A light efficient optically sectioning microscope

We describe a simple method by which optically sectioned images may be obtained. The system geometry is similar to that of a tandem scanning microscope but a one-dimensional grid pattern is used rather than an array of pinholes. This produces a composite image consisting of an optically sectioned image superimposed on a conventional image. A blank sector on the disc is used to provide a wide-field image. Image subtraction yields the optically sectioned image in real time.     

Optical sectioning in fluorescence microscopy

We review the origins of optical sectioning in fluorescence microscopy in terms of the structure of the illumination used to generate the fluorescence within the specimen. We note that the conventional microscope using essentially uniform illumination does not exhibit optical sectioning whereas the confocal microscope using point (many spatial frequencies) illumination does. We show that the optical sectioning strength of a confocal microscope is not optimal and discuss the advantages of using a single spatial frequency for the structure of the illumination and the detection. In this case the optical sectioning strength is shown to be up to 25% narrower than in the ideal confocal case.     

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.     

Orthogonal-plane fluorescence optical sectioning: Three-dimensional imaging of macroscopic biological specimens

An imaging technique called orthogonal-plane fluorescence optical sectioning (OPFOS) was developed to image the internal architecture of the cochlea. Expressions for the three-dimensional point spread function and the axial and lateral resolution are derived. Methodologies for tissue preparation and for construction, alignment, calibration and characterization of an OPFOS apparatus are presented. The instrument described produced focused, high-resolution images of optical sections of an intact, excised guineapig cochlea. The lateral and axial resolutions of the images were 10 and 26 μm, respectively, within a 1·5-mm field of view.     

Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts

Thin, uniformly fluorescing reference layers can be used to characterize the imaging conditions in confocal, or more general, sectioning microscopy. Through-focus datasets of such layers obtained by standard microscope routines provide the basis for the approach. A set of parameters derived from these datasets is developed for defining a number of relevant sectioned imaging properties. The main characteristics of a particular imaging situation can then be summarized in a Sectioned Imaging Property-chart or SIPchart. We propose the use of such charts for the characterization of imaging properties in confocal and multiphoton microscopy. As such, they can be the basis for comparison of sectioned imaging condition characteristics, quality control, maintenance or reproduction of sectioned imaging conditions and other applications. Such charts could prove useful in documenting the more relevant properties of the instrumentation used in microscopy studies. The method carries the potential to provide the basis for a general characterization of sectioned imaging conditions as the layers employed can be characterized and fabricated to standard specifications. A limited number of such thin, uniformly fluorescing layers is available from our group for this purpose. Extension of the method to multiphoton microscopy is discussed.     

Direct-view microscopy: optical sectioning strength for finite-sized, multiple-pinhole arrays

Direct-view microscopes use multiple-aperture arrays in the source and detector planes. We develop a theory for brightfleld and fluorescence direct-view microscopy which allows us to determine the optical sectioning strength for finite-sized, multiple-pinhole arrays with an arbitrary distribution of apertures. We specialize to the cases of square, hexagonal and interleaving Archimedean spiral arrays and consider the implications of the array configuration on both the optical sectioning strength and the light budget.     

基于奇值分解的三维光学切片显微图像恢复算法研究

阐述了光学切片显微技术的基本原理,分析了显微镜光学系统点扩展函数循环矩阵的奇异性造成荧光图像恢复质量大大降低所导致的病态问题,应用降质模糊矩阵的奇值分解方法,挑选出较大特征值,并将空域问题的求解通过傅立叶变换转到频率域,使计算的复杂度降低,最终得到较为理想的复原图像.     

Simultaneous multicolour imaging using quantum dot structured illumination microscopy

Multicolour structured illumination microscopy (SIM) is a powerful tool used for the investigation of the dynamic interaction between subcellular structures. Nevertheless, most of the multicolour SIM schemes are currently limited by conventional fluorescent dyes and wavelength-dependent optical systems, and can only sequentially record images of different colour channels instead of obtaining multicolour datasets simultaneously. To address these issues, we present a novel multicolour SIM scheme referred to as quantum dot structured illumination microscopy (QD-SIM). QD-SIM enables simultaneously excitation and collection of multicolour fluorescent signals. We also propose a theoretical analysis of the image formation in two-dimensional multicolour SIM to help combine the optically sectioned and super-resolution attributes of SIM. Based on this theory, QD-SIM enables optically sectioned, super-resolution, multicolour simultaneous imaging at a single plane.     

