A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120

We describe the construction and characterization of a laser‐line‐scanning microscope capable of detection of broad fluorescence spectra with a resolution of 1 nm. A near‐infrared femtosecond pulse train at 800 nm was illuminated on a line (one lateral axis, denoted as X axis) in a specimen by a resonant scanning mirror oscillating at 7.9 kHz, and total multi‐photon–induced fluorescence from the linear region was focused on the slit of an imaging polychromator. An electron‐multiplying CCD camera was used to resolve fluorescence of different colours at different horizontal pixels and fluorescence of different spatial positions in a specimen at different vertical pixels. Scanning on the other two axes (Y and Z) was achieved by a closed‐loop controlled sample scanning stage and a piezo‐driven objective actuator. The full widths at half maximum of the point‐spread function of the system were estimated to be 0.39–0.40, 0.33 and 0.56–0.59 μm for the X (lateral axis along the line‐scan), Y (the other lateral axis) and Z axes (the axial direction), respectively, at fluorescence wavelengths between 644 and 690 nm. A biological application of this microscope was demonstrated in a study of the sub‐cellular fluorescence spectra of thylakoid membranes in a cyanobacterium, Anabaena PCC7120. It was found that the fluorescence intensity ratio between chlorophyll molecules mainly of photosystem II and phycobilin molecules of phycobilisome (chlorophyll/phycobilin), in the thylakoid membranes, became lower as one probed deeper inside the cells. This was attributable not to position dependence of re‐absorption or scattering effects, but to an intrinsic change in the local physiological state of the thylakoid membrane, with the help of a transmission spectral measurement of sub‐cellular domains. The efficiency of the new line‐scanning spectromicroscope was estimated in comparison with our own point‐by‐point scanning spectromicroscope. Under typical conditions of observing cyanobacterial cells, the total exposure time became shorter by about 50 times for a constant excitation density. The improvement factor was proportional to the length of the line‐scanned region, as expected.     

EXAMINATION OF ORAL TISSUES BY FOURIER TRANSFORM SPECTRAL IMAGING FLUORESCENCE

Fourier transform imaging spectroscopy was combined with fluorescence microscopy and a cooled CCD detector for examination of human oral tissues. Oral tissue fragments, obtained from patients, were irradiated at 365 nm by a mercury lamp through the microscope objectives. Microscope images were transferred to an imaging Fourier transform spectrometer and to a CCD camera for simultaneous recording of the fluorescence spectra at each image pixel. Detailed information was observed at a microscopic resolution. Oral tissue fragments were also treated with aluminum phthalocyanine tetrasulfonatre (AlPcS₄) prior to irradiation and imaging. Since the latter is preferentially retained in proliferating vascular tissue such as oral tumors, its effect upon the fluorescence imaging is of practical importance. AlPcS₄ is highly soluble in biological solutions and has a strong absorbance at our excitation wavelength and a strong emission peak at λ = 680 nm; therefore, it was found suitable for detection of malignant tumors by this method. It was found that the proposed spectral imaging method, when combined with fluorescence labeling, allows for direct, in vivo, medical examination of oral tissues with detailed spatial resolution.     

Three-dimensional spectral imaging by Hadamard transform spectroscopy in a programmable array microscope

We report the acquisition and deconvolution of three-dimensional spectrally resolved images in a programmable array microscope implementing a Hadamard transform fluorescence spectroscopy system with adjustable spectral resolution. A stack of 16 two-dimensional spectral images was collected at 400 nm intervals along the optical axis. The specimen consisted of a polytene chromosome spread from Drosophila melanogaster doubly labelled for the Polyhomeotic protein by indirect immunofluorescence labelling with Alexa594 and for DNA with YOYO-1. The resulting four-dimensional data set consisted of the xyz spatial dimensions (898 × 255 × 16) with a 26-point spectrum at each spatial location. The total exposure time to the sample was 34 min. The system requires the acquisition of multiple images, and thus works best with fluorophores that are resistant to photobleaching. Image deconvolution reduced the amount of out-of-focus blur by up to a factor of 8, resulting in a dramatic improvement in the visualization of the chromosome backbone and localization of the specific Polyhomeotic domains.     

