基于SiPM和TCMPC的时间分辨拉曼散射测量技术研究

报道了一种基于硅光电信增管(SiPM)的时间相关多光子计数(TCMPC)技术并将其应用于时间分辨拉曼散射测量。相比于常规基于光电倍增管(PMT)或单光子雪崩二极管(SPAD)的时间相关单光子(TCSPC)技术,由于SiPM可以分辨信号脉冲的具体光子数,基于SiPM的TCMPC技术消除了信号脉冲包含的光子数必须小于等于1的限制,光子计数效率提高了10倍以上,大大节省了测量时间。此外,多光子测量比单光子测量能够得到更好的时间分辨率,时间分辨拉曼散射系统的仪器响应函数(IRF)从单光子81.4 ps缩短至双光子59.7 ps,因而可以用更窄的时间门限抑制荧光本底等噪声对拉曼散射测量的影响。使用TCMPC技术测量CCl₄在0.5和1.5 p.e.两个不同光子数阈值的拉曼峰的峰本比,后者较高的光子数阈值能进一步降低SiPM暗计数噪声的影响,增加了拉曼信号测量的信噪比,测量得到的CCl₄ 459 cm-1拉曼峰的峰本比是前者的6.4倍。将所述新的拉曼散射测量技术与基于PMT和锁相放大器(LIA)的传统拉曼散射测量技术进行了比较研究,前者由于可以使用仅有数十皮秒的测量门限,可以有效抑制荧光、环境杂散光和SiPM暗计数等噪声的影响,所得光谱具有更好的峰本比,测得CCl₄的459 cm-1拉曼峰和Si的一阶拉曼峰的峰本比分别是后者的3.9倍和5.5倍。     

Laser wavelength dependence of background fluorescence in Raman spectroscopic analysis of synovial fluid from symptomatic joints

Gout is a disease process where the nucleation and growth of crystals in the synovial fluid of joints elicit painful arthritis‐like symptoms. Raman spectroscopy is evolving as a potential diagnostic tool in identifying such crystals; however, attainment of sufficient Raman signal while overcoming the background fluorescence remains as a major challenge. The current study focused on assessing whether excitation in 532–700 nm range will provide greater signal intensity than the standard 785 nm while not being impeded by background fluorescence. We characterized the fluorescence spectra, absorption spectra and Raman spectra of synovial fluid from patients who presented ‘gout‐like symptoms’ (symptomatic) and controls (asymptomatic). A digestion and filtration method was developed to isolate crystals from synovial fluid while reducing the organic burden. Spectral profile and photobleaching dynamics during Raman spectroscopy were observed under an excitation wavelength range spanning 532 to 785 nm. Absorbance and fluorescence profiles indicated the digestion and filtration worked effectively to extract crystals from symptomatic synovial fluid without introducing additional fluorescence. Raman spectral analyses at 532 nm, 660 nm, 690 nm and 785 nm indicated that both asymptomatic and symptomatic samples had significant levels of fluorescence at excitation wavelengths below 700 nm, which either hindered the collection of Raman signal or necessitated prolonged durations of photobleaching. Raman‐based diagnostics were more feasible at the longest excitation wavelength of 785 nm without employing photobleaching. This study further demonstrated that a near‐infrared (NIR) OEM‐based lower‐cost Raman system at 785 nm excitation has sufficient sensitivity to identify crystals isolated from the synovial fluid. In conclusion, while lower excitation wavelengths provide greater signal, the fluorescence necessitates NIR wavelengths for Raman analysis of crystal species observed in synovial aspirates. Copyright © 2013 John Wiley & Sons, Ltd.     

