinsic lifetime obtained (18.2±0.9 ns) is in good agreement with published data. The decay rate was found to be proportional to concentration, which is indicative of collisional deactivation. The model allows the spatial resolution of a distributed sensing system to be improved for fluorophores with lifetimes that are longer than the resolution of the sensing system.     

Simultaneous use of time-resolved fluorescence and anti-stokes photoluminescence in a bioaffinity assay

A bioaffinity assay is described where anti-Stokes photoluminescence of inorganic lanthanide phosphors and time-resolved fluorescence of lanthanide chelates are measured from a single microtitration well without any disturbance from these label technologies to each other. Up-converting phosphor (UPC-phosphor) bioconjugate was produced by grinding the commercial, micrometer-sized UPC-phosphors to colloidal, submicrometer-sized phosphor particles and by attaching these phosphors to biomolecules. Experiments were carried out in standard 96-well microtitration plates to determine detection limits, linearity, and cross-talk of UPC-phosphor and europium chelate. In numbers of molecules the lower limits of detection for UPC-phosphor were roughly 3 x 10(3) particles in solution and 1 x 10(4) particles in solid phase, and for europium label same values were 9 x 10(6) and 9 x 10(7) molecules. Linearity of detection was for UPC-phosphor 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase and for europium label over 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase. The cross-talk between the two labels was practically nonexistent. In this study we show that up-converting anti-Stokes photoluminescent phosphors could be employed in bioaffinity assays as very potential labels with significant advantages either alone or together with long-lifetime lanthanide chelates.     

In situ time-resolved fluorescence spectroscopy in the frequency domain in capillary electrochromatography

In situ time-resolved fluorescence spectroscopy for capillary electrochromatography (CEC) is described in the frequency domain. Fluorescence decay of the solute molecules is collected directly in the packed stationary phase of the CEC capillary. The fluorescence lifetime profile of the solute molecules reveals the microenvironments they experience in the C18 chromatographic interface. A quartz flow cell and experimental optimization of the signal-to-noise ratio are described that enable the collection of high-quality decay data and subsequent calculation of fluorescence lifetime profiles of the solute molecules. The distribution of pyrene (PY), 1-pyrenemethanol (PY-MeOH), and 1-pyrenebutanol (PY-BuOH) into the C18 stationary phase and the solute-C18 phase interactions are probed, under separation conditions for CEC. All three molecules display a Gaussian distribution of lifetimes, consistent with an ensemble of heterogeneous microenvironments in the C18 stationary phase. The least polar molecule PY diffuses deeply into and interacts extensively with the C18 phase, experiencing high hydrophobicity and significant heterogeneity of microenvironments. The retention order of PY-MeOH, PY-BuOH, and PY in CEC is determined by their interactions with the stationary phase, revealed by their fluorescence lifetime distributions.     

RT-CW Operation of InGaN multi-quantum-well structure laser diodes

The continuous-wave operation of InGaN multi-quantum-well (MQW) structure laser diodes (LDs) was demonstrated at room temperature with a threshold current of 25 mA, a threshold voltage of 5.8 V, an output power of 30 mW and a high operating temperature of 100°C. The energy differences between the absorption and the emission energy of the InGaN MQW structure LDs were as large as 220 meV at RT. A deep localized state (the localization energy is >100 meV) was formed in the InGaN well layer due to the InGaN phase separation during the growth. Both the spontaneous emission and the stimulated emission of the LDs originated from these deep localized energy states. The far field pattern showed a higher order transverse mode of the entire 5-μm-thick epitaxial layer stack, with air and sapphire as the upper and lower cladding layers, respectively.     

Simulation of double quantum well GalnNAs laser diodes

The simulation of double quantum well (QW) GalnNAs ridge-waveguide (RW) lasers is performed over a wide range of cavity lengths and operating temperatures using a comprehensive in-house 2D laser simulator that takes into account all of the major device physics, including current spreading, capture escape processes, drift diffusion in the QW, 2D optical modes and fully resolved lasing spectra. The gain data used by the simulator were fitted to experimental gain spectra measured by the segmented contact method. The gain model includes the band-anticrossing model for the conduction band and a 4 x 4 kldrp model for the valence band. Using a carrier density-dependent and temperature-dependent linewidth broadening parameter, a good fit with experiment over a temperature range of 300-350 K was obtained. A Shockley-Read-Hall (SRH) lifetime of 0.5 ns and an Auger recombination coefficient of 1 x 10^-28 cm⁶/ s, were extracted from the calibration of the laser simulator to experimental device characteristics of broad-area (BA) devices. Using the same set of parameters for BA devices, except for a reduced SRH lifetime of 0.45 ns underneath the etch, 2D simulation results were found to agree well with the measured RW laser operating characteristics. The impact of the various recombination processes in the RW laser at threshold has also been identified using the calibrated laser simulator.     

Feasibility analysis of an epidermal glucose sensor based on time-resolved fluorescence

The goal of this study is to test the feasibility of using an embedded time-resolved fluorescence sensor for monitoring glucose concentration. Skin is modeled as a multilayer medium with each layer having its own optical properties and fluorophore absorption coefficients, lifetimes, and quantum yields obtained from the literature. It is assumed that the two main fluorophores contributing to the fluorescence at these excitation and emission wavelengths are nicotinamide adenine dinucleotide (NAD)H and collagen. The intensity distributions of excitation and fluorescent light in skin are determined by solving the transient radiative transfer equation by using the modified method of characteristics. The fluorophore lifetimes are then recovered from the simulated fluorescence decays and compared with the actual lifetimes used in the simulations. Furthermore, the effect of adding Poissonian noise to the simulated decays on recovering the lifetimes was studied. For all cases, it was found that the fluorescence lifetime of NADH could not be recovered because of its negligible contribution to the overall fluorescence signal. The other lifetimes could be recovered to within 1.3% of input values. Finally, the glucose concentrations within the skin were recovered to within 13.5% of their actual values, indicating a possibility of measuring glucose concentrations by using a time-resolved fluorescence sensor.