Experiments and quantitative analysis of frequency division multiplexing confocal fluorescence microscopy with UV excitation

In this paper, we experimentally demonstrated a two-channel frequency division multiplexing confocal fluorescence microscopy system using a UV laser as the excitation source. In our two-channel frequency division multiplexing confocal fluorescence system, the incoming laser beam was divided into two beams and each beam was modulated with an individual carrier frequency. These two laser beams were then spatially combined with a small angle and focused into two diffraction-limited spots on the targeted cell (rat neural cell) surface to generate fluorescent signal. As a result, the fluorescent signals from two spots of the rat neural cell surface can be demodulated and distinguished during data processing. Furthermore, a quantitative analysis on the cross-talk among different frequencies was provided as well. The experimental results confirm that the two-channel frequency division multiplexing confocal fluorescence technology can not only maintain the high spatial resolution, but also realize the multiple points detection simultaneously with high temporal resolution (within millisecond level range), which benefits the dynamic studies of living biological cells.     

大动态范围外延电阻淬灭型硅光电倍增器

外延电阻淬灭型硅光电倍增器(EQR SiPM)的特点是利用硅衬底外延层来制作器件淬灭电阻。为了进一步提高大动态范围EQR SiPM的光子探测效率,并且解决填充因子较低和增益较小等问题,在前期研究工作的基础上研制出微单元尺寸分别为15μm和7μm的EQR SiPM,有源区面积均是1 mm×1 mm。通过改变EQR SiPM的微单元尺寸优化填充因子,有效提高了探测效率与增益;其微单元密度分别是4400个/mm 2和23200个/mm 2,依然保持着较大的动态范围。室温条件下(20℃),工作在5 V过偏压的EQR SiPM至少可分辨13个光电子;15μm和7μm EQR SiPM的增益分别为5.1×105和1.1×105,在400 nm波长下的峰值光探测效率分别达到40%和34%。     

光纤Bragg光栅阵列的光时域反射探测技术研究

本文研究了采用光时域反射技术对串联光栅阵列的反射功率进行探测的新方法。在实验研究的基础上,对高反射率光栅在光时域反射测试系统中的“假峰”和“谐振峰”现象的产生根源进行了分析,并提出了适合于光时域反射测试系统的光栅反射率的具体要求。此外对光时域反射测试技术的空间分辨率、复用数量进行了分析,给出了综合考虑多种因素的光栅传感阵列的布置方案。光时域反射探测技术是一种新颖的光栅阵列测试技术,与传统的波长测试系统相比,可从时分复用角度大大增加复用数量。     

Wavelength division scanning for two-photon excitation fluorescence imaging

We investigate wavelength division scanning for two‐photon excitation fluorescence imaging. Two‐photon imaging using lateral wavelength division scanning is demonstrated. In addition, we theoretically analyse the spatial and temporal properties of a femtosecond laser beam focused by a Fresnel lens and investigate the feasibility of axial scanning using wavelength division.     

Magnetic resonance-coupled fluorescence tomography scanner for molecular imaging of tissue

A multichannel spectrally resolved optical tomography system to image molecular targets in small animals from within a clinical MRI is described. Long source/detector fibers operate in contact mode and couple light from the tissue surface in the magnet bore to 16 spectrometers, each containing two optical gratings optimized for the near infrared wavelength range. High sensitivity, cooled charge coupled devices connected to each spectrograph provide detection of the spectrally resolved signal, with exposure times that are automated for acquisition at each fiber. The design allows spectral fitting of the remission light, thereby separating the fluorescence signal from the nonspecific background, which improves the accuracy and sensitivity when imaging low fluorophore concentrations. Images of fluorescence yield are recovered using a nonlinear reconstruction approach based on the diffusion approximation of photon propagation in tissue. The tissue morphology derived from the MR images serves as an imaging template to guide the optical reconstruction algorithm. Sensitivity studies show that recovered values of indocyanine green fluorescence yield are linear to concentrations of 1 nM in a 70 mm diameter homogeneous phantom, and detection is feasible to near 10 pM. Phantom data also demonstrate imaging capabilities of imperfect fluorophore uptake in tissue volumes of clinically relevant sizes. A unique rodent MR coil provides optical fiber access for simultaneous optical and MR data acquisition of small animals. A pilot murine study using an orthotopic glioma tumor model demonstrates optical-MRI imaging of an epidermal growth factor receptor targeted fluorescent probe in vivo.     

