Dynamic three-dimensional visualization of collagen matrix remodeling and cytoskeletal organization in living corneal fibroblasts

The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing, as well as in tissue engineering. Three-dimensional (3-D) type I collagen matrices have been used extensively as an in vitro model for studying cell-induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the dynamic process of 3-D matrix remodeling at the cellular and subcellular level. We recently developed an experimental model for investigating cell-matrix mechanical interactions by plating green fluorescen protein (GFP)-zyxin transfected cells inside fibrillar collagen matrices and performing high-magnification time-lapse differential interference microscopy (DIC) and wide-field fluorescent imaging. In this study, we extend this experimental model by performing four-dimensional (4-D) reflected light and fluorescent confocal imaging (using either visible light or multiphoton excitation) of living corneal fibroblasts transfected to express GFP-zyxin or GFP-alpha-actinin, 18 h after plating inside 3-D collagen matrices. Reflected light confocal imaging allowed detailed visualization of the cells and the fibrillar collagen surrounding them. By overlaying maximum intensity projections of reflected light and GFP-zyxin or GFP-alpha-actinin images and generating stereo pair reconstructions, 3-D interactions between focal adhesions and collagen fibrils in living cells could be visualized directly. Focal adhesions were generally oriented parallel to the direction of collagen fibril alignment in front of the cell. Killing the cells induced relaxation of transient cell-induced tension on the matrix; however, significant permanent remodeling always remained. Time-lapse 3-D imaging demonstrated an active response to the Rho-kinase inhibitor Y-27632, as indicated by cell elongation, extracellular matrix relaxation, and extension of pseudopodial processes. It is interesting that, at higher cell densities, groups of collagen fibrils were compacted and aligned into straps between neighboring cells. Overall, the continued development and application of this new approach should provide important insights into the basic underlying biochemical and biomechanical regulatory mechanisms controlling matrix remodeling by corneal fibroblasts.     

Automated quantification and reconstruction of collagen matrix from 3D confocal datasets

The geometrical structure of fibrous extracellular matrix (ECM) impacts on its biological function. In this report, we demonstrate a new algorithm designed to extract quantitative structural information about individual collagen fibres (orientation, length and diameter) from 3D backscattered‐light confocal images of collagen gels. The computed quantitative data allowed us to create surface‐rendered 3D images of the investigated sample.     

Using the Hilbert transform for 3D visualization of differential interference contrast microscope images

Differential interference contrast (DIC) is frequently used in conventional 2D biological microscopy. Our recent investigations into producing a 3D DIC microscope (in both conventional and confocal modes) have uncovered a fundamental difficulty: namely that the phase gradient images of DIC microscopy cannot be visualized using standard digital image processing and reconstruction techniques, as commonly used elsewhere in microscopy. We discuss two approaches to the problem of preparing gradient images for 3D visualization: integration and the Hilbert transform. After applying the Hilbert transform, the dataset can then be visualized in 3D using standard techniques. We find that the Hilbert transform provides a rapid qualitative pre-processing technique for 3D visualization for a wide range of biological specimens in DIC microscopy, including chromosomes, which we use in this study.     

A simple method allowing DIC imaging in conjunction with confocal microscopy

Current optical methods to collect Nomarski differential interference contrast (DIC) or phase images with a transmitted light detector (TLD) in conjunction with confocal laser scanning microscopy (CLSM) can be technically challenging and inefficient. We describe for the first time a simple method that combines the use of the commercial product QPm (Iatia, Melbourne Australia) with brightfield images collected with the TLD of a CLSM, generating DIC, phase, Zernike phase, dark-field or Hoffman modulation contrast images. The brightfield images may be collected at the same time as the confocal images. This method also allows the calculation of contrast-enhanced images from archival data. The technique described here allows for the creation of contrast-enhanced images such as DIC or phase, without compromising the intensity or quality of confocal images collected simultaneously. Provided the confocal microscope is equipped with a motorized z-drive and a TLD, no hardware or optical modifications are required. The contrast-enhanced images are calculated with software using the quantitative phase-amplitude microscopy technique ( Barone-Nugent et al., 2002 ). This technique, being far simpler during image collection, allows the microscopist to concentrate on their confocal imaging and experimental procedures. Unlike conventional DIC, this technique may be used to calculate DIC images when cells are imaged through plastic, and without the use of expensive strain-free objective lenses.     

