Fluorescence lifetime imaging with picosecond resolution for biomedical applications

We describe a novel whole-field fluorescence lifetime imaging system, based on a time-gated image intensifier and a solid-state laser oscillator-amplifier, that images lifetime differences of less than 10 ps. This system was successfully applied to discrimination between biological tissue constituents.     

Construction and characterization of a frequency-domain fluorescence lifetime imaging microscopy system

The construction of a homodyne frequency domain fluorescence lifetime imaging microscope is described. The system consists of (i) an intensity-modulated laser excitation source, (ii) an epifluorescence microscope, (iii) a gain-modulated microchannel plate (MCP) image intensifier, and (iv) a slow-scan CCD camera. The phase and modulation homogeneity of the MCP image intensifier were determined at frequencies of 40, 100, 160, and 240 MHz. The detected modulation depths were 65, 52, 32, and 23%, respectively, and were highly homogeneously distributed. The phasedistribution image revealed iris effects at frequencies of 160 and 240 MHz but was homogeneous at lower frequencies. Lifetime imaging of a solution of the fluorescent flavoprotein lipoamide dehydrogenase demonstrated (i) the accuracy of the determined lifetimes (< 60 ps), (ii) the time resolution of the instrument (< 50 ps), and (iii) the average precision for single pixel fluorescence lifetimes (50 ps is feasible). The imaging of tiny fluorescent microspheres revealed that even in a volume of 0.3 x 10^-15 L, the standard error in the lifetimes can be as low as 79 ps. The spatial resolution of the instrument is estimated to be < 400 nm in the object plane at a 100 x magnification.     

Fiber-optic ring-down spectroscopy using a tunable picosecond gain-switched diode laser

Visible and near-infrared laser light pulses were coupled into two different types of optical fiber cavities. One cavity consisted of a short strand of fiber waveguide that contained two identical fiber Bragg gratings. Another cavity was made using a loop of optical fiber. In either cavity ∼40 ps laser pulses, which were generated using a custom-built gain-switched diode laser, circulated for a large number of round trips. The optical loss of either cavity was determined from the ring-down times. Cavity ring-down spectroscopy was performed on 200 pL volumes of liquid samples that were injected into the cavities using a 100 μm gap in the fiber loop. A detection limit of 20 ppm of methylene blue dye in aqueous solution, corresponding to a minimum absorptivity of εC<6 cm^-1, was realized. PACS 42.62.Fi; 42.81.-i     

Pulsed near-infrared laser diode excitation system for biomedical photoacoustic imaging

A pulsed laser diode system operating at 905 nm has been developed for the generation of photoacoustic signals in tissue. It was evaluated by measuring the photoacoustic waveforms generated in a blood vessel phantom comprising three dye-filled (mu(a)=1 mm(-1)) tubes of diameters 120-580 microm immersed to a maximum depth of 9 mm in a turbid liquid (mu'(s)=1 mm(-1)). The system was then combined with a cylindrical scanning system to obtain two-dimensional images of a tissue phantom. The signal-to-noise ratio of the detected signals in both cases and the image contrast in the latter suggest that such a system could provide a compact and inexpensive alternative to current excitation sources for superficial imaging applications.     

高功率半导体激光器的可靠性与寿命评价

介绍了高功率半导体激光器的结构特点、失效机理和热产生机制等方面的内容。就寿命评价方面展开讨论,详细分析了高功率半导体激光器寿命评价的难点、现有方法以及国内外发展状况,最后就寿命评价系统及寿命试验提出了一些建议。高功率半导体激光器在工业加工、国防航天等领域的巨大应用前景推动其可靠性与寿命的发展,而其可靠性的提高和寿命的延长会大大拓宽其应用领域。     

