Counting dendritic spines in brain tissue slices by image correlation spectroscopy analysis

Growth of new micrometre sized projections called dendritic spines in neurones has been linked to the encoding of long‐term memories in vertebrates. Numerous studies have been carried out at both the light and electron microscopy level to quantify dendritic spine densities in brain tissue in laboratory animals. Currently, such efforts using light microscopy have relied on manual counting of spines in confocal or two‐photon optical slice images of tissue containing fluorescently labelled spines. This manual approach can be slow and tedious, especially for samples with high spine densities. We introduce an alternative way of performing spine counting that uses an applied image intensity threshold followed by spatial image correlation spectroscopy (ICS) analysis. We investigated the effect of particle sizes above the diffraction limit on the autocorrelation analysis as well as the influence of background fluorescence. Our results show that, for well labelled cerebellar tissue samples imaged with a signal‐to‐noise ratio of 5 or greater, ICS‐based spine counts can be conducted with the same 15–20% precision as manual counting, but much more rapidly.     

Dynamic range and background filtering in raster image correlation spectroscopy

Raster-scan image correlation spectroscopy (RICS) enables researchers to measure molecular translational diffusion constants and concentrations from standard confocal laser scanning microscope images and is suitable for measuring a wide range of mobility, especially fast-diffusing molecules. However, as RICS analysis is based on the spatial autocorrelation function of fluorescence images, it is sensitive to the presence of fluorescent structures within the image. In this study, we investigate methods to filter out immobile or slow moving background structures and their impact on RICS results. Both the conventional moving-average subtraction-based method and cross-correlation subtraction-based method are rationalized and quantified. Simulated data and experimental measurements in living cells stress the importance of optimizing the temporal resolution of background filtering for reliable RICS measurements. Finally, the capacity of RICS analysis to separate two species is studied.     

Multicolour analysis and local image correlation in confocal microscopy

Multiparameter fluorescence microscopy is often used to identify cell types and subcellular organelles according to their differential labelling. For thick objects, the quantitative comparison of different multiply labelled specimens requires the three-dimensional (3-D) sampling capacity of confocal laser scanning microscopy, which can be used to generate pseudocolour images. To analyse such 3-D data sets, we have created pixel fluorogram representations, which are estimates of the joint probability densities linking multiple fluorescence distributions. Such pixel fluorograms also provide a powerful means of analysing image acquisition noise, fluorescence cross-talk, fluorescence photobleaching and cell movements. To identify true fluorescence co-localization, we have developed a novel approach based on local image correlation maps. These maps discriminate the coincident fluorescence distributions from the superimposition of noncorrelated fluorescence profiles on a local basis, by correcting for contrast and local variations in background intensity in each fluorescence channel. We believe that the pixel fluorograms are best suited to the quality control of multifluorescence image acquisition. The local image correlation methods are more appropriate for identifying co-localized structures at the cellular or subcellular level. The thresholding of these correlation maps can further be used to recognize and classify biological structures according to multifluorescence attributes.     

双光子受激发射损耗复合显微成像技术研究

鉴于双光子受激发射损耗（STED）复合显微镜在神经疾病临床诊断及脑科学研究中的重要作用,对双光子STED复合显微成像中多波长选通、多光束合束、关键技术指标等进行了研究,完成了复合显微镜样机系统集成研制和复合成像。该复合显微镜可以对荧光标记的样本进行扫描成像,具备红绿双色荧光扫描成像功能、双光子绿色荧光成像功能和STED超分辨绿色荧光成像功能。测试结果表明,该复合显微镜成像深度达到700 μm,分辨率优于60 nm。     

Quantitative measurement of the resolution and sensitivity of confocal microscopes using line-scanning fluorescence correlation spectroscopy

Spatial resolution and the sensitivity to detect a fluorophore are the two most important optical parameters that characterize a confocal microscope. However, these are rather difficult to estimate quantitatively. We show that fluorescence correlation spectroscopy (FCS) provides an easy and reliable measure of these quantities. We modify existing schemes for performing FCS on a commercial confocal microscope to carry out these measurements, and provide an analysis routine that can yield the relevant quantities. Our method does not require any modification of the confocal microscope, yet it yields a robust measure of the resolution and sensitivity of the instrument.     

