A white light confocal microscope for spectrally resolved multidimensional imaging

Spectrofluorometric imaging microscopy is demonstrated in a confocal microscope using a supercontinuum laser as an excitation source and a custom‐built prism spectrometer for detection. This microscope system provides confocal imaging with spectrally resolved fluorescence excitation and detection from 450 to 700 nm. The supercontinuum laser provides a broad spectrum light source and is coupled with an acousto‐optic tunable filter to provide continuously tunable fluorescence excitation with a 1‐nm bandwidth. Eight different excitation wavelengths can be simultaneously selected. The prism spectrometer provides spectrally resolved detection with sensitivity comparable to a standard confocal system. This new microscope system enables optimal access to a multitude of fluorophores and provides fluorescence excitation and emission spectra for each location in a 3D confocal image. The speed of the spectral scans is suitable for spectrofluorometric imaging of live cells. Effects of chromatic aberration are modest and do not significantly limit the spatial resolution of the confocal measurements.     

人眼安全的生物气溶胶短程遥测系统

建立了满足激光辐照人眼安全要求的便携式生物气溶胶短程遥测系统,用于实现有人员活动区域生物战剂气溶胶的实时预警监测。以高频调制的405nm半导体激光器为激发光源,200mm口径的卡塞格林望远系统为信号光收集系统,搭建了基于伪随机调制的便携型生物气溶胶激光雷达短程遥测系统。该系统具备平行偏振散射、垂直偏振散射光探测,以及450nm和530nm两个波段荧光探测等四个同步探测通道。以枯草芽孢杆菌、金黄色葡萄球菌和酵母菌为样本对该激光雷达系统进行了初步测试。外场试验表明,该生物气溶胶遥测系统的空间分辨率为1.5m,荧光通道的探测距离为100m;3种被测生物气溶胶种类识别的回代误报率分别为9%、11.5%和14.5%,交叉误报率为11.3%。该遥测系统的激光能量低于激光辐照人眼安全标准的要求,基本实现了生物气溶胶的甄别。     

Absolute photoluminescence quantum efficiency measurement of light-emitting thin films

We developed an integrated monochromatic excitation light source integrating sphere based detection system to accurately characterize the absolute photoluminescence quantum efficiency of commonly used polymer light emitting films without using a reference sample. Our methodology is similar to the method reported by de Mello et al. [Adv. Mater. 9, 230 (1997)] In this Note, we show that the absolute photoluminescence quantum efficiency might only be measured when an appropriate calibration of the spectral variation of the measurement system is done. This calibration is especially important when employing a short excitation wavelength (<400 nm) for common silicon-based detector.     

Use of a white light supercontinuum laser for confocal interference-reflection microscopy

Shortly after its development, the white light supercontinuum laser was applied to confocal scanning microscopy as a more versatile substitute for the multiple monochromatic lasers normally used for the excitation of fluorescence. This light source is now available coupled to commercial confocal fluorescence microscopes. We have evaluated a supercontinuum laser as a source for a different purpose: confocal interferometric imaging of living cells and artificial models by interference reflection. We used light in the range 460-700 nm where this source provides a reasonably flat spectrum, and obtained images free from fringe artefacts caused by the longer coherence length of conventional lasers. We have also obtained images of cytoskeletal detail that is difficult to see with a monochromatic laser.     

Total internal reflection fluorescence imaging using an upconverting cover slip for multicolour evanescent excitation

Total internal reflection fluorescence microscopy is well known as a means of studying surface‐bound structures in cell biology. It is usually measured either by coupling a light source to the sample using a prism or with a special objective where light passing through the periphery of the lens illuminates the contact region beyond the critical angle. In this study we present a new and simple approach to total internal reflection fluorescence microscopy where the sample is mounted on a cover slip prepared from a high‐index upconverting glass‐ceramic. Excitation of the cover slip with a low‐cost near‐infrared laser diode generates intense narrow‐band visible emission within the cover slip, some of which is totally internally reflected. This emission gives rise to an evanescent wave at the interface and hence can excite surface‐bound fluorescent species. Depending on the excitation conditions the cover slip can generate violet, green and red emission and hence can excite a wide range of fluorescent labels. Fluorescence emission from the sample can be detected in spectral regions where the direct emission from the cover slip is very weak. The advantages and limitations of the technique are discussed in comparison with conventional total internal reflection fluorescence microscopy measurements and prospects for novel total internal reflection fluorescence microscopy geometries are considered.     