Direct-view microscopy: experimental investigation of the dependence of the optical sectioning characteristics on pinhole-array configuration

We present a comparison between theoretical and experimental results for the axial response to a plane mirror specimen of the direct-view microscope employing multiple-pinhole arrays. The effects of pinhole size, pinhole spacing and array geometry are investigated in detail with a view to (i) achieving good optical sectioning characteristics and (ii) maximizing the amount of light available for imaging. The implications of our results for practical systems as regards pinhole-array design and fabrication are also discussed.     

Limitations on optical sectioning in live-cell confocal microscopy

In three-dimensional (3-D) live-cell microscopy, it has been common to treat cells as having a constant refractive index (RI). Although the variations in RI associated with the nucleus and other organelles were recognized from phase- and differential interference contrast (DIC) images, it was assumed that they were small and would not affect 3-D fluorescence images obtained using widefield/deconvolution, confocal of multiphoton imaging. This paper makes clear that this confidence was misplaced. Confocal images made using backscattered light (BSL) to image the flat, glass/water interfaces above and below living microscope specimens should reveal these structures as flat and featureless. That the image of the interface on the far side of the cells is neither flat nor featureless indicates that the "optical section" surface can be profoundly distorted by the RI irregularities associated with the presence of nuclei and other subcellar structures. This observation calls into question the reliability of images made using any of the current methods for performing 3-D light microscopy of living cells.     

Digital scanned laser light-sheet fluorescence lifetime microscopy with wide-field time-gated imaging

We develop a multidimensional fluorescence imaging technique by implementing a wide-field time-gated fluorescence lifetime imaging into digital scanned laser light-sheet microscopy (FLIM-DSLM) to measure 3D fluorescence lifetime distribution in mesoscopic specimens with high resolution. This is achieved by acquiring a series of time-gated images at different relative time delays with respect of excitation pulses at different depths. The lifetime is determined for each voxel by iteratively fitting to single exponential decay. The performance of the developed system is evaluated with the measurements of a lifetime reference Rhodamine 6G solution and a subresolution fluorescent bead phantom. We also demonstrate the application performances of this system to ex vivo and in vivo imaging of Tg(kdrl:EGFP) transgenic zebrafish embryos, illustrating the lifetime differences between the GFP signal and the autofluorescence signal. The results show that FLIM-DSLM can be used for sample size up to a few millimetres and can be utilised as a powerful and robust method for biomedical research, for example as a readout of protein–protein interactions via Förster resonance energy transfer.     

Out‐of‐focus background subtraction for fast structured illumination super‐resolution microscopy of optically thick samples

We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three‐dimensional (3D) samples that allows the use of two‐dimensional (2D) data processing. Indeed, obtaining super‐resolution images of thick samples is a difficult task if low spatial frequencies are present in the in‐focus section of the sample, as these frequencies have to be distinguished from the out‐of‐focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high‐resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out‐of‐focus content from the raw images. After this cleaning step, we can obtain super‐resolution images of optical sections in thick samples using a two‐beam harmonic illumination pattern and a limited number of raw images. This two‐step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two‐dimensional.     

Spatial calibration of structured illumination fluorescence microscopy using capillary tissue phantoms

Quantitative assessment of microvascular structure is relevant to the investigations of ischemic injury, reparative angiogenesis and tumor revascularization. In light microscopy applications, thick tissue specimens are necessary to characterize microvascular networks; however, thick tissue leads to image distortions due to out-of-focus light. Structured illumination confocal microscopy is an optical sectioning technique that improves contrast and resolution by using a grid pattern to identify the plane-of-focus within the specimen. Because structured illumination can be applied to wide-field (nonscanning) microscopes, the microcirculation can be studied by sequential intravital and confocal microscopy. To assess the application of structured illumination confocal microscopy to microvessel imaging, we studied cell-sized microspheres and fused silica microcapillary tissue phantoms. As expected, structured illumination produced highly accurate images in the lateral (X-Y) plane, but demonstrated a loss of resolution in the Z-Y plane. Because the magnitude of Z-axis distortion was variable in complex tissues, the silica microcapillaries were used as spatial calibration standards. Morphometric parameters, such as shape factor, were used to empirically optimize Z-axis software compression. We conclude that the silica microcapillaries provide a useful tissue phantom for in vitro studies as well as spatial calibration standard for in vivo morphometry of the microcirculation.     