Two-photon excitation fluorescence pH detection using 2,3-dicyanohydroquinone: a spectral ratiometric approach

The use of 2,3-dicyanohydroquinone (DCHQ) as an emission ratiometric probe of pH in vitro and in fibroblast cells was evaluated using two-photon excitation fluorescence microscopy (TPEFM). In addition, methods for spectrally calibrating the Zeiss LSM510 META spectroscopy system for TPEFM were also developed. The emissions of both the acid and base forms of DCHQ were detectable when using an 800-nm excitation in TPEFM, thereby allowing ratiometric determination of pH. These data suggest that, in contrast to most other emission ratiometric probes, both acid and base forms of DCHQ have similar two-photon cross-sectional areas at 800 nm. Acid (maximum at ∼457 nm) and base (maximum at ∼489 nm) DCHQ TPEFM emission spectra were similar to previously reported one-photon excitation emission spectra. Calibration curves for pH were successfully constructed using the ratio of DCHQ emission difference maxima at 460 nm and 512 nm in vitro and in cells. To our knowledge, DCHQ is currently the only effective emission ratiometric pH indicator for two-photon microscopy and may serve as a useful starting point for the development of other TPEFM ratiometric dyes for quantitative measurement of other cell parameters such as Ca²⁺, Mg²⁺ or Na⁺.     

Cluster analysis of soft X-ray spectromicroscopy data

Soft X-ray spectromicroscopy provides spectral data on the chemical speciation of light elements at sub-100 nm spatial resolution. When all chemical species in a specimen are known and separately characterized, existing approaches can be used to measure the concentration of each component at each pixel. In other cases (such as often occur in biology or environmental science), some spectral signatures may not be known in advance so other approaches must be used. We describe here an approach that uses principal component analysis to orthogonalize and noise-filter spectromicroscopy data. We then use cluster analysis (a form of unsupervised pattern matching) to classify pixels according to spectral similarity, to extract representative, cluster-averaged spectra with good signal-to-noise ratio, and to obtain gradations of concentration of these representative spectra at each pixel. The method is illustrated with a simulated data set of organic compounds, and a mixture of lutetium in hematite used to understand colloidal transport properties of radionuclides.     

超光谱成像仪的精细光谱定标

为了精细标定棱镜色散超光谱成像仪1024×80光谱像元的中心波长和响应带宽,建立了一套光谱定标装置,提出了实现1nm光谱定标精度的定标方法。首先,介绍了产生谱线弯曲与谱线倾斜的原因,确定了精细光谱定标的方法和数据处理算法;然后,利用光谱定标装置测定了全部光谱响应像元的离散单色光响应值,利用高斯方程拟合了相对光谱响应曲线;最后,建立了中心波长矩阵表和带宽矩阵表,采用多项式拟合算法确定了空间视场像元的色散方程和光谱通道谱线弯曲方程,实验测定了温度变化谱线漂移结果。另外,还对光谱定标精度对辐射定标精度的影响进行了分析。光谱定标结果表明:超光谱成像仪的光谱定标精度达到了±1nm,各谱段带宽平均为8.75nm;色散方程及谱线弯曲与设计结果相符,谱线弯曲值为14～19nm,平均值为17nm;1nm的定标精度对辐射定标精度的影响分别小于1%(3000K黑体)和0.25%(6000K黑体),满足超光谱成像仪1nm光谱定标精度的要求。     

