A novel approach for correlative light electron microscopy analysis

Correlative light and electron microscopy (CLEM) is a multimodal technique of increasing utilization in functional, biochemical, and molecular biology. CLEM attempts to combine multidimensional information from the complementary fluorescence light microscopy (FLM) and electron microscopy (EM) techniques to bridge the various resolution gaps. Within this approach the very same cell/structure/event observed at level can be analyzed as well by FLM and EM. Unfortunately, these studies turned out to be extremely time consuming and are not suitable for statistical relevant data. Here, we describe a new CLEM method based on a robust specimen preparation protocol, optimized for cryosections (Tokuyasu method) and on an innovative image processing toolbox for a novel type of multimodal analysis. Main advantages obtained using the proposed CLEM method are: (1) hundred times more cells/structures/events that can be correlated in each single microscopy session; (2) three‐dimensional correlation between FLM and EM, obtained by means of ribbons of serial cryosections and electron tomography microscopy (ETM); (3) high rate of success for each CLEM experiment, obtained implementing protection of samples from physical damage and from loss of fluorescence; (4) compatibility with the classical immunogold and immunofluorescence labeling techniques. This method has been successfully validated for the correlative analysis of Russel Bodies subcellular compartments. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Quantitative determination of the reduction of phototoxicity and photobleaching by controlled light exposure microscopy

Phototoxicity and photobleaching are major limitations in live-cell fluorescence microscopy. They are caused by fluorophores in an excited singlet or triplet state that generate singlet oxygen and other reactive oxygen species. The principle of controlled light exposure microscopy (CLEM) is based on non-uniform illumination of the field of view to reduce the number of excited fluorophore molecules. This approach reduces phototoxicity and photobleaching 2- to 10-fold without deteriorating image quality. Reduction of phototoxicity and photobleaching depends on the fluorophore distribution in the studied object, the optical properties of the microscope and settings of CLEM electronics. Here, we introduce the CLEM factor as a quantitative measure of reduction in phototoxicity and photobleaching. Finally, we give a guideline to optimize the effect of CLEM without compromising image quality.     

Correlative light and electron microscopy reveals discrepancy between gold and fluorescence labelling

Electron microscopy (EM) is traditionally employed as a follow‐up to fluorescence microscopy (FM) to resolve the cellular ultrastructures wherein fluorescently labelled biomolecules reside. In order to translate the information derived from FM studies to EM analysis, biomolecules of interest must be identified in a manner compatible with EM. Although fluorescent signals can serve this purpose when FM is combined with EM in correlative light and electron microscopy (CLEM), the traditional immunogold labelling remains commonly used in this context. In order to investigate how much these two strategies relate, we have directly compared the subcellular localization of on‐section fluorescence labelling with on‐section immunogold labelling. In addition to antibody labelling of LAMP‐1, bioorthogonal click labelling was used to localize soluble cysteine cathepsins or membrane‐associated sialylated glycans. We reveal and characterize the existence of inherent discrepancies between the fluorescence signal and the distribution of gold particles in particular in the case of membrane‐associated antigens.     

Correlative fluorescence and electron microscopy in tissues: immunocytochemistry

Correlative microscopy is a collection of procedures that rely upon two or more imaging modalities to examine the same specimen. The imaging modalities employed should each provide unique information and the combined correlative data should be more information rich than that obtained by any of the imaging methods alone. Currently the most common form of correlative microscopy combines fluorescence and electron microscopy. While much of the correlative microscopy in the literature is derived from studies of model cell culture systems we have focused, primarily, on correlative microscopy in tissue samples. The use of tissue, particularly human tissue, may add constraints not encountered in cell culture systems. Ultrathin cryosections, typically used for immunoelectron microscopy, have served as the substrate for correlative fluorescence and electron microscopic immunolocalization in our studies. In this work, we have employed the bifunctional reporter FluoroNanogold. This labeling reagent contains both a fluorochrome and a gold-cluster compound and can be imaged by sequential fluorescence and electron microscopy. This approach permits the examination of exactly the same sub-cellular structures in both fluorescence and electron microscopy with a high level of spatial resolution.     

Four‐dimensional telomere analysis in recordings of living human cells acquired with Controlled Light Exposure Microscopy

Telomeres are the complex end structures that confer functional integrity and positional stability to human chromosomes. Telomere research has long been dominated by length measurements and biochemical analyses. Recently, interest has shifted towards the role of their three‐dimensional organization and dynamics within the nuclear volume. In the mammalian interphase nucleus, there is increasing evidence for a telomeric configuration that is non‐random and is cell cycle and cell type dependent. This has functional implications for genome stability. Objective and reproducible representation of the spatiotemporal organization of telomeres, under different experimental conditions, requires quantification by reliable automated image processing techniques. In this paper, we describe methods for quantitative telomere analysis in cell nuclei of living human cells expressing telomere‐binding fusion proteins. We present a toolbox for determining telomere positions within the nucleus with subresolution accuracy and tracking telomeres in 4D controlled light exposure microscopy (CLEM) recordings. The use of CLEM allowed for durable imaging and thereby improved segmentation performance considerably. With minor modifications, the underlying algorithms can be expanded to the analysis of other intranuclear features, such as nuclear bodies or DNA double stranded break foci.     

