基于激光诱导叶绿素荧光寿命成像技术的植物荧光特性研究

针对植物荧光遥感探测中信号易受干扰的问题, 提出了一种用于评估植物生长状况及环境监测的荧光寿命成像技术. 采用凹透镜对355 nm波长的激光扩束, 再照射植物激发叶绿素荧光, 由增强型电荷耦合器件接收荧光信号. 采用时间分辨测量法, 连续用相同激光脉冲照射植物以激发相同的荧光信号, 同时不断改变激光脉冲触发探测器启动的延时时间, 从而能够得到完整的离散荧光信号分布图像. 对植物特定位置点产生的离散荧光信号进行拟合, 再运用一种改进型的迭代解卷积法可反演高精度的荧光寿命; 进而反演图像各点的荧光寿命以生成植物的荧光寿命分布图. 该方法所绘制的荧光寿命图比荧光强度图能更准确地反映植物内部的叶绿素含量, 并对活体植物叶绿素荧光寿命的物理特性进行了初步研究, 证明叶绿素荧光寿命与植物生理状态存在一定关联; 并且叶绿素荧光寿命与活体植物所处环境存在着复杂的关系. 未来将与生物物理学家们合作, 继续探寻叶绿素荧光寿命与植物生存环境的关系.     

Calcium imaging using fluorescence lifetimes and long-wavelength probes

We describe imaging of calcium concentrations using the long-wavelength Ca²⁺ indicators, Calcium Green, Orange, and Crimson. The lifetimes of these probes were measured using the frequency-domain method and were found to increase from 50% to severalfold in response to calcium. The two-dimensional images of the calcium concentration were obtained using a new apparatus for fluorescence lifetime imaging (FLIM). We also describe procedures to correct for the position-dependent frequency response of the gain-modulated image intensifier used in the FLIM apparatus. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra. Using the FLIM method, calcium imaging is possible using probes which display changes in lifetime in response to calcium. Consequently, calcium imaging is possible with excitation wavelengths ranging from 488 to as long as 620 nm, where autofluorescence and/or photochemical damage is minimal. These probes are also suitable for calcium measurements of single cells using lifetime-based flow cytometry.     

大视场声光可调谐滤波器成像光谱仪光学设计

为了在15视场角范围内获得工作谱段范围在400 nm~ 900 nm声光可调谐滤波器(AOTF)成像光谱仪系统的二维空间信息以及一维光谱信息,设计了一种应用于AOTF 成像光谱仪的光学系统。介绍AOTF的工作原理,根据AOTF 成像光谱仪总体方案,对前置系统及后置成像系统进行了设计。设计中前置系统采用倒置的伽利略望远镜结构,后置成像系统采用改进的库克三片式结构。最终完成了一个焦距为19.311 mm,F数为12.3,在34 lp/ mm 的空间频率下各视场调制传递函数( MTF) 均值大于0.5的光学系统。     

基于声光可调谐滤光器的显微光谱成像技术

为了解决传统声光可调谐滤光器(AOTF)成像模糊的缺点,设计了一种新的AOTF。该器件通过在传统的AOTF的出射孔后面放置一个自行设计的等边色散棱镜来实现对衍射光的色散展宽进行补偿,明显地提高了成像的对比度和空间分辨率。将此器件附加在传统光学显微镜上,获得了一种新型的光谱显微成像仪器。其光谱分辨率在575nm波长处为4.2nm、成像空间分辨率为2μm、图像采集速度为毫秒量级。为基于AOTF的光谱成像技术在生物医学等领域的更广泛的应用奠定了基础。     

眼底视网膜色素上皮层细胞脂褐素及氧化黑色素自体荧光寿命成像研究

利用一种基于时间相关单光子计数器的双光子激发荧光寿命显微成像技术，对猪眼底视网膜色素上皮层细胞内的脂褐素和氧化黑色素颗粒的空间分布及其荧光寿命特性进行了研究，尤其对于这些色素颗粒在光致氧化环境中的荧光寿命差异进行了分析.结果表明，利用荧光寿命测量能有效区分视网膜色素上皮层细胞中的多组分荧光团，利用荧光寿命的衰减参数可分辨正常及异常的荧光现象.该方法有望发展成为一种用于眼科临床诊断及病理学研究的高灵敏度的工具，对眼底细胞随年龄增长的衰老机理的研究具有重要的意义.     