A Fourier transform Raman spectrometer with visible laser excitation

We present the development and performance of a Fourier transformation (FT)‐based Raman spectrometer working with visible laser (532 nm) excitation. It is generally thought that FT‐Raman spectrometers are not viable in the visible range where shot noise limits the detector performance and therein they are outperformed by grating based, dispersive ones. We show that contrary to this common belief, the recent advances of high‐performance interference filters makes the FT‐Raman design a valid alternative to dispersive Raman spectrometers for samples which do not luminesce. We critically compare the performance of our spectrometer to two dispersive ones: a home‐built single channel and a state‐of‐the‐art charge coupled device‐based instruments. We demonstrate a similar or even better sensitivity than the charge coupled device‐based dispersive spectrometer particularly when the laser power density is considered. The instrument possesses all the known advantages of the FT principle of spectral accuracy, high throughput, and economic design. We also discuss the general considerations, which helps the community reassess the utility of the different Raman spectrometer designs. Copyright © 2015 John Wiley & Sons, Ltd.     

Temporal filtering with fast ICCD cameras in Raman studies

A common problem when applying Raman scattering in applied research is spectral interference from laser‐induced fluorescence. Extensive work has been invested in developing spectral and polarization filters as well as modulation schemes to refine spontaneous Raman signals. This current work, however, focuses on utilizing the temporal domain using a picosecond laser system and ICCD cameras with relatively short decay of the camera gate to prevent the fluorescence tail from being captured in Raman experiments. Further, the approach of using an ICCD camera to perform temporal filtering is compared to earlier proposed detection schemes using streak cameras or Kerr gates. The temporal‐filtering scheme is evaluated in a spectroscopic investigation where a background subtraction algorithm is presented. The temporal‐filtering scheme was also evaluated for Raman imaging of a levitated water droplet surrounded by fluorescing toluene vapor. Furthermore, the temporal‐filter detection scheme was simulated in order to provide straight forward evaluation tools to estimate the potential of performing temporal filtering with a laser/camera system considering: laser‐pulse duration, time jitter, camera‐gate characteristics, gate delay times, fluorescence lifetimes, and relative signal strength between the Raman and fluorescence signal. The fluorescence signal was modeled with a closed two‐level system, and the simulated results were compared to results from an investigation of the rising slope of toluene fluorescence. These evaluation tools and experimental investigations may serve as guidelines for planning and performing Raman measurements in situations where traditional filter‐rejection schemes are insufficient. Copyright © 2013 John Wiley & Sons, Ltd.     

High-resolution study of x-ray resonant Raman scattering at the K edge of silicon

We report on the first high-resolution measurements of the K x-ray resonant Raman scattering (RRS) in Si. The measured x-ray RRS spectra, interpreted using the Kramers-Heisenberg approach, revealed spectral features corresponding to electronic excitations to the conduction and valence bands in silicon. The total cross sections for the x-ray RRS at the 1s absorption edge and the 1s-3p excitation were derived. The Kramers-Heisenberg formalism was found to reproduce quite well the x-ray RRS spectra, which is of prime importance for applications of the total-reflection x-ray fluorescence technique.     

Non‐invasive probing of pharmaceutical capsules using transmission Raman spectroscopy

We demonstrate how transmission Raman geometry can be effectively used for non‐invasive probing of the content of pharmaceutical capsules. This approach is particularly beneficial in situations where the conventional Raman backscattering method is hampered or fails because of excessive surface Raman or fluorescence signals emanating from the capsule shell material, which pollute the much weaker subsurface Raman signals with undesired noise. It is demonstrated that such interfering signals can be effectively suppressed by the transmission geometry. The ability to avoid surface fluorescence and Raman signals in conjunction with the superior, bulk‐probing properties of the transmission Raman geometry provides an analytical technique ideally suited for fast on‐line process control monitoring applications in pharmaceutical industry where rapid, chemically specific bulk analysis is required. Copyright © 2007 John Wiley & Sons, Ltd.     