Even illumination in total internal reflection fluorescence microscopy using laser light

In modern fluorescence microscopy, lasers are a widely used source of light, both for imaging in total internal reflection and epi-illumination modes. In wide-field imaging, scattering of highly coherent laser light due to imperfections in the light path typically leads to nonuniform illumination of the specimen, compromising image analysis. We report the design and construction of an objective-launch total internal reflection fluorescence microscopy system with excellent evenness of specimen illumination achieved by azimuthal rotation of the incoming illuminating laser beam. The system allows quick and precise changes of the incidence angle of the laser beam and thus can also be used in an epifluorescence mode.     

Application of a laser scalpel to sectioning fragile tissues prior to optical sectioning microscopy and 3D reconstruction

A method is described for the cutting of fragile material with a laser scalpel which minimizes damage to friable materials, making the interior of structures accessible for optical sectioning microscopy or for high resolution X-ray microtomography followed by 3D reconstruction.     

Advances in laser sources for confocal and multiphoton microscopy

The illumination source for all high-resolution, optical sectioning, scanning microscopes is crucially important to the overall performance of the system. We examine advances that have been made in laser sources for both confocal and multiphoton microscopy where the emphasis has been on the development of potentially low-cost, easy to use sources. Growing interest in temporally and spatially resolved techniques has directed laser research towards addressing these challenges. We present the most recent developments in sources for confocal and multiphoton microscopy along with the considerations that should be made when a new source is being considered.     

Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy

Layer‐by‐layer technique is used to adsorb a uniform ultrathin layer of fluorescently labelled polyelectrolytes on a glass cover slip. Due to their thickness, uniformity and fluorescence properties, these ultrathin layers may serve as a simple and applicable standard to directly measure the z‐response of different scanning optical microscopes. In this work we use ultrathin layers to measure the z‐response of confocal, two‐photon excitation and 4Pi laser scanning microscopes. Moreover, due to their uniformity over a wide region, i.e. cover slip surface, it is possible to quantify the z‐response of the system over a full field of view area. This property, coupled with a bright fluorescence signal, enables the use of polyelectrolyte layers for representation on sectioned imaging property charts: a very powerful method to characterize image formation properties and capabilities (z‐response, off‐axis aberration, spherical aberration, etc.) of a three‐dimensional scanning system. The sectioned imaging property charts method needs a through‐focus dataset taken from such ultrathin layers. Using a comparatively low illumination no significant bleaching occurs during the excitation process, so it is possible to achieve long‐term monitoring of the z‐response of the system. All the above mentioned properties make such ultrathin layers a suitable candidate for calibration and a powerful tool for real‐time evaluation of the optical sectioning capabilities of different three‐dimensional scanning systems especially when coupled to sectioned imaging property charts.     

Continuous serial thin sectioning for electron microscopy

The process of serial sectioning for electron microscopy has been refined such that loss of thin sections is kept below 0.1% and the series is continued at will. The method relies on microscopic control of all manipulative steps, Formvar casting on plate glass for coated slot grids, coating of the block with contact cement for reliable ribboning, pickup by a one-step method with grid support in the diamond knife trough, staining in LKB grid holders, gentle treatment of grids in the electron microscope, and a slight modification to the microscope for safe grid withdrawal. The results are particularly applicable to the reconstruction of neuronal microcircuits and larger volumes of neuropil.     

Image calibration in fluorescence microscopy

A fluorescence image calibration method is presented based on the use of standardized uniformly fluorescing reference layers. It is demonstrated to be effective for the correction of non‐uniform imaging characteristics across the image (shading correction) as well as for relating fluorescence intensities between images taken with different microscopes or imaging conditions. The variation of the illumination intensity over the image can be determined on the basis of the uniform bleaching characteristics of the layers. This permits correction for the latter and makes bleach‐rate‐related imaging practical. The significant potential of these layers for calibration in quantitative fluorescence microscopy is illustrated with a series of applications. As the illumination and imaging properties of a microscope can be evaluated separately, the methods presented are also valuable for general microscope testing and characterization.     

Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach

Practical applications of structured illumination microscopy (SIM) often suffer from various artefacts that result from imprecise instrumental hardware and certain bleaching properties of the sample. These artefacts can be observed as residual stripe patterns originating from the illumination grating. We investigated some significant causes of these artefacts and developed a correction approach that can be applied to images after acquisition. Most of the artefacts can be attributed to changes in illumination and detection intensities during acquisition. The proposed correction algorithm has been shown to be functional on noisy image data, and produces exceptional, artefact‐free results in everyday laboratory work.