Autofluorescence removal using a customized filter set

Quantitative fluorescence microscopy is severely hindered by intrinsic autofluorescence (AF). Endogenous fluorescent molecules in tissue and cell samples emit fluorescence that often dominates signals from specific dyes. This makes AF removal critical to the development and practice of quantitative fluorescence microscopy. In this study, we showed that AF signal could be separated from specific signal using a customized filter set. The filter set used the same excitation and beam splitter as the standard filter set, but the emission filter was red‐shifted 40–60 nm from the peak of the specific dye. This filter set configuration collected mostly AF with minimum contribution from the specific dye. A linear transformation of AF images was required to correct for the difference in exposure and filter configuration. The constants (slope and intercept) in linear transformation were obtained through a pixel to pixel comparison between AF images (no staining) obtained by the standard filter set and the customized AF filter set. After staining of specific dye, the standard filter collecting target dye spectra was used to capture both target signal and AF, whereas customized filter was used to capture only AF. AF removal was accomplished by subtracting the linear transformed AF image from the image obtained from the standard filter. To validate our approach, we examined weak staining of androgen receptor in an AF abundant prostate tissue sample. Our method revealed a similar but cleaner nuclear staining of androgen receptor in a specimen, when compared to a traditional autofluorescence removal method. Microsc. Res. Tech., 76:1007–1015, 2013. © 2013 Wiley Periodicals, Inc.     

Comparison of hyperspectral classification methods for the analysis of cerium oxide nanoparticles in histological and aqueous samples

Hyperspectral imaging (HSI) and classification are established methods that are being applied in new ways to the analysis of nanoscale materials in a variety of matrices. Typically, enhanced darkfield microscopy (EDFM)‐based HSI data (also known as image datacubes) are collected in the wavelength range of 400–1000 nm for each pixel in a datacube. Utilising different spectral library (SL) creation methods, spectra from pixels in the datacube corresponding to known materials can be collected into reference spectral libraries (RSLs), which can be used to classify materials in datacubes of experimental samples using existing classification algorithms. In this study, EDFM‐HSI was used to visualise and analyse industrial cerium oxide (CeO₂; ceria) nanoparticles (NPs) in rat lung tissues and in aqueous suspension. Rats were exposed to ceria NPs via inhalation, mimicking potential real‐world occupational exposures. The lung tissues were histologically prepared: some tissues were stained with hematoxylin and eosin (H&E) and some were left unstained. The goal of this study was to determine how HSI and classification results for ceria NPs were influenced by (1) the use of different RSL creation and classification methods and (2) the application of those methods to samples in different matrices (stained tissue, unstained tissue, or aqueous solution). Three different RSL creation methods – particle filtering (PF), manual selection, and spectral hourglass wizard (SHW) – were utilised to create the RSLs of known materials in unstained and stained tissue, and aqueous suspensions, which were then used to classify the NPs in the different matrices. Two classification algorithms – spectral angle mapper (SAM) and spectral feature fitting (SFF) – were utilised to determine the presence or absence of ceria NPs in each sample. The results from the classification algorithms were compared to determine how each influenced the classification results for samples in different matrices. The results showed that sample matrix and sample preparation significantly influenced the NP classification thresholds in the complex matrices. Moreover, considerable differences were observed in the classification results when utilising each RSL creation and classification method for each type of sample. Results from this study illustrate the importance of appropriately selecting HSI algorithms based on specific material and matrix characteristics in order to obtain optimal classification results. As HSI is increasingly utilised for NP characterisation for clinical, environmental and health and safety applications, this investigation is important for further refining HSI protocols while ensuring appropriate data collection and analysis.     