Fiber laser based two-photon FRET measurement of calmodulin and mCherry-E(0)GFP proteins

The speed and accuracy of Förster resonance energy transfer (FRET) measurements can be improved by rapidly alternating excitation wavelengths between the donor and acceptor fluorophore. We demonstrate FRET efficiency measurements based on a fiber laser and photonic crystal fiber as the source for two‐photon excitation (TPE). This system offers the potential for rapid wavelength switching with the benefits of axial optical sectioning and improved penetration depth provided by TPE. Correction of FRET signals for cross excitation and cross emission was achieved by switching the excitation wavelength with an electrically controlled modulator. Measurement speed was primarily limited by integration times required to measure fluorescence. Using this system, we measured the FRET efficiency of calmodulin labeled with Alexa Fluor 488 and Texas Red dyes. In addition, we measured two‐photon induced FRET in an E⁰GFP‐mCherry protein construct. Results from one‐photon and two‐photon excitation are compared to validate the rapid wavelength switched two‐photon measurements. Microsc. Res. Tech. 75:837–843, 2012. © 2011 Wiley Periodicals, Inc.     

Flexible lock-in detection system based on synchronized computer plug-in boards applied in sensitive gas spectroscopy

We present a flexible and compact, digital, lock-in detection system and its use in high-resolution tunable diode laser spectroscopy. The system involves coherent sampling, and is based on the synchronization of two data acquisition cards running on a single standard computer. A software-controlled arbitrary waveform generator is used for laser modulation, and a four-channel analog/digital board records detector signals. Gas spectroscopy is performed in the wavelength modulation regime. The coherently detected signal is averaged a selected number of times before it is stored or analyzed by software-based, lock-in techniques. Multiple harmonics of the modulation signal (1f, 2f, 3f, 4f, etc.) are available in each single data set. The sensitivity is of the order of 10(-5), being limited by interference fringes in the measurement setup. The capabilities of the system are demonstrated by measurements of molecular oxygen in ambient air, as well as dispersed gas in scattering materials, such as plants and human tissue.     

Two‐photon microscopy of deep intravital tissues and its merits in clinical research

Multiphoton excitation laser scanning microscopy, relying on the simultaneous absorption of two or more photons by a molecule, is one of the most exciting recent developments in biomedical imaging. Thanks to its superior imaging capability of deeper tissue penetration and efficient light detection, this system becomes more and more an inspiring tool for intravital bulk tissue imaging. Two‐photon excitation microscopy including 2‐photon fluorescence and second harmonic generated signal microscopy is the most common multiphoton microscopic application. In the present review we take diverse ocular tissues as intravital samples to demonstrate the advantages of this approach. Experiments with registration of intracellular 2‐photon fluorescence and extracellular collagen second harmonic generated signal microscopy in native ocular tissues are focused. Data show that the in‐tandem combination of 2‐photon fluorescence and second harmonic generated signal microscopy as two‐modality microscopy allows for in situ co‐localization imaging of various microstructural components in the whole‐mount deep intravital tissues. New applications and recent developments of this high technology in clinical studies such as 2‐photon‐controlled drug release, in vivo drug screening and administration in skin and kidney, as well as its uses in tumourous tissues such as melanoma and glioma, in diseased lung, brain and heart are additionally reviewed. Intrinsic emission two‐modal 2‐photon microscopy/tomography, acting as an efficient and sensitive non‐injurious imaging approach featured by high contrast and subcellular spatial resolution, has been proved to be a promising tool for intravital deep tissue imaging and clinical studies. Given the level of its performance, we believe that the non‐linear optical imaging technique has tremendous potentials to find more applications in biomedical fundamental and clinical research in the near future.     

Compact non‐contact total emission detection for in vivo multiphoton excitation microscopy

We describe a compact, non‐contact design for a total emission detection (c‐TED) system for intra‐vital multiphoton imaging. To conform to a standard upright two‐photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. The non‐contact design of this device allows for maximal light collection without disrupting the physiology of the specimen being examined. Tests were conducted on exposed tissues in live animals to examine the emission collection enhancement of the c‐TED device compared to heavily optimized objective‐based emission collection. The best light collection enhancement was seen from murine fat (5×–2× gains as a function of depth), whereas murine skeletal muscle and rat kidney showed gains of over two and just under twofold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a twofold gain throughout the entire volume of an intact embryo (approximately 150 μm deep). Direct measurement of bleaching rates confirmed that the lower laser powers, enabled by greater light collection efficiency, yielded reduced photobleaching in vivo. The potential benefits of increased light collection in terms of speed of imaging and reduced photo‐damage, as well as the applicability of this device to other multiphoton imaging methods is discussed.     