Combination of multiphoton and reflective confocal imaging of cornea

We combine reflective confocal microscopy with multiphoton microscopy to form a minimally invasive technique to observe the cornea. The two imaging modalities allow detection of complementary information from the cornea. The autofluorescence signal shows the cytoplasm of epithelial cells, and the second harmonic generation signal is used to detect collagen, found mostly in the stroma of the cornea. The reflective confocal imaging allows detection of epithelial cells and keratocytes in the stroma. The system is first tested on bovine cornea. Assessment of the result on the bovine eye will be used to evaluate the potential of the system as a technique for in vivo clinical application.     

Multimodal and non‐linear optical microscopy applications in reproductive biology

A plethora of optical techniques is currently available to obtain non‐destructive, contactless, real time information with subcellular spatial resolution to observe cell processes. Each technique has its own unique features for imaging and for obtaining certain biological information. However none of the available techniques can be of universal use. For a comprehensive investigation of biological specimens and events, one needs to use a combination of bioimaging methods, often at the same time. Some modern confocal/multiphoton microscopes provide simultaneous fluorescence, fluorescence lifetime imaging, and four‐dimensional imaging. Some of them can also easily be adapted for harmonic generation imaging, and to permit cell manipulation technique. In this work we present a multimodal optical workstation that extends a commercially available confocal microscope to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools. The nonlinear microscopy capabilities were added to the commercial confocal microscope by exploiting all the flexibility offered by the manufacturer. The various capabilities of this workstation as applied directly to reproductive biology are discussed. Microsc. Res. Tech. 79:567–582, 2016. © 2016 Wiley Periodicals, Inc.     

Total internal reflection microscopy for live imaging of cellular uptake of sub-micron non-fluorescent particles

This paper presents a simple, high-resolution, non-fluorescent imaging technique called total internal reflection microscopy (TIRM) and demonstrates its potential application to real-time imaging of live cellular events. In addition, a novel instrument is introduced that combines the simplicity of TIRM with the specificity afforded by dual-colour total internal reflection fluorescence (TIRF) microscopy and allows sequential imaging with the two modalities. The key design considerations necessary to apply these imaging modes in a single instrument are discussed. The application of TIRM alone yielded high-resolution live images of cell adherence to a poly- l -lysine modified substrate, whereby fine cellular structures are imaged. Non-fluorescent imaging of the uptake of sub-micron–sized polymeric particles by live cells is also demonstrated. Finally, images of fluorescently labelled cells were obtained in TIRF mode, sequentially to images obtained of the same cell in TIRM mode. Visual information gained using TIRF is compared with TIRM to demonstrate that the level of cell structure information obtainable with our total internal reflection microscope is comparable with the TIRF technique.     

Quantification of micrometre-sized porosity in quasicrystals using coherent synchrotron radiation imaging

The ability to image phase distributions with high spatial resolution is a key capability of microscopy systems. Consequently, the development and use of phase microscopy has been an important aspect of microscopy research and development. Most phase microscopy is based on a form of interference. Some phase imaging techniques, such as differential interference microscopy or phase microscopy, have a low coherence requirement, which enables high‐resolution imaging but in effect prevents the acquisition of quantitative phase information. These techniques are therefore used mainly for phase visualization. On the other hand, interference microscopy and holography are able to yield quantitative phase measurements but cannot offer the highest resolution. A new approach to phase microscopy, quantitative phase‐amplitude microscopy (QPAM) has recently been proposed that relies on observing the manner in which intensity images change with small defocuses and using these intensity changes to recover the phase. The method is easily understood when an object is thin, meaning its thickness is much less than the depth of field of the imaging system. However, in practice, objects will not often be thin, leading to the question of what precisely is being measured when QPAM is applied to a thick object. The optical transfer function formalism previously developed uses three‐dimensional (3D) optical transfer functions under the Born approximation. In this paper we use the 3D optical transfer function approach of Streibl not for the analysis of 3D imaging methods, such as tomography, but rather for the problem of analysing 2D phase images of thick objects. We go on to test the theoretical predictions experimentally. The two are found to be in excellent agreement and we show that the 3D imaging properties of QPAM can be reliably predicted using the optical transfer function formalism.     