Automatic segmentation of fluorescence lifetime microscopy images of cells using multiresolution community detection—a first study

Inspired by a multiresolution community detection based network segmentation method, we suggest an automatic method for segmenting fluorescence lifetime (FLT) imaging microscopy (FLIM) images of cells in a first pilot investigation on two selected images. The image processing problem is framed as identifying segments with respective average FLTs against the background in FLIM images. The proposed method segments a FLIM image for a given resolution of the network defined using image pixels as the nodes and similarity between the FLTs of the pixels as the edges. In the resulting segmentation, low network resolution leads to larger segments, and high network resolution leads to smaller segments. Furthermore, using the proposed method, the mean‐square error in estimating the FLT segments in a FLIM image was found to consistently decrease with increasing resolution of the corresponding network. The multiresolution community detection method appeared to perform better than a popular spectral clustering‐based method in performing FLIM image segmentation. At high resolution, the spectral segmentation method introduced noisy segments in its output, and it was unable to achieve a consistent decrease in mean‐square error with increasing resolution.     

Time-domain whole-field fluorescence lifetime imaging with optical sectioning

A whole-field time-domain fluorescence lifetime imaging (FLIM) microscope with the capability to perform optical sectioning is described. The excitation source is a mode-locked Ti:Sapphire laser that is regeneratively amplified and frequency doubled to 415 nm. Time-gated fluorescence intensity images at increasing delays after excitation are acquired using a gated microchannel plate image intensifier combined with an intensified CCD camera. By fitting a single or multiple exponential decay to each pixel in the field of view of the time-gated images, 2-D FLIM maps are obtained for each component of the fluorescence lifetime. This FLIM instrument was demonstrated to exhibit a temporal discrimination of better than 10 ps. It has been applied to chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment. Initially, standard fluorescent dyes were studied and then this FLIM microscope was applied to the imaging of biological tissue, successfully contrasting different tissues and different states of tissue using autofluorescence. To demonstrate the potential for real-world applications, the FLIM microscope has been configured using potentially compact, portable and low cost all-solid-state diode-pumped laser technology. Whole-field FLIM with optical sectioning (3D FLIM) has been realized using a structured illumination technique.     

Spectral Fluorescence Imaging for Quantitative Diagnosis and Mapping of Dental Fluorosis Affected Tooth Surfaces

Abstract

A new analytical method for quantitative diagnosis of dental fluorosis is suggested. The method is based on the spectral modifications in the fluorescence light emitted from affected enamel surfaces, relative to normal dental enamel. Imaging of the tooth surface is obtained using an imaging Fourier transform fluorescence spectrophotometer. This technique provides simultaneous fluorescence spectra at all pixels of the examined area. Images and the corresponding spectra were acquired at various tooth regions, and it was shown that normal, white opaque, brown discolored, and pitted tooth surfaces have different distinct spectral features which characterize the different degrees of the observed pathology of dental fluorosis. Criteria for quantitative assessment of the dental fluorosis stages were suggested. These were based on the measured spectral emission ratio at two wavelengths and comparison with the spectrum of normal enamel. The suggested parameters were validated against clinical observation of numerous samples affected by dental fluorosis at various pathological stages. Besides the introduction of the quantitative assessment, the method is suggested for detection of early changes in the characteristics of the tooth surface in dental fluorosis. The associated mapping capability allows for morphological characterization, which provides additional important clinical information.     

Time-gated total internal reflection fluorescence spectroscopy (TG-TIRFS): application to the membrane marker laurdan

A novel setup for total internal reflection fluorescence microscopy with spectral and temporal (nanosecond) resolution was used to measure the emission spectra of the membrane marker laurdan either selectively within the plasma membrane or in whole living cells, depending on the incident angle of the excitation light. With increasing temperature, the intensity of the fluorescence band around 490 nm increased in comparison with the band around 440 nm, which has previously been assigned to a phase transition of membrane lipids from gel to liquid crystalline phase. For a better separation of the overlapping spectral bands, time‐gated detection with a delay of 10–15 ns with respect to the exciting laser pulse was used. As a parameter of membrane dynamics the so‐called generalized polarization GP = (I₄₄₀ ? I₄₉₀)/(I₄₄₀ + I₄₉₀) was evaluated at temperatures between 24 and 41 °C and variable angles of the incident light permitting to excite laurdan molecules either within the plasma membrane or in the whole cell. A decrease of the GP values by ≈ 0.2 units between 28 and 41 °C indicated an increase in membrane fluidity or a decrease in membrane stiffness with increasing temperature. In addition, higher GP values were observed for the plasma membrane as compared with intracellular membranes, probably due to a higher amount of cholesterol. Because properties of the plasma membrane have a large influence on the uptake or release of certain pharmaceutical agents or metabolites, the direct assessment of the dynamics of the plasma membrane by total internal reflection fluorescence spectroscopy appears to be important for pharmacology.     