荧光褪色效应与拉曼光谱基线干扰消除

荧光是直接测定的拉曼光谱中背景的最主要来源,需要采用真实、准确的方法消除,以得到纯净的拉曼响应。基线拟合消除和查找荧光贡献扣除是解决背景问题的两条思路,目前多采用基线拟合方法,其优点是满足用户“视觉”要求,无需额外硬件,但并非机理或实质上的解释,因而难以保证数据的真实性与合理性;查找荧光的方法,更为真实,但是目前提出的方法,需要增加光源等额外设计和成本。另外,在实验方法上,也有采用消荧光剂和长时间照射漂白的,存在操作繁琐、效率低等不足。利用稳定体系中拉曼和荧光的时间差异解决体系中荧光问题。在微小的时间段内,例如几个毫秒,激发光不会导致体系性质发生显著变化,荧光具有寿命周期,会随激发时间延长强度下降的“褪色”,“褪色”的强度差异可以被认为是整体荧光的一个微元;与此同时,由于体系组成未发生显著变化,拉曼光对于短时间照射可以保持稳定。利用此差异可以区分出混合信号中的荧光和拉曼光。根据该原理,提出了荧光褪色差分法(FBDA),实现拉曼光谱的背景校正。方法的主要步骤：测量微小时刻内的多张直接拉曼光谱,求取系列光谱的差分,对差分值作高频滤波降噪,可获得荧光强度微元;然后,多个荧光微元平均归一化后,得到荧光强度单元。以拉曼光谱2 000～2 500 cm-1的静默区,即通常不会出现拉曼信号的频段为基准,对荧光单元作逆差分,逆差分累计值与原始光谱在此频段一致时,得到整体荧光响应;最终,从原始光谱中扣除荧光成分,完成背景扣除和基线校正。以盐酸二甲双胍片的拉曼测量为例,说明和讲解了所提出的原理和方法,验证方法的有效性。与目前效果较好的基线校正方法(不对称最小二乘和自适应迭代再加权惩罚偏最小二乘)进行了对比,表明FBDA方法更为客观真实,FBDA的另一个优势是不需要额外的设计和成本,所有数据都是在现有设备直接采集和完成。需要说明的是,微小时刻光谱差异的要求,可以确保FBDA光谱实时性,长时间的光谱差异,将会影响结果的准确性;另外,对于光化学反应体系和其他非荧光引起的复杂背景,FBDA的适用性有待改善。     

Implementation of a novel low‐noise InGaAs detector enabling rapid near‐infrared multichannel Raman spectroscopy of pigmented biological samples

Pigmented tissues are inaccessible to Raman spectroscopy using visible laser light because of the high level of laser‐induced tissue fluorescence. The fluorescence contribution to the acquired Raman signal can be reduced by using an excitation wavelength in the near infrared range around 1000 nm. This will shift the Raman spectrum above 1100 nm, which is the principal upper detection limit for silicon‐based CCD detectors. For wavelengths above 1100 nm indium gallium arsenide detectors can be used. However, InGaAs detectors have not yet demonstrated satisfactory noise level characteristics for demanding Raman applications. We have tested and implemented for the first time a novel sensitive InGaAs imaging camera with extremely low readout noise for multichannel Raman spectroscopy in the short‐wave infrared (SWIR) region. The effective readout noise of two electrons is comparable to that of high quality CCDs and two orders of magnitude lower than that of other commercially available InGaAs detector arrays. With an in‐house built Raman system we demonstrate detection of shot‐noise limited high quality Raman spectra of pigmented samples in the high wavenumber region, whereas a more traditional excitation laser wavelength (671 nm) could not generate a useful Raman signal because of high fluorescence. Our Raman instrument makes it possible to substantially decrease fluorescence background and to obtain high quality Raman spectra from pigmented biological samples in integration times well below 20 s. Copyright © 2015 John Wiley & Sons, Ltd.     

Temperature-insensitive strain sensor based on four-wave mixing using Raman fiber Bragg grating laser sensor with cooperative Rayleigh scattering

A temperature-insensitive strain sensor based on Four-Wave Mixing (FWM) using two Raman fiber Bragg grating (FBG) lasers with cooperative Rayleigh scattering is proposed. Two FBG were used to form two linear cavities laser sensors based on Raman amplification combined with cooperative Rayleigh scattering. Due to the very low dispersion coefficient of the fiber, it is possible to obtain the FWM using the two lasers. This configuration allows the operation as a temperature-insensitive strain sensor where both sensors have the same sensitivity to temperature but only one of the FBG laser is sensitive to strain. The difference between the wavelengths of the signal sensor and the converted signal presents a strain coefficient sensitivity of 2?pm/??? with insensitivity to temperature. The FWM efficiency is also dependent on the applied strain, but it is temperature independent, presenting a maximum sensibility of 0.01?dB/???.     