眼底视网膜色素上皮层细胞脂褐素及氧化黑色素自体荧光寿命成像研究

利用一种基于时间相关单光子计数器的双光子激发荧光寿命显微成像技术,对猪眼底视网膜色素上皮层细胞内的脂褐素和氧化黑色素颗粒的空间分布及其荧光寿命特性进行了研究,尤其对于这些色素颗粒在光致氧化环境中的荧光寿命差异进行了分析.结果表明,利用荧光寿命测量能有效区分视网膜色素上皮层细胞中的多组分荧光团,利用荧光寿命的衰减参数可分辨正常及异常的荧光现象.该方法有望发展成为一种用于眼科临床诊断及病理学研究的高灵敏度的工具,对眼底细胞随年龄增长的衰老机理的研究具有重要的意义. 关键词： 双光子激发荧光 荧光寿命成像 视网膜色素上皮层     

荧光寿命显微成像技术及应用的最新研究进展

由于荧光寿命不受探针浓度、激发光强度和光漂白效应等因素影响,荧光寿命显微成像技术(fluorescence lifetime imaging microscopy, FLIM)在监测微环境变化、反映分子间相互作用方面具有高特异性、高灵敏度、可定量测量等优点,近年来已被广泛应用于生物医学等领域.然而,尽管FLIM的发明和发展已历经数十年时间,其在实际应用中仍然面临着许多挑战.例如,其成像分辨率受衍射极限限制,而其成像速度与成像质量和寿命测量精度则存在相互制约的关系.近几年来,相关硬件和软件的快速发展及其与其他光学技术的结合,极大地推动了FLIM技术及其应用的新发展.本文简要介绍了基于时域和频域的不同寿命探测方法的FLIM技术的基本原理及特点,在此基础上概述了该技术的最新研究进展,包括其成像性能的提升和在生物医学应用中的研究现状,详细阐述了近几年来研究者们通过硬件和软件算法的改进以及与自适应光学、超分辨成像技术等新型光学技术的结合来提升FLIM的成像速度、寿命测量精度、成像质量和空间分辨率等方面所做的努力,以及FLIM在生物医学基础研究、疾病诊断与治疗、纳米材料的生物医学研究等方面的应用,最后对其未来发展趋势进行了展望.     

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.      

Investigation of defect luminescence from multicrystalline Si wafer solar cells using X‐ray fluorescence and luminescence imaging

Multicrystalline silicon wafer solar cells reveal performance‐ reducing defects by luminescence. X‐ray fluorescence spectra are used to investigate the elemental constituents from regions of solar cells yielding reverse‐bias or sub‐bandgap luminescence from defects. It is found that a higher concentration of metals is present in regions yielding reverse‐bias electroluminescence than in regions yielding sub‐bandgap electroluminescence. This suggests, dislocations do not create strong breakdown currents in the absence of impurity precipitates.

     

基于时间相关单光子计数的荧光寿命成像技术

采用时域法中的时间相关单光子计数方法记录荧光寿命,时间相关单光子计数采用多波长通道同时记录荧光光子数,可以提高计数效率和信息量,还可以在稳态图像中分离不同荧光团,形成4维图像。并采用多光子激发技术,利用长波长光源发出的两个或多个光子可以激发出一个短波长的光子。多个光子必须几乎同时到达激发点, 才能提供被激发分子足够的能量以产生荧光。多光子激发波长较长, 生物组织对其散射减小,因而可以穿透到更深层的组织,从而提高荧光成像深度和空间分辨力,并减少对活体样品的损伤。     

Frequency-domain fluorescence lifetime imaging for endoscopic clinical cancer photodetection: Apparatus design and preliminary results

We describe a new fluorescence imaging device for clinical cancer photodetection in hollow organs in which the tumor/normal tissue contrast is derived from the fluorescence lifetime of endogenous or exogenous fluorochromes. This fluorescence lifetime contrast gives information about the physicochemical properties of the environment which are different between normal and certain diseased tissues. The excitation light from a CW laser is modulated in amplitude at a radio frequency by an electrooptical modulator and delivered by an optical fiber through an endoscope to the hollow organ. The image of the tissue collected by the endoscope is separated in two spectral windows, one being the backscattered excitation light and the other the fluorescence of the fluorochrome. Each image is then focused on the photocathode of image intensifiers (II) whose optical gain is modulated at the same frequency as the excitation intensity, resulting in homodyne phase-sensitive images. By acquiring stationary phase-sensitive frames at different phases between the excitation and the detection, it is possible to calculate in quasi-real time the apparent fluorescence lifetime of the corresponding tissue region for each pixel. A result obtained by investigating the endogenous fluorochromes present in the mucous membrane of an excised human bladder is presented to illustrate this method and most of the optical parameters which are of major importance for this photodetection modality have been evaluated.     