X-ray omni microscopy

The science of wave‐field phase retrieval and phase measurement is sufficiently mature to permit the routine reconstruction, over a given plane, of the complex wave‐function associated with certain coherent forward‐propagating scalar wave‐fields. This reconstruction gives total knowledge of the information that has been encoded in the complex wave‐field by passage through a sample of interest. Such total knowledge is powerful, because it permits the emulation in software of the subsequent action of an infinite variety of coherent imaging systems. Such ‘virtual optics’, in which software forms a natural extension of the ‘hardware optics’ in an imaging system, may be useful in contexts such as quantitative atom and X‐ray imaging, in which optical elements such as beam‐splitters and lenses can be realized in software rather than optical hardware. Here, we develop the requisite theory to describe such hybrid virtual‐physical imaging systems, which we term ‘omni optics’ because of their infinite flexibility. We then give an experimental demonstration of these ideas by showing that a lensless X‐ray point projection microscope can, when equipped with the appropriate software, emulate an infinite variety of optical imaging systems including those which yield interferograms, Zernike phase contrast, Schlieren imaging and diffraction‐enhanced imaging.     

The importance of microscopy in studying the wear behaviour of ceramic surfaces

This paper demonstrates how careful microscopy of worn ceramic surfaces can be used to provide information on the mechanisms of material removal. This information is necessary as a critical complement to wear-rate data obtainable from simple wear tests alone (e.g. lapping with diamond grits). Scanning electron microscopy has been extensively used to investigate the changing appearance of worn surfaces as plasticity and fracture processes compete as materials removal and redistribution mechanisms. Examples of the use of secondary electron imaging at different surface tilts, back-scattered electron imaging and stereo imaging are shown. Further, transmission electron microscopy of samples specially prepared to contain the worn surface layer can reveal the presence of phase changes accompanying wear. Furthermore, observations have been made of instances whereby brittle fracture has unexpectedly occurred as a result of repeated plastic deformation of surfaces at low contact severities. Some conclusions are drawn regarding the influence of specimen microstructure, abrasive grit size and environment of the wear of glass-bonded (debased) alumina and titania materials.     

Correlative high-resolution morphologic analysis of the three-dimensional organization of human chromosomes

A correlative morphologic analysis was carried out on isolated metaphase chromosomes by means of field emission in-lens scanning electron microscopy (FEISEM) and atomic force microscopy (AFM). Whereas FEISEM provides ultra-high resolution power and allows the surface analysis of biological structures free of any conductive coating, the AFM allows imaging of biological specimens in ambient as well as in physiologic conditions. The analysis of the same samples was made possible by the use of electrical conductive and light transparent ITO glass as specimen holder. Further preparation of the specimen specific for the instrumentation was not required. Both techniques show a high correlation of the respective morphologic information, improving their reciprocal biological significance. In particular, the biological coat represents a barrier for surface morphologic analysis of chromosome spreads and it is sensitive to protease treatment. The chemical removal of this layer permits high-resolution imaging of the chromatid fibers but at the same time alters the chromosomal dimension after rehydration. The high-resolution level, necessary to obtain a precise physical mapping of the genome that the new instruments such as FEISEM and AFM could offer, requires homogeneously cleaned samples with a high grade of reproducibility. A correlative microscopical approach that utilizes completely different physical probes provides complementary useful information for the understanding of the biological, chemical, and physical characteristics of the samples and can be applied to optimize the chromosome preparations for further improvement of the knowledge about spatial genome organization.     

A protocol for registration and correction of multicolour STED superresolution images

Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut‐shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co‐localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross‐correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co‐staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user‐friendly criteria, which – if fulfilled – support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co‐localisation analyses. Although the reference protocol is discussed exemplarily for two‐colour STED imaging, it can be readily expanded to three or more colours and STED channels.     

An open‐source deconvolution software package for 3‐D quantitative fluorescence microscopy imaging

Deconvolution techniques have been widely used for restoring the 3‐D quantitative information of an unknown specimen observed using a wide‐field fluorescence microscope. Deconv , an open‐source deconvolution software package, was developed for 3‐D quantitative fluorescence microscopy imaging and was released under the GNU Public License. Deconv provides numerical routines for simulation of a 3‐D point spread function and deconvolution routines implemented three constrained iterative deconvolution algorithms: one based on a Poisson noise model and two others based on a Gaussian noise model. These algorithms are presented and evaluated using synthetic images and experimentally obtained microscope images, and the use of the library is explained. Deconv allows users to assess the utility of these deconvolution algorithms and to determine which are suited for a particular imaging application. The design of Deconv makes it easy for deconvolution capabilities to be incorporated into existing imaging applications.     