Multiphoton confocal microscopy using a femtosecond Cr:forsterite laser

With its output wavelength covering the infrared penetrating window of most biological tissues at 1,200-1,250 nm, the femtosecond Cr:forsterite laser shows high potential to serve as an excellent excitation source for the multiphoton fluorescence microscope. Its high output power, short optical pulse width, high stability, and low dispersion in fibers make it a perfect replacement for the currently widely used Ti:sapphire laser. In this paper, we study the capability of using a femtosecond Cr:forsterite laser in multiphoton scanning microscopy. We have performed the multiphoton excited photoluminescence spectrum measurement on several commonly used bioprobes using the 1,230 nm femtosecond pulses from a Cr:forsterite laser. Efficient fluorescence can be easily observed in these bioprobes through two-photon or three-photon excitation processes. These results will assist in the selection of dichroic beam splitter and band pass filters in a multiphoton microscopic system. We have also performed the autofluorescence spectrum measurement from chlorophylls in live leaves of the plant Arabidopsis thaliana excited by 1,230 nm femtosecond pulses from the Cr:forsterite laser. Bright luminescence from chlorophyll, centered at 673 and 728 nm, respectively, can be easily observed. Taking advantage of the bright two-photon photoluminescence from chlorophyll, we demonstrated the two-photon scanning paradermal and cross-sectional images of palisade mesophyll cells in live leaves of Arabidopsis thaliana.     

Frequency-domain fluorescence microscopy with the LED as a light source

We describe a frequency-domain lifetime fluorometer based on a microscope and a modulated light-emitting diode (LED) excitation source (370/460 nm), which operates in the frequency range 120 Hz–250 MHz. We collected multifrequency phase and modulation fluorescence responses from cellular areas as small as 10–15 µm in diameter. We also collected fluorescence lifetime data from cells stained by a lipophilic coumarin sensitized europium fluorophore, Coum-Eu, with a millisecond lifetime, and Ru(bpy)₂phe-C₁₂, with microsecond lifetime. Nanosecond lifetimes from native nuclei stained with SYTO 14 and SYTO 16 probes were measured as well. We demonstrate that a simple LED excitation source can, for many applications, successfully replace complex and expensive laser systems, which have been used for cellular frequency-domain lifetime measurements. As the LEDs are very stable with low noise, it will be possible to image even smaller sample areas using brighter LEDs. With availability of modulated LEDs emitting at several wavelengths covering almost the entire visible spectrum it is easy to assemble a system for the fluorophore of choice. The ability to select an excitation source for a given fluorophore and low price make such an excitation source even more practical.     

全固态复合内腔和频570nm连续波黄光激光器

研制了全固态连续波570nm黄光激光器,黄激光分别由两片Nd:YAG的1444nm和946nm谱线非线性和频产生,两条谱线分别对应各自的晶体能级跃迁4F3/2-4I15/2和4F3/2-4I9/2。实验采用复合腔结构,利用KTP晶体II类临界相位进行内腔和频。测量了570nm黄激光输出功率随泵浦功率的变化,结果表明,当注入到两片Nd:YAG晶体的泵浦功率分别为24W和15W时,获得了560mW的连续波570nm黄激光输出,其4h功率稳定度优于±2.8%。在输出功率为560mW时,采用光束质量分析仪测量了激光输出光斑质量,结果显示,在570nm最大和频激光输出时的光束质量因子M2为2.3。所提出的复合内腔和频技术可为新波长激光器的发展提供参考。     

激光诱导荧光水质监测系统的研制

提出以Nd：YAG激光器的三倍频355nm波长激光为激发光源,以单个光电倍增管（PMT）为探测器,以水体污染监测为目的的激光诱导荧光系统的研制。采用光电倍增管高速双脉冲门控与单次脉冲触发数据采样相结合新技术,有效减少了户外环境下背景光的影响,提高了系统的测量精确度。试验结果表明,该系统能接收水体表面受激发射的荧光信号,探测到的荧光信号经信号处理后与水体受污染程度有良好的相关性。     

Autofluorescence spectroscopy and imaging of Platymonas subcordiformis irradiated by diode laser based on LSCM