Three-dimensional analysis of cell nucleus structures visualized by confocal scanning laser microscopy

Studies of the three-dimensional (3-D) organization of cell nuclei are becoming increasingly important for the understanding of basic cellular events such as growth and differentiation. Modern methods of molecular biology, including in situ hybridization and immunofluorescence, allow the visualization of specific nuclear structures and the study of spatial arrangements of chromosome domains in interphase nuclei. Specific methods for labelling nuclear structures are used to develop computerized techniques for the automated analysis of the 3-D organization of cell nuclei. For this purpose, a coordinate system suitable for the analysis of tri-axial ellipsoidal nuclei is determined. High-resolution 3-D images are obtained using confocal scanning laser microscopy. The results demonstrate that with these methods it is possible to recognize the distribution of visualized structures and to obtain useful information regarding the 3-D organization of the nuclear structure of different cell systems.     

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.     

Confocal differential interference contrast (DIC) microscopy: including a theoretical analysis of conventional and confocal DIC imaging

A technique for obtaining differential interference contrast (DIC) imaging using a confocal microscope system is examined and its features compared to those of existing confocal differential phase contrast (DPC) techniques as well as to conventional Nomarski DIC. A theoretical treatment of DIC imaging is presented, which takes into account the vignetting effect caused by the finite size of the lens pupils. This facilitates the making of quantitative measurements in DIC and allows the user to identify and select the most appropriate system parameters, such as the bias retardation and lateral shear of the Wollaston prism.     

Restoration of confocal images for quantitative image analysis

Quantitative studies of three-dimensional (3-D) structure of microscopic objects have been made possible through the introduction of microscopic volume imaging techniques, most notably the confocal fluorescence microscope (CFM). Although the CFM is a true volume imager, its specific imaging properties give rise to distortions in the images and hamper subsequent quantitative analysis. Therefore, it is a prerequisite that confocal images are restored prior to analysis. The distortions can be divided into several categories: attenuation of areas in the image due to self-absorption, bleaching effects, geometrical effects and distortions due to diffraction effects. Of these, absorption and diffraction effects are the most important. This paper describes a method aimed at the correction of diffraction-induced distortions. All the steps necessary in restoring confocal images are discussed, including a novel method to measure instrumental properties on a routine basis. To test the restoration procedure an image of a fluorescent planar object was restored. The results show a considerable improvement in the z-resolution and no ringing artefacts. The relevance of the method for image analysis is demonstrated by a comparison of results of applying 3-D texture analysis to restored and unrestored images of a synthetic object. Furthermore, the method can be successfully applied to noisy fluorescence images of biological objects, such as interphase cell nucei.     

Extended confocal microscopy of myocardial laminae and collagen network

Ventricular myocardium has a complex three-dimensional structure which has previously been inferred from two-dimensional images. We describe a technique for imaging the 3D organization of myocytes in conjunction with the collagen network in extended blocks of myocardium. Rat hearts were fixed with Bouin's solution and perfusion-stained with picrosirius red. Transmural blocks from the left ventricular free wall were embedded in Agar 100 resin and mounted securely in an ultramicrotome chuck. Confocal fluorescence laser scanning microscopy was used to obtain 3D images to a depth of 60 μm in a contiguous mosaic across the surface. Approximately 50 μm was then cut off the surface of the block with an ultramicrotome. This sequence was repeated 20 times. Images were assembled and registered in 3D to form an extended volume 3800 × 800 × 800 μm³ spanning the heart wall from epicardium to endocardium. Examples are given of how digital reslicing and volume rendering methods can be applied to the resulting dataset to provide quantitative structural information about the 3D organization of myocytes, extracellular collagen matrix and blood vessel network of the heart.     