Novel detectors for fluorescence lifetime imaging on the picosecond time scale

Simultaneous acquisition of time and space information on the picosecond time scale became feasible with a recent advance in microchannel-plate photomultiplier-tube (MCP-PMT) technology: we present two novel MCP-PMT detectors for time- and space-correlated single-photon counting (TSCSPC), featuring a space-sensitive delay-line (DL) anode and quadrant anode (QA), respectively. The linear DL-MCP-PMT is characterized by a spatial instrument response function (IRF) of 100-Μm FWHM, resulting in 200 space channels, whereas the QA-MCP-PMT is a 2D imager with 400 x 400 pixels at 40-Μm resolution. The detectors have a temporal IRF of 75 ps (DL) and 80 ps (QA) FWHM, sufficient for 10 ps time resolution, at a dynamic range of 10⁵ of the uncooled detector. A throughput of 10⁵ cps is possible; in the imaging mode without timing, the QA-detector can achieve 10⁶ cps. We present time-resolved spectroscopy of DNA probes (DAPI, TOTO, C350) in solution, in micelles, complexed to DNA, protein, and fixed cells. Aging of DAPI stock solutions is reported. A polarity model for the photophysics of DAPI is proposed. First microscope lifetime images on the picosecond time scale show a clear potential for dynamic stray-light rejection and kinetic discrimination of probe-protein and probe-DNA complexes.     

Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging

We demonstrate stimulated emission depletion (STED) microscopy implemented in a laser scanning confocal microscope using excitation light derived from supercontinuum generation in a microstructured optical fiber. Images with resolution improvement beyond the far-field diffraction limit in both the lateral and axial directions were acquired by scanning overlapped excitation and depletion beams in two dimensions using the flying spot scanner of a commercially available laser scanning confocal microscope. The spatial properties of the depletion beam were controlled holographically using a programmable spatial light modulator, which can rapidly change between different STED imaging modes and also compensate for aberrations in the optical path. STED fluorescence lifetime imaging microscopy is demonstrated through the use of time-correlated single photon counting.     

Rapid infrared wavelength access with a picosecond PPLN OPO synchronously pumped by a mode-locked diode laser

We theoretically and experimentally investigate wavelength tuning of synchronously pumped optical parametric oscillators (OPOs) on changing the cavity length or the pump-repetition rate. Conditions for rapid and wide-range wavelength access are derived. Using an OPO pumped directly by a mode-locked diode-laser master-oscillator power-amplifier (MOPA) system, an all-electronically controlled access to near- and mid-infrared wavelengths is demonstrated. The singly (signal) resonant OPO is based on periodically poled lithium niobate (PPLN) and emits 8 ps idler pulses at a repetition rate of 2.5 GHz in the wavelength range 1986 to 2348 nm (signal: 1530 to 1737 nm). Wavelength tuning over 114 nm (signal) and 189 nm (idler) is achieved solely by electronically varying the repetition rate of the diode-laser oscillator over 720 kHz. By controlling the repetition rate with a programmable driver, an arbitrary emission sequence of the OPO on two wavelength channels is generated, with access times as short as 10 μs. 11 OPO wavelengths equally spaced in the range 1627–1689 nm (signal) or 2054–2154 nm (idler) could be addressed. Received: 6 September 2000 / Revised version: 16 March 2001 / Published online: 23 May 2001     

Tomographic imaging by two-wave mixing with a wavelength-scanning laser diode

A method of tomographic imaging is proposed in which two-wave mixing in a photorefractive crystal is used with wavelength scanning of a laser diode and phase modulation of the pump beam. This method provides full optical processing and is effective for weak light from objects because of the use of two-wave mixing. The depth resolution of the method was ~1 cm when the wavelength-scanning width was ~0.02 nm .     

Atomic gallium laser spectroscopy with violet/blue diode lasers

We describe the operation of two GaN-based diode lasers for the laser spectroscopy of gallium at 403 nm and 417 nm. Their use in an external cavity configuration has enabled us to investigate of absorption spectroscopy in a gallium hollow cathode. We have analyzed the Doppler-broadened profiles, accounting for hyperfine and isotope structure and extracting both the temperature and density of the neutral atomic sample produced in the glow discharge. We have also built a setup to produce a thermal atomic beam of gallium. By using the GaN-based diode lasers, we have studied the laser-induced fluorescence and hyperfine-resolved spectra of gallium. PACS 42.55.Px; 42.60.-v; 32.30.-r; 03.75.Be     

UV and blue picosecond pulse generation by a nitrogen-laser-pumped distributed feedback dye laser

Tunable picosecond pulse generation in the 362–420 nm spectral range is reported. The laser is simple in construction and can be easily constructed from relatively inexpensive optical and mechanical parts.     