CWDM技术在光电组合汇流环中的应用

设计了一种新型的可多路光信号同时传输的光电组合汇流环系统.该汇流环系统中光信号传输主要采用CWDM(粗波分复用)技术结合单芯光滑环来完成,具有高速率、多信道、扩展性强等特点.经过实验验证,该光纤汇流环传输系统可在各种军标要求的环境下稳定地传输多路光信号,并且信号稳定、可靠性高,在未来的雷达信号传输中具有广泛应用.     

High Throughput Wear Debris Detection in Lubricants Using a Resonance Frequency Division Multiplexed Sensor

With a long time goal of detecting signs of potential machine failure, we demonstrate a proof-of-principle multiplexed, multichannel, inductive pulse sensor based on resonant frequency division multiplexing for high throughput detection of micro-scale metallic debris in lubricants. In the four-channel sensor, each sensing coil is connected to a specific external capacitance to form a parallel LC circuit that has a unique resonant frequency. Only one combined sinusoidal excitation signal consisting of four frequencies components that are close to the 4 sensing channels’ resonant frequencies was applied to the sensor, and only one combined voltage response was measured. Because each sensing channel exhibited a peak amplitude at its resonant frequency, the signals for each individual channel were recovered from the combined response by taking the spectrum components at each resonant frequency with an improved signal-to-noise ratio. Inductance change for each channel was then calculated from signals of individual channels. Testing results show that the use of resonant frequency division multiplexing allows simultaneous detection of debris in lubricants using only one set of detection electronics; for the four-channel sensor, there is a 300 % increase in throughput. The resonant frequency division multiplexing concept can be potentially applied to a multichannel oil debris sensor with a large number of sensing channels to achieve a very high throughput, which is necessary for online health monitoring of rotating and reciprocal mechanical components.     

Frequency multiplexed superconducting quantum interference device readout of large bolometer arrays for cosmic microwave background measurements

A technological milestone for experiments employing transition edge sensor bolometers operating at sub-Kelvin temperature is the deployment of detector arrays with 100s-1000s of bolometers. One key technology for such arrays is readout multiplexing: the ability to read out many sensors simultaneously on the same set of wires. This paper describes a frequency-domain multiplexed readout system which has been developed for and deployed on the APEX-SZ and South Pole Telescope millimeter wavelength receivers. In this system, the detector array is divided into modules of seven detectors, and each bolometer within the module is biased with a unique ～MHz sinusoidal carrier such that the individual bolometer signals are well separated in frequency space. The currents from all bolometers in a module are summed together and pre-amplified with superconducting quantum interference devices operating at 4 K. Room temperature electronics demodulate the carriers to recover the bolometer signals, which are digitized separately and stored to disk. This readout system contributes little noise relative to the detectors themselves, is remarkably insensitive to unwanted microphonic excitations, and provides a technology pathway to multiplexing larger numbers of sensors.     

基于盖格-雪崩光电二极管的光子计数成像

考虑用传统的成像技术检测光生电荷信号时易受模数转换噪声干扰,本文提出了一种基于单光子灵敏雪崩光电二极管(GM-APD)的光子计数成像方法。该方法以单光子灵敏GM-APD作为探测单元,以全数字化方式实现微弱光学信号的有效检测。建立了GM-APD光子流响应模型,利用马尔科夫更新过程分析了光子探测盲区对GM-APD瞬态响应的影响,得到了GM-APD输出的数字脉冲频率与光子流密度之间的关联表达式。搭建了二维扫描成像实验验证装置,通过实验得到了不同密度光子流条件下的光子计数图像。采用标准化互信息量分析得到,在光子流密度为3.0×104count/s的条件下,该实验装置仍然可以实现可视成像;当光子流密度达到2.7×105 count/s时,标准化互信息量大于0.5;实现了在低光子流密度时采用光子计数方式对目标物体的有效成像。     

基于信息融合技术的交通量检测算法研究

针对智能交通系统中交通流数据采集及处理实时性和准确性的要求,设计了基于多传感器信息融合技术的交通量检测系统。首先构造了基于时间和空间特性的两级融合结构;然后分别设计了空间信息融合算法和时间信息融合算法,在分析融合算法特点及使用的基础上,对算法进行优化;最后对系统进行仿真实验。经过实验验证,该系统实时性强、准确性高,满足智能交通系统的需要。     