Helium ion microscopy for high-resolution visualization of the articular cartilage collagen network

The articular cartilage collagen network is an important research focus because network disruption results in cartilage degeneration and patient disability. The recently introduced helium ion microscope (HIM), with its smaller probe size, longer depth of field and charge neutralization, has the potential to overcome the inherent limitations of electron microscopy for visualization of collagen network features, particularly at the nanoscale. In this study, we evaluated the capabilities of the helium ion microscope for high-resolution visualization of the articular cartilage collagen network. Images of rabbit knee cartilage were acquired with a helium ion microscope; comparison images were acquired with a field emission scanning electron microscope (FE-SEM) and a transmission electron microscope (TEM). Sharpness of example high-resolution helium ion microscope and field emission scanning electron microscope images was quantified using the 25-75% rise distance metric. The helium ion microscope was able to acquire high-resolution images with unprecedented clarity, with greater sharpness and three-dimensional-like detail of nanoscale fibril morphologies and fibril connections, in samples without conductive coatings. These nanoscale features could not be resolved by field emission scanning electron microscopy, and three-dimensional network structure could not be visualized with transmission electron microscopy. The nanoscale three-dimensional-like visualization capabilities of the helium ion microscope will enable new avenues of investigation in cartilage collagen network research.     

Quantitative determination of the mineral distribution in different collagen zones of calcifying tendon using high voltage electron microscopic tomography

High voltage electron microscopic tomography was used to make the first quantitative determination of the distribution of mineral between different regions of collagen fibrils undergoing early calcification in normal leg tendons of the domestic turkey, Meleagris gallopavo. The tomographic 3-D reconstruction was computed from a tilt series of 61 different views spanning an angular range of +/- 60 degrees in 2 degrees intervals. Successive applications of an interactive computer operation were used to mask the collagen banding pattern of either hole or overlap zones into separate versions of the reconstruction. In such 3-D volumes, regions specified by the mask retained their original image density while the remaining volume was set to background levels. This approach was also applied to the mineral crystals present in the same volumes to yield versions of the 3-D reconstructions that were masked for both the crystal mass and the respective collagen zones. Density profiles from these volumes contained a distinct peak corresponding only to the crystal mass. A comparison of the integrated density of this peak from each profile established that 64% of the crystals observed were located in the collagen hole zones and 36% were found in the overlap zones. If no changes in crystal stability occur once crystals are formed, this result suggests the possibilities that nucleation of mineral is preferentially and initially associated with the hole zones, nucleation occurs more frequently in the hole zones, the rate of crystal growth is more rapid in the hole zones, or a combination of these alternatives. All lead to the conclusion that the overall accumulation of mineral mass is predominant in the collagen hole zones compared to overlap zones during early collagen fibril calcification.     

A comparison study of detecting gold nanorods in living cells with confocal reflectance microscopy and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two‐photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two‐photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto‐second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 μW 800 nm fs have a relatively poor resolution, whereas the 50 μW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.     

Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy

The orientation of fibrils within biological tissues is of primary importance. In this study, we propose a simple method based on second harmonic generation (SHG) microscopy to map, pixel by pixel, the orientation of the symmetry axis of the second‐order nonlinear susceptibility tensor of fibrils that produce SHG. The method uses only four images acquired at specific polarizations of the input laser beam, and can be easily and cheaply implemented on a confocal microscope. In addition to orientation informations, the method also provides polarization independent images and estimations of the ratio of the nonlinear susceptibility components. We demonstrate the relevance of our concept by studying the orientation fields of the collagen meshwork in a healthy rat liver that provides well separated fibrils. By correlating the mean orientation of the nonlinear susceptibility to the fibril orientation itself for many fibril segments, and using circular statistics, it is shown that both orientations are truly parallel at the fibril scale. Our polarimetric method allows to map fibril orientation fields, independently of individual fibril contrast in the SHG image.     

Modelling of three-dimensional fluorescence images of muscle fibres: an application of the three-dimensional optical transfer function

Striated muscle fibres can be modelled by a simple geometry, which has allowed three-dimensional (3-D) images in conventional and confocal microscopes to be calculated. This model is useful for comparing different imaging methods and represents a simple example of an application of the 3-D optical transfer function (OTF) for the system. The rejection of out-of-focus blur is demonstrated, and the effects of fibre thickness and confocal pinhole size on image contrast are investigated. The effects of using a simple filter for image enhancement are studied, elucidating the characteristics of the OTF.     