Laser system based on a picosecond dye laser with combined feedback for spectroscopy

A laser system comprising a synchronously pumped picosecond dye laser with combined cavity-distributed feedback and a two-stage dye amplifier is described. The dependence of the laser pulse duration on the detuning of the cavity length, the pumping level of the active medium, and the pulse number in the pulse train was investigated. It is shown that the combination of the two types of feedback provides more than ten-fold shortening of the dye laser ultrashort pulse duration. B. I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, 70, F. Skorina Ave., Minsk, 220072, Belarus. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 65, No. 1, pp. 47–55, January–February, 1998.     

Scanning emitter lifetime imaging microscopy for spontaneous emission control

We report an experimental technique to map and exploit the local density of optical states of arbitrary planar nanophotonic structures. The method relies on positioning a spontaneous emitter attached to a scanning probe deterministically and reversibly with respect to its photonic environment while measuring its lifetime. We demonstrate the method by imaging the enhancement of the local density of optical states around metal nanowires. By nanopositioning, the decay rate of a pointlike source of fluorescence can be reversibly and repeatedly changed by a factor of 2 by coupling it to the guided plasmonic mode of the wire.     

Fluorescence feedback control of a diode pumped solid state laser

We implement active feedback control in a Q-switched diode-pumped Nd:YVO₄ laser by monitoring the fluorescence intensity from the laser crystal. When the initial inversion level indicated by the detected fluorescence has reached a predetermined value, Q-switching is initiated. The Q-switched pulse energy is stabler with our feedback scheme based on the fluorescence intensity than that with the conventional Q-switching when pumping source is not stable.     

Theoretical analysis of S1-state lifetime measurements of dyes with picosecond laser pulses

Various methods for the determination of the S₁-state lifetime of dye solutions (laser dyes and modelocking dyes) are analysed. A general model of interaction of laser light with dye molecules is presented and reduced to a dye energy level scheme of six levels. Fluorescence emission, light amplification and absorption recovery techniques are investigated theoretically and their limitations revealed. The determination of the S₁-state lifetime of saturable absorbers by single picosecond pulse bleaching experiments is very thoroughly discussed. The influence of various laser and dye parameters on the bleaching experiments are analysed numerically. The results are compared with isotropic steady state two- and three-level dye models.     

Fluorescence lifetime imaging in biosciences: technologies and applications

The biosciences require the development of methods that allow a non-invasive and rapid investigation of biological systems. In this aspect, high-end imaging techniques allow intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e., wide-field, confocal one-photon and two-photon microscopy, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e., steady-state techniques based on the analysis of the total fluorescence signal originating from the sample, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macroscopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at the molecular level. The intrinsic disadvantages of steady-state techniques are countered by using time-resolved techniques. Among these fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosciences, especially for fast intravital studies are discussed in this work.     

Fluorescence lifetime imaging in biosciences: technologies and applications

The biosciences require the development of methods that allow a non-invasive and rapid investigation of biological systems. In this aspect, high-end imaging techniques allow intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e., wide-field, confocal one-photon and two-photon microscopy, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e., steady-state techniques based on the analysis of the total fluorescence signal originating from the sample, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macroscopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at the molecular level. The intrinsic disadvantages of steady-state techniques are countered by using time-resolved techniques. Among these fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosciences, especially for fast intravital studies are discussed in this work.      

激光与二维多势阱系统相互作用下的谐波发射

采用二维渐近边界条件和辛算法数值求解了激光和二维多势阱系统互作用的二维含时Schrödinger方程的无穷空间初值问题。计算了二维多势阱系统分别在线偏振激光和圆偏振激光作用下的谐波发射,得到不同模型发射谐波谱的特点,说明多势阱环境有利于高次谐波的发射。     

Broadly tunable UV-blue picosecond pulsed laser and its application for biological imaging

We have achieved a rapid and random wavelength tuned picosecond pulsed laser and a widely tunable UV-blue picosecond pulsed laser by using the intracavity second harmonic generation of the laser. The tuning range was from 384 to 434 nm with picosecond pulse oscillation. In addition, we demonstrated biological imaging using a fluorescent protein excited by the widely tunable UV-blue picosecond pulsed laser. We found that the laser is suitable for biological imaging using the fluorescent protein as an excitation light source without damages.