双通道原子相干性转移效率与拉曼单光子失谐的关系研究

研究了原子相干性在双通道间的转移效率与拉曼单光子失谐的关系。在冷原子四能级Tripod型系统中,首先利用EIT动力学过程将光信号存储在原子的一个记忆通道上。然后通过拉曼双光子过程,我们进行了原子记忆在两个通道间相干转移的研究,结果表明,在一定的拉曼光强下,原子相干性在双通道间的转移效率随着拉曼单光子失谐发生变化,最大的转移效率可达到94%。     

The use of diode array detectors in conjunction with continuous wave gas lasers for anti-Stokes Raman spectroscopy

The Raman spectrum of any molecule consists of two mirror-image signals, th e Stokes and anti-Stokes Raman spectra. In most cases, unless highly specific sampling conditions are used, the anti-Stokes signal is much weaker than that of the Stokes. The recent application of intensified diode array detectors to Raman spectroscopy has produced a marked increase in the sensitivity of the technique which makes a study of the anti-Stokes spectrum potentially more rewarding than it has been to date. The present study has shown that, although of limited use for general purposes, there are some specific instances where the anti-Stokes spectrum can be of considerable practical use. Such applications are to extend the operating range of the Raman spectrometer, to study photodegradable samples and for the analysis of samples which exhibit strong fluorescence.     

Signal-to-noise ratio of Raman signal measured by multichannel detectors

Raman spectroscopy has been widely used to characterize the physical properties of two-dimensional materials(2 DMs). The signal-to-noise ratio(SNR or S/N ratio) of Raman signal usually serves as an important indicator to evaluate the instrumental performance rather than Raman intensity itself. Multichannel detectors with outstanding sensitivity, rapid acquisition speed and low noise level have been widely equipped in Raman instruments for the measurement of Raman signal. In this mini-review, we first introduce the recent advances of Raman spectroscopy of 2 DMs. Then we take the most commonly used CCD detector and IGA array detector as examples to overview the various noise sources in Raman measurements and analyze their potential influences on SNR of Raman signal in experiments. This overview can contribute to a better understanding on the SNR of Raman signal and the performance of multichannel detector for numerous researchers and instrumental design for industry, as well as offer practical strategies for improving spectral quality in routine measurement.     

Gemstones and noble metals adorning the sceptre of the Faculty of Science of Charles University in Prague: integrated analysis by Raman and XRF handheld instruments

A commonly marketed handheld Raman spectrometer showed excellent possibilities in being used as a key instrument for unambiguous identification of gemstones mounted in the sceptre of the Faculty of Science of Charles University in Prague from the mid‐20s of the 20th century. Numerous SiO₂ forms including chalcedonies intermixed with moganites (e.g. moss agates and carnelians), amethysts, citrines as well as garnets (pyrope–almandines) were identified. The estimation of the garnet type was based on obtained Raman parameters. The individual minerals constituting the lapis lazulis could not be discerned because of very high fluorescence they exhibited in the fingerprint region of the Raman spectrum, nevertheless the positions of the observed peaks in the 1000–2000 cm^–1 area were in very good agreement with the values of commercially available lapis lazuli pigment. The noble metals of the sceptre were studied by means of X‐ray fluorescence analysis, which confirmed the presence of silver alloys and gilding. The comparison of silver alloys' semiquantitative analysis with the expected fineness, denoted by the hallmarks, indicated silvering. This has been later confirmed by the newly discovered restoration documentation. Portable handheld Raman and X‐ray fluorescence instruments represent an ideal tool for studying historical artefacts, where an in situ investigation in museums or similar sites is obligatory. Copyright © 2012 John Wiley & Sons, Ltd.     