近红外光谱成像系统在液体安检中的应用

易燃易爆液体的实时、远程和动态探测对于保障公共安全具有重要意义,在公安、民航和海关等领域应用前景广阔。研制了基于液晶可调滤光片的近红外光谱成像系统,通过波长优选算法大幅降低了光谱通道数,有效提高了扫描成像速度和数据处理速度,可实现对低速运动的液体危险品的远程和实时检测。利用该系统开展了不同环境下对易燃易爆液体的检测实验,结果表明该系统对静态目标检测精度为100%,对速度小于0.2 m/s的运动目标检测精度高于95%。     

Spectral characteristics of luminescence of solutions of complex molecules under conditions of electronic excitation transfer to a saturable acceptor in the case of inhomogeneous broadening of electronic energy levels

Spectral characteristics of solutions of complex molecules under conditions of inhomogeneous broadening of energy levels are considered in the case when the nonlinear dependence of the population of molecular states on the excitation intensity is determined not by saturation of molecular levels but by exchange of the electronic excitation energy with the environment. Calculations have shown that the dependence of the position of the fluorescence spectrum on the excitation frequency is nonmonotonic in solutions of the type and varies substantially with the excitation intensity. Belarusian State University, 4, F. Skorina Ave., Minsk, 220072, Belarus. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 64, No. 2, pp. 164–168, March–April, 1997.     

可见光液晶光谱成像系统研究

利用向列相液晶材料的电控双折射效应,研制了用于可见光波段光谱调谐的大口径液晶可调滤光片,透过光谱测试表明,该滤光片在可见光波段(420~700 nm)以20 nm光谱分辨率可实现连续调谐和任意波长选择。利用该滤光片搭建了一种小型光谱成像系统,并在实验室内对若干样品进行了测试,结果表明,该系统可同时实现高图像分辨率和高光谱分辨率光谱成像,具备高、超光谱分辨率光谱成像的应用潜力,在生物医学、环境保护和资源探测等领域具有较好的应用前景。     

拟威布尔分布密度函数在荧光寿命成像数据分析中的应用

荧光寿命法成像技术(FLIM)是一种非常有效、功能强大且能用来分析复杂生物组织和细胞分子的成像技术。传统的荧光寿命成像的数据分析,按某些具有不同寿命、离散的单参量指数模型来描述荧光衰减过程。在生物组织这样既复杂又不均匀的样品中,虽然多参量指数模型能提供比单参量指数模型对实验数据更好的拟合效果,但是离散多参量的假定往往是随意的。提出了拟威布尔分布密度函数可能是生物荧光分子团衰减动力过程的真实再现,并且通过计算证明,对于某些生化感兴趣的荧光分子团的多槽基面效价测定样品的数据,相对于单参量指数与多参量指数衰减函数有更好的一致性。同时讨论了将该荧光衰减模型应用于荧光寿命成像的前景。     

Validity of retinal oxygen saturation analysis: Hyperspectral imaging in visible wavelength with fundus camera and liquid crystal wavelength tunable filter

The purpose of this paper was to investigate the feasibility of a newly developed hyperspectral fundus imaging camera with a liquid crystal tunable filter. The intensities of different wavelengths of light transmitted through an artery, vein, and the area surrounding these vessels and reflected out were measured, and the differential spectral absorptions were analyzed. Measurements were made from 16 normal eyes and from two artificial capillaries. The ratios of absorption (ROA) of arteries to veins from 500 to 580 nm (range 1) and from 600 to 720 nm (range 2) were calculated. For all eyes, the ROA_range1 was larger than ROA_range2. The ROA obtained from the artificial capillary filled with blood saturated with oxygen or nitrogen was similar to that of simulated data of oxy- and deoxyhemoglobin extinction rate. Most ROAs of human eyes were lower than those of the simulated data and the artificial capillaries. Oxygen saturation analysis by hyperspectral fundus imaging of retinal vessels were qualitatively in agreement with thein vitro analysis or simulated values. However, further improvements are necessary to evaluate the oxygen saturation quantitatively in the retinal blood vessels.     

Natural color low-level-light EBAPS imaging system based on three-color LCTF北大核心CSCD

The high-sensitivity electron bombarded active pixel sensor (EBAPS) not only has the characteristics of high gain and high sensitivity of vacuum imaging devices, but also has the digital characteristics of solid-state imaging devices, which greatly improves the level of night vision imaging. A natural color low-level-light EBAPS imaging system was built based on three-color liquid crystal tunable filter (LCTF). According to the characteristics of low-level-light images, the color enhancement processing such as grayscale stretching, white balance, and color correction was performed on the color images obtained by the system. The experimental results show that the system can realize the natural low-illumination color imaging that reflects the color characteristics of the scene itself, which can effectively improve the observation effect of the characteristics of the target scene at night, and can realize the color imaging under 5×10−4 lx illumination. © 2022 Editorial office of Journal of Applied Optics. All rights reserved.     