An engineered microenvironment for multidimensional microscopy of live cells

Multidimensional imaging (MD) of live cells is gaining importance in biomedical research as the commercial availability of confocal, nonlinear optical microscopes, environmental chambers, and specific fluorescence probes grows. One crucial aspect of the MD live cell imaging involves the proper immobilization of cells, which refers to the rapid and sufficient immobilization of cells on the microscope stage, neither disrupting the cellular structure and functions nor affecting the optical properties of the cells and the environments. Conventional cell immobilization methods glue the anchoring cells to coated surfaces, but such methods require centrifugation or extended incubation and are not suitable for cells in suspension. Most of the current three-dimensional (3-D) gels either exhibit unsatisfactory optical properties or have adverse effects on cell functions in culture. Recently, an engineered 3-D microcapsule has been developed that involves the complex coacervation of a positively charged collagen and a negatively charged polymer of 2-hydroxyethyl methacrylate--methacrylic acid--methyl methacrylate (HEMA-MMA-MAA). Hence, confocal imaging of live cells in this engineered 3-D microenvironment was investigated for its optical properties and cellular function compatibility. We report here that this microenvironment facilitates efficient cell immobilization, exhibits good optical properties, and can preserve cellular structures and functions, which will be useful in MD imaging of live cells for various applications.     

Imaging modes for bilateral confocal scanning microscopy

The bilateral scanning approach to confocal microscopy is characterized by the direct generation of the image on a two-dimensional (2-D) detector. This detector can be a photographic plate, a CCD detector or the human eye, the human eye permitting direct visualization of the confocal image. Unlike Nipkow-type systems, laser light sources can be used for excitation. A design called a carousel has been developed, in which the bilateral confocal scan capability can be added to an existing microscope so that rapid exchange and comparison between confocal and non-confocal imaging conditions is possible. The design permits independent adjustment of confocal sectioning properties with lateral resolutions better than, or, in the worst case equivalent to, those available in conventional microscopy. The carousel can be considered as a stationary optical path in which certain imaging conditions, such as confocality, are defined and operate on part of the imaging field. The action of the bilateral scan mirror then extends this image condition over the whole field. A number of optical arrangements for the carousel are presented which realize various forms of confocal fluorescence and reflection imaging, with point, multiple point or slit confocal detection arrangements. Through the addition of active elements to the carousel direct stereoscopic, ratio, time-resolved and other types of imaging can be achieved, with direct image formation on a CCD, eye or other 2-D detectors without the need to modify the host microscope. Depending on the photon flux available, these imaging modes can run in real-time or can use a cooled CCD at (very) low light level for image integration over an extended period.     

The significance of 3-D transfer functions in confocal scanning microscopy

The three-dimensional (3-D) transfer function is a useful concept for describing image formation in confocal scanning microscopy. From it we can derive the corresponding 2-D transfer function for in-focus imaging. In confocal transmission this can be derived analytically. The 1-D transfer function for on-axis imaging, which can be expressed in an analytical form even for confocal fluorescence with differing wavelengths of excitation and fluorescence, can be derived from the 3-D transfer function. The 2-D transfer function for in-focus imaging in confocal fluorescence microscopy with a finite-sized detector is also presented, which is shown to exhibit sign changes and can therefore result in reversals of image contrast.     

Three‐dimensional visualization of dermal skin structure using confocal laser scanning microscopy

The properties and performance of collagen‐based materials may be affected by the collagen fibre bundle pattern, orientation and weave. The aim of this study was to develop and apply methods to visualize the dermis using confocal laser scanning microscopy from thin tissue sections stained with haematoxylin and eosin. The data was processed to allow three‐dimensional (3‐D) visualization on a PC and using a 3‐D immersive technology system. The 3‐D visualization of the confocal microscope image stacks allowed the evaluation of the collagen macromolecular structure including the collagen fibre bundles. The methods developed provide a novel way of viewing complex organic structures with further potential applications in the medical field.