Study of tryptophan assisted synthesis of gold nanoparticles by combining UV–Vis, fluorescence, and SERS spectroscopy

We developed a rapid and non-toxic method for the preparation of colloidal gold nanoparticles (GNPs) by using tryptophan (Trp) as reducing/stabilizing agent. We show that the temperature has a major influence on the kinetics of gold ion reduction and the crystal growth, higher temperatures favoring the synthesis of anisotropic nanoparticles (triangles and hexagons). The as-synthesized nanostructures were characterized by UV–Vis absorption spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD), fluorescence, and surface-enhanced Raman scattering (SERS) spectroscopy. The UV–Vis measurements confirmed that temperature is a critical factor in the synthesis process, having a major effect on the shape of the synthesized GNPs. Moreover, fluorescence spectroscopy was able to monitor the quenching of the Trp fluorescence during the in situ synthesis of GNPs. Using Trp as molecular analyte to evaluate the SERS efficiency of as-prepared GNPs at different temperatures, we demonstrated that the Raman enhancement of the synthesized gold nanoplates is higher than that of the gold spherical nanoparticles.     

基于叶片及冠层叶绿素参数的冬小麦籽粒蛋白质含量预测研究

小麦籽粒蛋白质含量是衡量小麦营养品质的重要指标,实现小麦品质快速的预测预报对于粮食收购部门和加工企业具有重要意义。研究基于作物叶绿素/氮素速测仪SPAD及Multiplex 3,获取冬小麦不同生育期叶片及冠层叶绿素参数,从小麦个体及群体参量两方面进行冬小麦收获期籽粒蛋白质含量及蛋白产量的预测研究。试验于2012年4—6月在国家精准农业研究示范基地开展,研究结果表明,冬小麦返青至灌浆初期,小麦冠层氮素密度与籽粒蛋白质含量的相关性优于叶片氮素含量与蛋白质含量的相关性,灌浆中期两者与籽粒蛋白质含量相关性差别不大;小麦叶片SPAD值与叶片氮素含量相关性总体优于其与冠层氮素密度的相关性,而叶绿素荧光参数SFR_G, SFR_R与冠层氮素密度的相关性优于其与叶片氮素含量的相关性;叶片SPAD与籽粒蛋白质含量的相关性在拔节期最弱,在灌浆中期最强,小麦冠层叶绿素荧光参数SFR_G, SFR_R与籽粒蛋白质含量相关性在返青至拔节期不显著,但孕穗期开始显著相关,在灌浆中期相关性最强且明显优于同期叶片SPAD与籽粒蛋白质含量的相关性;冬小麦籽粒蛋白产量与叶片SPAD值在小麦孕穗期至灌浆期显著相关,与SFR_G和SFR_R在小麦灌浆期显著相关;研究基于灌浆中期SPAD值及SFR_R值,构建了冬小麦籽粒蛋白质含量及籽粒蛋白产量的预测模型,其中,籽粒蛋白质含量预测模型复相关指数分别为0.426和0.497,模型标准误差分别为0.060%和0.055%,籽粒蛋白产量预测模型复相关指数分别为0.366和0.386,模型标准误差分别为125.367和123.454 kg·ha-1。研究表明,利用叶片SPAD值及冠层叶绿素荧光信息,在小麦收获前进行品质的快速预测是可行的。     

A robust method for automated background subtraction of tissue fluorescence

This paper introduces a new robust method for the removal of background tissue fluorescence from Raman spectra. Raman spectra consist of noise, fluorescence and Raman scattering. In order to extract the Raman scattering, both noise and background fluorescence must be removed, ideally without human intervention and preserving the original data. We describe the rationale behind our robust background subtraction method, determine the parameters of the method and validate it using a Raman phantom against other methods currently used. We also statistically compare the methods using the residual mean square (RMS) with a fluorescence‐to‐signal (F/S) ratio ranging from 0.1 to 1000. The method, ‘adaptive minmax’, chooses the subtraction method based on the F/S ratio. It uses multiple fits of different orders to maximize each polynomial fit. The results show that the adaptive minmax method was significantly better than any single polynomial fit across all F/S ratios. This method can be implemented as part of a modular automated real‐time diagnostic in vivo Raman system. Copyright © 2007 John Wiley & Sons, Ltd.     