Video fluorescence microscopic techniques to monitor local lipid and phospholipid molecular order and organization in cell membranes during hypoxic injury

Digitized video microscopy is rapidly finding uses in a number of fields of biological investigation because it allows quantitative assessment of physiological functions in intact cells under a variety of conditions. In this review paper, we focus on the rationale for the development and use of quantitative digitized video fluorescence microscopic techniques to monitor the molecular order and organization of lipids and phospholipids in the plasma membrane of single living cells. These include (1) fluorescence polarization imaging microscopy, used to measure plasma membrane lipid order, (2) fluorescence resonance energy transfer (FRET) imaging microscopy, used to detect and monitor phospholipid domain formation, and (3) fluorescence quenching imaging microscopy, used to spatially map fluid and rigid lipid domains. We review both the theoretical as well as practical use of these different techniques and their limits and potential for future developments, and provide as an illustrative example their application in studies of plasma membrane lipid order and topography during hypoxic injury in rat hepatocytes. Each of these methods provides complementary information; in the case of hypoxic injury, they all indicated that hypoxic injury leads to a spatially and temporally heterogeneous alteration in lipid order, topography, and fluidity of the plasma membrane. Hypoxic injury induces the formation of both fluid and rigid lipid domains; the formation of these domains is responsible for loss of the plasma membrane permeability barrier and the onset of irreversible injury (cell death). By defining the mechanisms which lead to alterations in lipid and phospholipid order and organization in the plasma membrane of hypoxic cells, potential sites of intervention to delay, prevent, or rescue cells from hypoxic injury have been identified. Finally, we briefly discuss fluorescence lifetime imaging microscopy (FLIM) and its potential application for studies monitoring local lipid and phospholipid molecular order and organization in cell membranes.     

CVD grown 2D MoS2 layers: A photoluminescence and fluorescence lifetime imaging study

In this letter, we report on the fluorescence lifetime imaging and accompanying photoluminescence properties of a chemical vapour deposition (CVD) grown atomically thin material, MoS₂. µ‐Raman, µ‐photoluminescence (PL) and fluorescence lifetime imaging microscopy (FLIM) are utilized to probe the fluorescence lifetime and photoluminescence properties of individual flakes of MoS₂ films. Usage of these three techniques allows identification of the grown layers, grain boundaries, structural defects and their relative effects on the PL and fluorescence lifetime spectra. Our investigation on individual monolayer flakes reveals a clear increase of the fluorescence lifetime from 0.3 ns to 0.45 ns at the edges with respect to interior region. On the other hand, investigation of the film layer reveals quenching of PL intensity and lifetime at the grain boundaries. These results could be important for applications where the activity of edges is important such as in photocatalytic water splitting. Finally, it has been demonstrated that PL mapping and FLIM are viable techniques for the investigation of the grain‐boundaries.

     

Li~+,Zn~(2+),Mg~(2+)掺杂Lu_2O_3∶Er~(3+)荧光粉的制备及发光特性

用微波高温固相法合成了Er~(3+)单掺Lu_2O_3,Li~+与Er~(3+)共掺Lu_2O_3及Li~+,Zn~(2+),Mg~(2+)掺杂Lu_2O_3∶Er~(3+)的荧光粉。实验表明金属离子Li~+、Zn~(2+)、Mg~(2+)、Er~(3+)掺杂Lu_2O_3,不影响Lu_2O_3的立方晶相。扫描电子显微镜测量表明,Li~+掺杂可以有效改善粉体的分散性和形貌,Li~+,Zn~(2+),Mg~(2+)共掺杂获得的粉体颗粒分布更加均匀,粒径范围为80～100 nm。379 nm激发下,Li~+与Er~(3+)共掺样品发光较单掺Er~(3+)样品在565 nm处的发光增强了4.5倍,而Li~+、Zn~(2+)、Mg~(2+)与Er~(3+)共掺样品较其发光增强5.3倍。980 nm激发下,Li~+与Er~(3+)共掺样品,Li~+、Zn~(2+)、Mg~(2+)与Er~(3+)共掺样品的发光分别比单掺Er~(3+)样品在565 nm处发光增强23倍与39倍,在662 nm处发光强度分别增强20倍与43倍。379 nm激发下,较单掺Er~(3+)的样品,掺杂Li~+的样品和Li~+,Zn~(2+),Mg~(2+)和Er~(3+)共掺的样品荧光寿命均有所增加,而Zn~(2+)、Er~(3+)共掺及Mg~(2+)、Er~(3+)共掺样品的荧光寿命则有所缩短。