Autofluorescence spectra and optical imaging of Platymonas subcordiformis after irradiation of diode laser were observed via laser scanning confocal microscopy (LSCM). With 488 nm Ar(+) laser excitation, the horizontal and vertical dimensions of a cup-shaped chloroplast of the irradiation group increased about 10% compared with the control group. The fluorescence spectra were similar between irradiation group and control group with a maximum fluorescence band around 682 nm, whereas the former has a higher intensity. Image of a small circular substance with stronger two-photon autofluorescence (TPA) was obtained when using two-photon excitation wavelength of 800 nm in single-channel mode. Further analysis by the 800 nm excitation based on two independent-channels mode showed an emission band of the small circular substance around 376-505 nm, which corresponded to the eyespot of P. subcordiformis. In lambda scanning mode, with two-photon wavelength of 800 nm excitation, six fluorescence peaks that are located at 465, 520, 560, 617, 660 and 680 nm were observed; the fluorescence intensity of the irradiation group was higher than that of the control group, especially at 520, 560 and 617 nm. As a conclusion, diode laser irradiation can promote chloroplast growth of P. subcordiformis cells in the form of expanding area and the increasing content of protein, phospholipids and chlorophyll. LSCM, especially TPA imaging based on femtosecond laser excitation, provides a nondestructive, real-time and accurate method to study changes of living algal cells under laser irradiation and other environmental factors.     

A Laser Ranging System Using Chaotic Light

A novel laser ranging system using chaotic laser as probe light source is designed and presented.The system is made up of five components:chaotic light source,transmitter,receiver,data acquisition unit and data processing unit.Chaotic light is generated by an 808 nm,500 mW,single-mode laser diode with optical feedback cavity.Single target detection and multi-target detection are experimentally realized by correlating the chaotic reference light and the reflected or backscattered probe light.The performances,including the resolution of 18 cm within at least 130 m range and the sensitivity of-20 dB,are achieved and analyzed.     

基于光子晶体光纤参量振荡器的CARS成像光源研究

研究了一种基于光子晶体光纤参量振荡器的相干反斯托克斯拉曼散射(CARS)成像光源。该光源采用中心波长为1 030nm的皮秒光纤激光器作为泵浦源,以无截止单模光子晶体光纤作为参量增益,通过搭建光参量振荡器,实现平均功率为10mW的参量激光输出。在色散滤波效应的作用下,该光源输出波长可实现连续可调,调谐范围为782～793nm,对应的波数覆盖范围为2 901～3 078cm-1。该光源产生的两束激光脉冲可实现空间自同步、时间自重合,用于CARS成像测量时,无需空间、时间对准,有望推动CARS成像光源向全光纤化、小型化的方向发展。     

980nm高稳定度激光泵浦源控制系统

设计、制作了一款980nm高稳定度激光泵浦源控制系统,以满足掺铒光纤放大器(EDFA)稳定工作的需要。首先,以恒流激励原理设计了控制系统的驱动单元。接着,使用半导体制冷器(TEC)作为泵浦源的温度控制手段,设计了控制系统的温度控制单元。为了验证控制系统的有效性,选用一款激光泵浦模块组成了完整的激光泵浦源系统。最后,对激光泵浦源的激光输出进行了实验,研究了光功率与驱动电流的关系,以及系统的光功率稳定度与光谱稳定性等。对系统进行了相关测试实验,结果显示:应用了本控制系统的激光泵浦源的激光输出中心波长为975.2nm,光功率可达600mW,短期光功率稳定度为±0.008dB,长期光功率稳定度为±0.05dB,比同类激光泵浦源具有更高的稳定度。得到的结果表明:所设计的激光泵浦源控制系统满足设计要求,具有一定的实用价值。     

多功能灵敏固相时间分辨荧光免疫分析仪设计

针对固相时间分辨荧光光谱的测量,设计出一种全新的固相时间分辨荧光免疫分析系统.使用氮分子激光器作为激发光源,采用积分球和单色仪相结合的荧光收集结构,使杂散光对样品荧光的影响降到最低;用光电倍增管进行光电转换,在500～700 nm范围实现了高分辨荧光光谱测量;利用数字方式实现取样积分功能,提高了系统的信噪比.系统可实现荧光寿命、时间分辨荧光光谱、物质浓度的自动测量,仪器的检测灵敏度可达10 - 12 moL/L,线性范围为10-12～10-9 mol/L,稳定性相对误差小于3％,荧光光谱分辨为0.5nm.     

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.     