Maximum pixel spectrum: a new tool for detecting and recovering rare, unanticipated features from spectrum image data cubes

A new software tool, the maximum pixel spectrum, detects rare events within a spectrum image data cube, such as that generated with electron‐excited energy‐dispersive X‐ray spectrometry in a scanning electron microscope. The maximum pixel spectrum is a member of a class of ‘derived spectra’ that are constructed from the spectrum image data cube. Similar to a conventional spectrum, a derived spectrum is a linear array of intensity vs. channel index that corresponds to photon energy. A derived spectrum has the principal characteristics of a real spectrum so that X‐ray peaks can be recognized. A common example of a derived spectrum is the summation spectrum, which is a linear array in which the summation of all pixels within each energy plane gives the intensity value for that channel. The summation spectrum is sensitive to the dominant features of the data cube. The maximum pixel spectrum is constructed by selecting the maximum pixel value within each X‐ray energy plane, ignoring the remaining pixels. Peaks corresponding to highly localized trace constituents or foreign contaminants, even those that are confined to one pixel of the image, can be seen at a glance when the maximum pixel spectrum is compared with the summation spectrum.     

High throughput operando studies using Fourier transform infrared imaging and Raman spectroscopy

A prototype high throughput operando (HTO) reactor designed and built for catalyst screening and characterization combines Fourier transform infrared (FT-IR) imaging and Raman spectroscopy in operando conditions. Using a focal plane array detector (HgCdTe focal plane array, 128x128 pixels, and 1610 Hz frame rate) for the FT-IR imaging system, the catalyst activity and selectivity of all parallel reaction channels can be simultaneously followed. Each image data set possesses 16 384 IR spectra with a spectral range of 800-4000 cm(-1) and with an 8 cm(-1) resolution. Depending on the signal-to-noise ratio, 2-20 s are needed to generate a full image of all reaction channels for a data set. Results on reactant conversion and product selectivity are obtained from FT-IR spectral analysis. Six novel Raman probes, one for each reaction channel, were specially designed and house built at Pacific Northwest National Laboratory, to simultaneously collect Raman spectra of the catalysts and possible reaction intermediates on the catalyst surface under operando conditions. As a model system, methanol partial oxidation reaction on silica-supported molybdenum oxide (MoO3SiO2) catalysts has been studied under different reaction conditions to demonstrate the performance of the HTO reactor.     

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。     

Microscopic spectral imaging using a video camera

A technique is described which permits the simultaneous acquisition of multiple fluorescent emission and/or absorption spectra from discrete regions of a specimen under microscopic observation. The instrument consists of a modified inverted microscope, an optical diffraction grating, a silicon intensified target (SIT) camera, and a digital video image processor. Observation of the zero diffraction order of the grating with the SIT camera permits an optical slice of the specimen to be selected by positioning the region of interest over the entrance slit of the grating housing. To obtain the spectral characteristics of this optical slice, the grating is rotated to impinge the first order diffraction on the camera. The video image of this first order diffraction maintains spatial integrity along the slit's long axis and provides spectral dispersion on the perpendicular axis. Thus, each of the horizontal video lines along the long axis of the slit represents a spectral analysis of the corresponding spatial location within the specimen. The spectral resolution (0.2 nm/channel) of each video line is determined by the resolution of the camera system in conjunction with the resolution of the grating. The image processing system acquires and processes all 500 spectra in 33 ms and permits the accurate localization of the source of each spectrum in the slice. This type of topological spectral analysis permits the determination of both spatial and spectral characteristics of intrinsic or extrinsic chromophores within the specimen. In addition, this technique permits the detection of and the possible correction for photobleaching, light scattering and image plane effects. The application of this technique to the study of single cells is discussed and an example of the technique in determining the fluorescence spectra of acridine orange within the nucleus of an intact mammalian blastocyst is described.     