Raman spectroscopy and microscopy of individual cells and cellular components

Raman spectroscopy provides the unique opportunity to nondestructively analyze chemical concentrations in individual cells on the submicrometer length scale without the need for optical labels. This enables the rapid assessment of cellular biochemistry inside living cells, and it allows for their continued analysis. Here, we review recent developments in the analysis of single cells, subcellular compartments, and chemical imaging based on Raman spectroscopy. Spontaneous Raman spectroscopy provides for the full spectral assessment of cellular biochemistry, while coherent Raman techniques, such as coherent anti‐Stokes Raman scattering is primarily used as an imaging tool comparable to confocal fluorescence microscopy. These techniques are complemented by surface‐enhanced Raman spectroscopy, which provides higher sensitivity and local specificity, and also extends the techniques to chemical indicators, i.e. pH sensing. We review the strengths and weaknesses of each technique, demonstrate some of their applications and discuss their potential for future research in cell biology and biomedicine.     

食品中三聚氰胺的光谱检测与分析技术研究进展

三聚氰胺是豆、乳类制品中的非法食品添加剂,曾作为蛋白质的廉价代替物被非法添加进奶粉等食品中,造成了严重的社会危害,极大地威胁人民生命财产安全。目前光谱技术已成为识别和定量检测非法食品添加剂的有效手段,为质量监管部门提供了可靠的研究方法和鉴定依据。光谱检测技术的时效性、无损性和准确性提高了食品中三聚氰胺的检测效率,促进了精准化、自动化食品质量检测的发展。近年来有大量研究围绕着三聚氰胺的光谱检测新技术,如开发新型增强底物或传感器,降低三聚氰胺的检测限,提高检测精度;开发更加便携的自动化光谱快检设备,降低检测成本,提高检测效率。这些光谱技术各具优势,但很难形成标准化、统一化的检测规范,使得各种光谱检测技术仅仅停留在试验阶段,无法应用于实战。另一方面,随着人工智能与模式识别技术的发展,光谱数据分析方法在近年来也有着长足的进步,各种光谱预处理和数据建模方法被不断提出,大大提高了光谱检测技术的灵敏性和稳定性。综述了近十年光谱技术（拉曼光谱、近红外光谱、荧光光谱、光谱成像等）在三聚氰胺检测中的应用现状,总结了不同仪器检测限、定量范围和样品前处理方法;分析了各种光谱预处理和光谱数据建模方法在不同光谱数据中的适用性,归纳出这些方法的优劣与适配的仪器,并对其应用前景和研究趋势进行了展望。     

Wide area illumination Raman scheme for simple and nondestructive discrimination of seawater cultured pearls

Raman spectroscopy, along with discriminant partial least squares (PLS), was successfully used to discriminate among three different groups of cultured pearls (fresh water, Akoya and South seawater). The discrimination between Akoya and South seawater pearls using XRF (X‐ray fluorescence), one of the most frequently adopted analytical methods in pearl analysis, has been especially difficult owing to their similar mineral compositions. The selective Raman features helped in effectively discriminating between these two pearl groups. The difference in the intensities of the CaCO₃ bands of Akoya and South seawater pearls provided a valuable clue. Along with the selective Raman feature, a reproducible Raman spectral collection achieved using a wide area illumination (WAI) scheme played an important role in the determination of the pearl groups, although the pearls were hard‐surfaced, round, solid samples of different sizes and surface shapes. Unwanted spectral variation originating from sensitivity to sample placement relative to the focal plane and from unsuccessful sample representation due to the probing of a localized area, factors that could possibly deteriorate Raman reproducibility, were substantially lessened using the WAI scheme. ATR (attenuated total reflection) IR spectroscopy requiring direct contact with the pearl could be inadequate for discrimination or classification where large numbers of repeating and reproducible measurements are required. Copyright © 2009 John Wiley & Sons, Ltd.