Analysis of fluorescence from algae fossils of the Neoproterozoic Doushantuo formation of China by confocal laser scanning microscope

Chinese algae fossils can provide unique information about the evolution of the early life. Thin sections of Neoproterozoic algae fossils, from Guizhou, China, were studied by confocal laser scanning microscopy, and algae fossils were fluorescenced at different wavelengths when excited by laser light of 488 nm, 476 nm, and 568 nm wavelength. When illuminated by 488 nm laser light, images of the algae fossils were sharper and better defined than when illuminated by 476 nm and 568 nm laser light. The algae fossils fluoresce at a wide range of emission wavelengths. The three-dimensional images of the fluorescent algae fossils were compared with the transmission images taken by light microscope. We found that the fluorescence image of the confocal laser scanning microscope in a single optical section could pass for the transmission image taken by a light microscope. We collected images at different sample depths and made a three-dimensional reconstruction of the algae fossils. And on the basis of the reconstruction of the three-dimensional fluorescent images, we conclude that the two algae fossils in our present study are red algae.     

激光扫描共聚焦光谱成像系统

在激光扫描共聚焦显微成像技术基础上引入了光谱成像技术以便区分生物组织中的不同荧光成分。采用分光棱镜对荧光进行光谱展开,在光谱谱面处设置两个可移动缝片形成出射狭缝,两个步进电机带动安装其上的两个缝片设置系统在整个工作波长（400~700 nm）内的光谱带宽,其最小光谱带宽优于5 nm。用488 nm激光和低压汞灯实际测量了几条谱线对应的狭缝位置并和理论值做了比较,结果显示实际狭缝位置和理论值的差值均小于0.1 mm。在全光谱和50 μm出射狭缝（对应2.5 nm光谱带宽）对老鼠肾脏组织进行了共聚焦光谱成像实验,获得了老鼠肾脏组织中DAPI标定的细胞核图像和Alexa Fluor^®488标定的肾脏小球曲管图像,实现了对老鼠肾脏组织不同成分的区分。实验结果表明：提出的系统能够进行共聚焦光谱成像,扩大了共聚焦显微镜的适用范围。     

多波长激发高分辨率微型拉曼光谱仪设计

针对不同激光波长激发测试样品所需拉曼光谱范围的差异性问题,同时为了保证拉曼光谱仪的小型化及高分辨率需求,提出一种以Czerny-Turner光路结构为基础的微型拉曼光谱仪,通过Zemax光学设计软件对光谱仪的准直镜、聚焦镜、柱面镜、光栅以及CCD的倾角及距离进行了优化。该仪器激光波长为633 nm,光谱范围为640～800 nm。进一步优化光栅旋转角度并配合聚焦镜,可使此光学系统同时适用于激光波长532 nm、光谱范围540～650 nm和激光波长785 nm、光谱范围790～1 000 nm两个波段。拉曼光谱仪分辨率为0.1 nm,该光谱仪在保证高分辨率的情况下解决了不同波段范围光学结构差异性大而导致光机设计很难整合在一起的问题。     

Apparatus for the measurement of small absorption change kinetics at 820 nm in the nanosecond range after a ruby laser flash

An apparatus for the measurement of kinetics of small absorption changes (DeltaA approximately 10(-3)-10(-4)) at 820 nm in a biological sample in the nanosecond range after a short high-intensity ruby laser flash (200 mJ, 8 ns) is described. The laser-induced fluorescence artefact is greatly reduced by the use of a solid-state laser diode as source of measuring light, in combination with a microscope lens and a diaphragm. The signal-to-noise ratio is enhanced by the use of an avalanche photodetector connected to a laboratory-made low-noise and interference-free amplifier. The signals are processed by a transient digitizer in combination with a signal averager. The response time of the apparatus is about 20 ns. With some modifications the apparatus can be used to measure absorption changes in a broad spectral range.     

基于液芯波导激光诱导荧光检测器的缝管自动进样毛细管电泳仪的研制及其在DNA分析中的应用

介绍一种自行研制的毛细管电泳仪。以半导体激光器作为激发光源,使用液芯波导石英毛细管同时作为电泳分离通道和荧光检测光路,结合基于取样探针、缺口型样品圆盘两者的缝管自动进样系统,以激光诱导荧光的检测方式实现对两种常用DNA分子标记物（DNA Marker）快速、低耗、准确的有效检测。该仪器具有结构简单、体积小、操作方便等特点。