Two-photon fluorescence lifetime imaging microscopy of macrophage-mediated antigen processing

Two-photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage-mediated endocytosis, intracellular vacuolar encapsulation, and protease-dependent processing. Fluorescein-conjugated bovine serum albumin (FITC–BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time-dependent changes in fluorescence properties. Using two-photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0.5 ns increased to α 3.0 ns. Control experiments using fluorescein conjugated poly- l -lysine and poly- d -lysine demonstrated that the increase in fluorescence parameters observed with FITC–BSA were due to intracellular proteolysis since addition of the inert d -isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC–dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two-photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.     

Two-photon excited lifetime imaging of autofluorescence in cells during UV A and NIR photostress

By monitoring coenzyme autofluorescence modifications. as an indicator of cell damage. the cellular response to femtosecond near-infrared (NIR) radiation (two-photon absorption) was compared with exposure to low-power UV A radiation (one-photon absorption). Excitation radiation from a tunable Ti-sapphire laser. focused through highnumerical- aperture microscope optics. provided diffractionlimited mlcrobeams of an adjustable peak power. Laser scanning NIR microscopy was used to detect spatially the intracellular distribution of fluorescent coenzymes by fluorescence intensity imaging as well as fluorescence lifetime imaging (_T-mapping). Upon the onset of UV or NIR exposure. Chinese hamster ovary cells exhibited blue/green autofluorescence witq a mean lifetime of 2·2 ns. which was attributed to NAD(P)H in mitochondria. Exposure to 365 nm radiation from a high-pressure mercury lamp (1 m W. 300 J cm^-2 ) resulted in oxidative stress correlated with increased autofluorescence intensity. onset of nuclear fluorescence. and a fluorescence lifetime decrease. The cellular response to femtosecond NIR micro beams depended significantly on peak power. Peak powers above a threshold value of about 0·5kW (average power: 6mW). 0·55kW (7mW) and 0·8kW (lOmW) at 730nm. 760nm and 800nm. respectively. resulted in the onset of short-lived luminescence with higher intensity (100x) than the intracellular NAD(P)H fluorescence. This luminescence. accompanied by destruction of cellular morphology. was localized and occurred in the mitochondrial region. In contrast. beams at a power of less than 0·5 kW allowed nondestructive fluorophore detection with high spatial and temporal resolution without modification of cellular redox state or cell morphology.     

Organelle transport in melanophores analyzed by white light image correlation spectroscopy

Intracellular transport of organelles, vesicles and proteins is crucial in all eukaryotic cells, and is accomplished by motor proteins that move along cytoskeletal filaments. A widely used model of intracellular transport is Xenopus laevis melanophores. These cells help the frog to change color by redistributing melanin-containing organelles in the cytoplasm. The high contrast of the pigment organelles permits changes in distribution to be observed by ordinary light microscopy; other intracellular transport systems often require fluorescence labeling. Here we have developed white light Image Correlation Spectroscopy (ICS) to monitor aggregation and dispersion of pigment. Hitherto in ICS, images of fluorescent particles from Confocal Laser Scanning Microscopy (CLSM) have been used to calculate autocorrelation functions from which the density can be obtained. In the present study we show that ICS can be modified to enable analysis of light-microscopy images; it can be used to monitor pigment aggregation and dispersion, and distinguish between different stimuli. This new approach makes ICS applicable not only to fluorescent but also to black-and-white images from light or electron microscopy, and is thus very versatile in different studies of movement of particles on the membrane or in the cytoplasm of cells without potentially harmful fluorescence labeling and activation.     

Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation

A scanning microscope utilizing two-photon excitation in combination with fluorescence lifetime contrast is presented. The microscope makes use of a tunable femtosecond titanium:sapphire laser enabling the two-photon excitation of a broad range of fluorescent molecules, including UV probes. Importantly, the penetration depth of the two-photon exciting (infra)red light is substantially greater than for the corresponding single-photon wavelength while photobleaching is significantly reduced. The time structure of the Ti:Sa laser can be employed in a straightforward way for the realization of fluorescence lifetime imaging. The fluorescence lifetime is sensitive to the local environment of the fluorescent molecule. This behaviour can be used for example to quantify concentrations of ions, such as pH and Ca²⁺, or pO₂ and pCO₂. In the set-up presented here the fluorescence lifetime imaging is accomplished by time-gated single photon counting. The performance and optical properties of the microscope are investigated by a number of test measurements on fluorescent test beads. Point-spread functions calculated from measurements on 230-nm beads using an iterative restoration procedure compare well with theoretical expectations. Lifetime imaging experiments on a test target containing two different types of test bead in a fluorescent buffer all with different lifetimes (2.15 ns, 2.56 ns and 3.34 ns) show excellent quantitative agreement with reference values obtained from time correlated single photon counting measurements. Moreover, the standard deviation in the results can be wholly ascribed to the photon statistics. Measurements of acridine orange stained biofilms are presented as an example of the potential of two-photon excitation combined with fluorescence lifetime contrast. Fluorescence lifetime and intensity images were recorded over the whole sample depth of 100 μm. Fluorescence intensity imaging is seriously hampered by the rapid decrease of the fluorescence signal as a function of the depth into the sample. Fluorescence lifetime imaging on the other hand is not affected by the decrease of the fluorescence intensity.     