谱线弯曲对成像光谱仪辐射信号采集的影响

为了研究谱线弯曲对棱镜色散成像光谱仪光谱辐射信号采集的影响。首先,给出探测器像元采集到的辐射能量的表达式。然后,结合复合棱镜的色散特性,在可见近红外光谱范围（400～1000nm）内,计算当光谱偏离量为0.01d、0.1d和0.5d（d为探测器像元尺寸）时系统采集到的辐射能量与没有谱线弯曲情况下系统采集到的辐射能量的归一化差值,衡量谱线弯曲下系统辐射测量的变化。结果表明：谱线弯曲引起的探测器上的光谱偏离导致系统辐射信号采集发生变化,与没有谱线弯曲的情况相比,采集到的景物辐射信号在大气吸收带的边缘出现明显的偏差,且信号的差值随光谱偏离量的增大而增大,当光谱分辨率提高时,一些较弱的吸收峰附近也会出现明显的信号偏差。对于光谱分辨率平均为10nm的成像光谱仪,谱线弯曲量应控制在0.3nm以内。     

A setup for remote recording of the spectrum of laser-induced fluorescence from crowns of woody plants

A setup intended for remote studies of the chlorophyll content in woody plants is described. Fluorescence spectra from an object under study excited by laser radiation at a wavelength of 532 nm are recorded in the spectral range 600–850 nm. The performance characteristics of the setup allow acquisition of the spectra of short-term fluorescence of nanosecond duration, which are normalized to the maximum value of the signal of the reference channel. An MДP-23 monochromator is used as an element of spectral selection. Examples of recording a return pulse and the fluorescence spectrum from a Scotch pine are presented.     

快照式光谱光场成像技术

针对快照式多维成像系统难以实现高维成像的问题,本文提出了一种快照式光谱光场成像方法,使用单个探测器实现了对目标场景光谱光场信息的快速获取.该方法将聚焦光场成像结构引入到快照式超光谱成像傅里叶变换光谱仪中,首先获取混叠了目标场景光场信息和干涉信息的原始图像,然后使用基于卷积神经网络的信息重建算法,将光场信息和干涉信息从原...     

Reconstruction of the absorption spectrum of an object spot from the colour values of the corresponding pixel(s) in its digital image: the challenge of algal colours

A novel procedure for deriving the absorption spectrum of an object spot from the colour values of the corresponding pixel(s) in its image is presented. Any digital image acquired by a microscope can be used; typical applications are the analysis of cellular/subcellular metabolic processes under physiological conditions and in response to environmental stressors (e.g. heavy metals), and the measurement of chromophore composition, distribution and concentration in cells. In this paper, we challenged the procedure with images of algae, acquired by means of a CCD camera mounted onto a microscope. The many colours algae display result from the combinations of chromophores whose spectroscopic information is limited to organic solvents extracts that suffers from displacements, amplifications, and contraction/dilatation respect to spectra recorded inside the cell. Hence, preliminary processing is necessary, which consists of in vivo measurement of the absorption spectra of photosynthetic compartments of algal cells and determination of spectra of the single chromophores inside the cell. The final step of the procedure consists in the reconstruction of the absorption spectrum of the cell spot from the colour values of the corresponding pixel(s) in its digital image by minimization of a system of transcendental equations based on the absorption spectra of the chromophores under physiological conditions.