Mid infrared microspectroscopic mapping and imaging: a bio-analytical tool for spatially and chemically resolved tissue characterization and evaluation of drug permeation within tissues

The combination of the two classical biophysical methods, microscopy and infrared spectroscopy, has led to the development of a potent analytical technology termed infrared microspectroscopy. It combines high lateral resolution as obtained by microscopy and the chemical identification of the sample components by infrared spectroscopy. The two approaches mainly utilized in microspectroscopy are the mapping and the imaging techniques, which are introduced and presented. Especially, since the development of so called focal plane array detectors, which are implemented in the imaging methods (microspectroscopic imaging) has become a promising bio-analytical tool for ultrastructural medical diagnostics, due to the fact that the time required for analyzing a sample has been reduced dramatically and the lateral resolution improved to approximately 4 microm. Mid infrared microscopy allows a direct access to spatially resolved molecular and structural information of the analyzed area. The image contrast is generated on the basis of the tissue's intrinsic biochemical composition. The current investigation shows how mid infrared microspectroscopic mapping and imaging is used for the bio-analytical characterization and identification of specific molecular components of a tissue sample at high lateral resolution of a few microns (approaching the mid infrared diffraction limit). Furthermore, the potential of these methods for monitoring the penetration and distribution of drugs within biological tissues are presented. Due to the fact, that mid infrared microspectroscopy is a noninvasive, nondestructive technique for the analyzed sample, requiring no complicated and time consuming staining procedures, it is a convenient method for histological and pathological investigations, allowing the generation of a huge amount of biochemical information not yet available with other nonvibrational techniques. The strength of the presented microscopic technique is the fact that the infrared images are directly comparable to outcomes of classical histological staining procedures and can be interpreted by nonspectroscopists.     

A comparison of imaging methodologies for 3D tissue engineering

Imaging of cells in two dimensions is routinely performed within cell biology and tissue engineering laboratories. When biology moves into three dimensions imaging becomes more challenging, especially when multiple cell types are used. This review compares imaging techniques used regularly in our laboratory in the culture of cells in both two and three dimensions. The techniques reviewed include phase contrast microscopy, fluorescent microscopy, confocal laser scanning microscopy, electron microscopy, and optical coherence tomography. We compare these techniques to the current "gold standard" for imaging three-dimensional tissue engineered constructs, histology.     

Three-dimensional imaging in fluorescence by confocal scanning microscopy

The improved resolution and sectioning capability of a confocal microscope make it an ideal instrument for extracting three-dimensional information especially from extended biological specimens. The imaging properties, also with finite detection pinholes are considered and a number of biological applications demonstrated.     

Differential scanning tunnelling microscopy

We review the principle of differential imaging and its application to scanning tunnelling microscopy (STM). It is shown that placing a lateral dither on an STM tip at high frequency provides the means for transfering topographic information to a frequency range where noise is small. Differential STM imaging on graphite and gold is demonstrated. A simple relation between the differential image and the conventional topographic image is described.     

Correlative microscopy: a potent tool for the study of rare or unique cellular and tissue events

Biological studies have relied on two complementary microscope technologies – light (fluorescence) microscopy and electron microscopy. Light microscopy is used to study phenomena at a global scale to look for unique or rare events, and it also provides an opportunity for live imaging, whereas the forte of electron microscopy is the high resolution. Traditionally light and electron microscopy observations are carried out in different populations of cells/tissues and a 'correlative' inference is drawn. The advent of true correlative light-electron microscopy has allowed high-resolution imaging by electron microscopy of the same structure observed by light microscopy, and in advanced cases by video microscopy. Thus a rare event captured by low-resolution imaging of a population or transient events captured by live imaging can now also be studied at high resolution by electron microscopy. Here, the potential and difficulties of this approach, along with the most impressive breakthroughs obtained by these methods, are discussed.     

Comparison of holotomographic microscopy and coherence-controlled holographic microscopy

Quantitative phase imaging (QPI) is a powerful tool for label-free visualisation of living cells. Here, we compare two QPI microscopes – the Telight Q-Phase microscope and the Nanolive 3D Cell Explorer-fluo microscope. Both systems provide unbiased information about cell morphology, such as individual cell dry mass, perimeter and area. The Q-Phase microscope uses artefact-free, coherence-controlled holographic imaging technology to visualise cells in real time with minimal phototoxicity. The 3D Cell Explorer-fluo employs laser-based holotomography to reconstruct 3D images of living cells, visualising their internal structures and dynamics. Here, we analysed the strengths and limitations of both microscopes when examining two morphologically distinct cell lines – the cuboidal epithelial MDCK cells which form multicellular clusters and solitary growing Rat2 fibroblasts. We focus mainly on the ability of the devices to generate images suitable for single-cell segmentation by the built-in software, and we discuss the segmentation results and quantitative data generated from the segmented images. We show that both microscopes offer slightly different advantages, and the choice between them depends on the specific requirements and goals of the user.