荧光相关光谱的原理、技术及应用

本文介绍荧光相关光谱的原理、技术以及其在生命科学和化学领域内的应用。     

Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope

Raster image correlation spectroscopy (RICS) is a new and novel technique for measuring molecular dynamics and concentrations from fluorescence confocal images. The RICS technique extracts information about molecular dynamics and concentrations from images of living cells taken on commercial confocal systems. Here we develop guidelines for performing the RICS analysis on an analogue commercial laser scanning confocal microscope. Guidelines for typical instrument settings, image acquisition settings and analogue detector characterization are presented. Using appropriate instrument/acquisition parameters, diffusion coefficients and concentrations can be determined, even for highly dynamic dye molecules in solution. Standard curves presented herein demonstrate the ability to detect protein concentrations as low as ～ 2 nM. Additionally, cellular measurements give accurate values for the diffusion of paxillin‐enhanced‐green fluorescent protein (EGFP), an adhesion adaptor molecule, in the cytosol of the cell and also show slower paxillin dynamics near adhesions where paxillin interacts with immobile adhesion components. Methods are presented to account for bright immobile structures within the cell that dominate spatial correlation functions; allowing the extraction of fast protein dynamics within and near these structures. A running average algorithm is also presented to address slow cellular movement or movement of cellular features such as adhesions. Finally, methods to determine protein concentration in the presence of immobile structures within the cell are presented. A table is presented giving guidelines for instrument and imaging setting when performing RICS on the Olympus FV300 confocal and these guidelines are a starting point for performing the analysis on other commercial confocal systems.     

Two-photon scanning microphotolysis for three-dimensional data storage and biological transport measurements

Scanning microphotolysis is a method that permits the user to select, within the scanning field of a confocal microscope, areas of arbitrary geometry for photobleaching or photoactivation. Two-photon absorption, by contrast, confers on laser scanning microscopy a true spatial selectivity by restricting excitation to very small focal volumes. In the present study the two methods were combined by complementing a laser scanning microscope with both a fast programmable optical switch and a titan sapphire laser. The efficiency and accuracy of fluorescence photobleaching induced by two-photon absorption were determined using fluorescein-containing polyacrylamide gels. At optimal conditions a single scan was sufficient to reduce the gel fluorescence by ≈40%. Under these conditions the spatial accuracy of photobleaching was 0.5±0.1 μm in the lateral ( x y ) and 3.5±0.5 μm in the axial ( z ) direction, without deconvolution accounting for the optical resolution. Deconvolution improved the accuracy values by ≈30%. The method was applied to write complex three-dimensional patterns into thick gels by successively scanning many closely spaced layers, each according to an individual image mask. Membrane transport was studied in a model tissue consisting of human erythrocyte ghosts carrying large transmembrane pores and packed into three-dimensional arrays. Upon equilibration with a fluorescent transport substrate single ghosts could be selectively photobleached and the influx of fresh transport substrate be monitored. The results suggest that two-photon scanning microphotolysis provides new possibilities for the optical analysis and manipulation of both technical and biological microsystems.     

Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy

The imaging performance in single-photon (1-p) and two-photon (2-p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of the three-dimensional (3-D) point spread function and the 3-D optical transfer function. Images of fluorescent sharp edges and layers are modelled, giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2-p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3-D optical transfer function has the strongest response along the axial direction.     

Two-photon fluorescence surface wave microscopy

This paper demonstrates the principle of two-photon surface wave microscopy with a view to applications on biological samples. We describe a modified scanning optical microscope, which uses specially prepared coverslips. These coverslips are designed to support the propagation of surface waves capable of large field enhancements. We also discuss the beam conditioning necessary to ensure efficient use of the available illumination. Two-photon surface wave fluorescent excitation is demonstrated on fluorescent nanospheres, demonstrating a point spread function width of ≈220 nm at an illumination wavelength of 925 nm. The potential of non-linear surface wave excitation for both fluorescence and harmonic imaging microscopy is discussed.     