压缩传感用于极弱光计数成像

为解决灵敏度达到单光子水平的面阵探测器件其单位像素上灵敏度有限和测量数多等问题,研制了具有极高灵敏度的成像系统来实现欠采样的极弱光成像探测。该成像系统基于光子计数成像技术和压缩感知理论,利用数字微镜器件(DMD)完成随机空间光调制,通过单光子点探测器收集光子,以计数形式记录下光强值。然后,利用算法重建出极弱光照明下的图像。文中设计了相关实验,研究了测量数、光强极弱程度和测量时间对成像质量的影响。最后,引入了图像质量评价标准和系统信噪比,分析对比了实验数据。结果表明,当测量数高于信号总维度的19.5%时,系统能完美成像,信噪比可低至2.843 8dB,DMD单位像素上的平均光子数可低于1.106count/s,成像的关键在于信号的波动大于噪声的波动。该成像系统基本满足了极弱光成像探测在光强、灵敏度和采样数等方面的要求。     

Ultrafast photon counting applied to resonant scanning STED microscopy

To take full advantage of fast resonant scanning in super‐resolution stimulated emission depletion (STED) microscopy, we have developed an ultrafast photon counting system based on a multigiga sample per second analogue‐to‐digital conversion chip that delivers an unprecedented 450 MHz pixel clock (2.2 ns pixel dwell time in each scan). The system achieves a large field of view (～50 × 50 μm) with fast scanning that reduces photobleaching, and advances the time‐gated continuous wave STED technology to the usage of resonant scanning with hardware‐based time‐gating. The assembled system provides superb signal‐to‐noise ratio and highly linear quantification of light that result in superior image quality. Also, the system design allows great flexibility in processing photon signals to further improve the dynamic range. In conclusion, we have constructed a frontier photon counting image acquisition system with ultrafast readout rate, excellent counting linearity, and with the capacity of realizing resonant‐scanning continuous wave STED microscopy with online time‐gated detection.     

High-time-resolution pump-probe system with broadband detection for the study of time-domain vibrational dynamics

We present an ultrafast transient absorption spectroscopy system in the visible combining high-sensitivity broadband detection with extreme temporal resolution. The instrument is based on an ultrabroadband sub-10 fs optical parametric amplifier coupled to an optical multichannel analyzer with fast electronics, enabling single-shot detection at 1 kHz repetition rate. For a given pump-probe delay tau, we achieve a differential transmission (DeltaTT) sensitivity of the order of 10(-4) over the lambda(pr)=490-720 nm probe wavelength range by averaging over 1000 shots, allowing the acquisition of complete two-dimensional DeltaTT (lambda(pr),Tau) maps within a few minute measurement time. We present application examples highlighting the capability of this instrument to observe ultrafast dynamical processes, follow impulsively excited vibrational motions with frequency as high as 3000 cm(-1) (11 fs period), and determine the probe wavelength dependence of amplitude and phase of the oscillations.     

用于光纤光栅传感的低成本高分辨率多通道波长解调仪

本文报道一种用于光纤光栅传感的低成本高分辨率多通道波长解调仪。基于薄膜滤波技术的密集波分复用模块已被广泛用于光纤通信系统中，在本文中，采用已商品化的薄膜密集波分复用模块同时完成了以波分复用方式连接的多个光纤光栅传感器的信号分离与波长解调功能，研究了一种结构简单、低成本、高分辨率的光纤光栅传感系统。该传感系统的特点是可用于多通道动态测量。本文将具体介绍一种旧通道传感系统，该系统在3kHz频率下的动态应变的分辨率优于1nε√Hz。     

Multispectral fluorescence lifetime imaging by TCSPC

We present a fluorescence lifetime imaging technique with simultaneous spectral and temporal resolution. The technique is fully compatible with the commonly used multiphoton microscopes and nondescanned (direct) detection. An image of the back-aperture of the microscope lens is projected on the input of a fiber bundle. The input of the fiber bundle is circular, and the output is flattened to match the input slit of a spectrograph. The spectrum at the output of the spectrograph is projected on a 16-anode PMT module. For each detected photon, the encoding logics of the PMT module deliver a timing pulse and the number of the PMT channel in which the photon was detected. The photons are accumulated by a multidimensional time-correlated single photon counting (TCSPC) process. The recording process builds up a four-dimensional photon distribution over the times of the photons in the excitation pulse period, the wavelengths of the photons, and the coordinates of the scan area. The method delivers a near-ideal counting efficiency and is capable of resolving double-exponential decay functions. We demonstrate the performance of the technique for autofluorescence imaging of tissue.     

Multiphoton microscopy in life sciences

Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three‐dimensional fluorescence imaging based on non‐resonant two‐photon or three‐photon fluorophor excitation requires light intensities in the range of MW cm^?2 to GW cm^?2, which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi‐gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non‐invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth‐resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non‐invasive fluorophore loading into single living plant cells. Non‐destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis‐like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two‐photon excitation process rather than a one‐photon or three‐photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two‐photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid‐induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm^?2 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non‐invasive NIR laser surgery within a living cell or within an organelle is therefore possible.