Gold nanorods as probes in two‐photon fluorescence correlation spectroscopy: Emerging applications and potential artifacts

Owing to the highly efficient two‐photon fluorescence of gold nanorods and very short fluorescence lifetime compared with the rotational correlation time, the rotation and diffusion of a single gold nanorod can be easily observed by two‐photon fluorescence correlation spectroscopy (TP‐FCS). This property, along with the previous successful use as a contrast agent in two‐photon fluorescence imaging, suggests a potential application in TP‐FCS as well. Although the FCS measurement becomes highly efficient with gold nanorods as probes, the amplitude and temporal decay of the measured correlation functions depend critically on excitation power. Here, we investigate various photophysical processes of gold nanorods to determine the cause of such a sensitive power dependency. This understanding provides a basis for choosing appropriate FCS models to recover reasonable physical parameters. Although the correlation function amplitude G(0) is 32 times lower when the excitation power increases from 20 µW to 1.12 mW, the application of a saturation‐modified FCS model yields very good fit to each data set and the fitted concentration of 0.64 nM is comparable to the 0.7 nM given by the inductively coupled plasma mass spectrometry measurement. The FCS assay appears to be an efficient method for the quantification of gold nanorods when correctly interpreted. However, even with the saturation considered in the fitting model, the fitted rotational and translational diffusion rates are getting faster as the power increases. This indicates that other effects such as photothermal effects may raise the local temperature, and thus increasing the rotational and translational diffusion rate. Microsc. Res. Tech. 76:882–889, 2013. © 2013 Wiley Periodicals, Inc.     

二维异核相关实验方法的比较研究

本工作在实验的基础上对几种同类功能的二维异核相关实验的优势和局限进行讨论。结果反映核磁共振具有实验方法多样化的技术特点,并重要指出:在实际工作中,为获取丰富和可靠NMR谱图信息,核磁共振工作者应该根据实验目的、样品特点和拟解决的问题对实验方法与实验参数进行合理的选择。     

Reversible photoswitching enables single-molecule fluorescence fluctuation spectroscopy at high molecular concentration

We demonstrate that photoswitchable markers enable fluorescence fluctuation spectroscopy at high molecular concentration. Reversible photoswitching allows precise control of the density of fluorescing entities, because the equilibrium between the fluorescent ON- and the dark OFF-state can be shifted through optical irradiation at a specific wavelength. Depending on the irradiation intensity, the concentration of the ON-state markers can be up to 1,000 times lower than the actual concentration of the labeled molecular entity. Photoswitching expands the range of single-molecule detection based experiments such as fluorescence fluctuation spectroscopy to large entity concentrations in the micromolar range.     

Fluorescence photobleaching-based image standardization for fluorescence microscopy

A method is presented for the standardization of images acquired with fluorescence microscopy, based on the knowledge of spatial distributions proportional to the microscope's absolute excitation intensity and fluorescence detection efficiency distributions over the image field. These distributions are determined using a thin fluorescent test layer, employed under practically mono-exponential photobleaching conditions. It is demonstrated that these distributions can be used for (i) the quantitative evaluation of differences between both the excitation intensity and the fluorescence detection efficiency of different fluorescence microscopes and (ii) the standardization of images acquired with different microscopes, permitting the deduction of quantitative relationships between images obtained under different imaging conditions.     

一种基于参考图频谱相关的模糊参数判别方法

对于模糊图像的复原,模糊模型参数判别的准确性是一关键,针对大尺度模糊图像有效细节信息的丢失,噪声等干扰对模型特征的减弱,以及实拍图像的频谱特征不明显等问题,提出了一种基于参考图像频谱相关性的模糊参数判别方法,将一幅清晰图像按照不同模糊参数进行模糊处理,并与待复原图的频谱进行相关性分析计算,以获得准确的模型参数。实验结果证明,该方法能够适应大尺度散焦模糊,具有较强的抗噪性并且适应实拍的散焦模糊图像复原。     

Chromatic two-photon excitation fluorescence imaging

We report on a chromatic axial scanning method for two-photon excitation fluorescence imaging. Effective axial scanning is achieved by incorporating a Fresnel lens in the system, which has large chromatic aberration and can therefore focus the excitation beam to different axial positions depending on its wavelength. We experimentally demonstrated this technique and used it to image the cross-section of fluorescent microspheres.