膨胀显微成像技术的原理及应用

膨胀显微成像技术（expansion microscopy,ExM）是一种新型超分辨成像技术。该技术借助可膨胀水凝胶均匀地物理放大生物样本,在常规光学成像条件下实现超分辨成像。ExM适用于细胞、组织切片等多种类型生物样本。蛋白质、核酸、脂质等生物大分子均可借助ExM进行超分辨成像。ExM可与共聚焦显微镜、光片显微镜、超高分辨显微镜联合使用,进一步提高成像分辨率。近年来,多种从基础ExM拓展而来的衍生技术进一步促进了该技术的实际应用。本文综述了ExM及其衍生技术的基本原理、ExM与不同成像技术联用的研究进展及ExM在不同类型生物样本中的应用进展,并对ExM技术的发展前景做出展望。     

Opportunities for biomineralization research using multiscale computed X-ray tomography as exemplified by bone imaging

Biominerals typically have complex hierarchical structures traversing many length scales. This makes their structural characterization complicated, since it requires 3D techniques that can probe full specimens at down to nanometer-resolution, a combination that is difficult – if not impossible – to achieve simultaneously. One challenging example is bone, a mineralized tissue with a highly complex architecture that is replete with a network of cells. X-ray computed tomography techniques enable multiscale structural characterization through the combination of various equipment and emerge as promising tools for characterizing biominerals. Using bone as an example, we discuss how combining different X-ray imaging instruments allow characterizing bone structures from the nano- to the organ-scale. In particular, we compare and contrast human and rodent bone, emphasize the importance of the osteocyte lacuno-canalicular network in bone, and finally illustrate how combining synchrotron X-ray imaging with laboratory instrumentation for computed tomography is especially helpful for multiscale characterization of biominerals.     

Chemical taphonomy of biomineralized tissues

Biomineralized tissues are chemically altered after death, and this diagenetic alteration can obscure original biological chemical features or provide new chemical information about the depositional environment. To use the chemistry of fossil biominerals to reconstruct biological, environmental or taphonomic information, a solid appreciation of biomineralization, mineral diagenesis and biomineral–water interaction is needed. Here, I summarize the key recent developments in the fields of biomineralization and post‐mortem trace element exchange that have significant implications for our understanding of the diagenetic behaviour of biominerals and the ways in which biomineral chemistry can be used in palaeontological and taphonomic research.     

有机荧光团及荧光标记细胞的阴极荧光成像研究

目的 阴极荧光（CL）成像是一种以电子束为激发源的高分辨荧光成像技术,但生物材料对电子束的敏感性限制了CL技术在生命科学中的广泛应用。为了研究和发展CL技术在生物样品中的应用,本文旨在通过探究电子辐照引起碳基材料的结构损伤、有机基团的降解及荧光猝灭等问题,深入理解电子源对有机荧光团的激发特性。方法 本研究应用扫描电镜（SEM）和阴极荧光谱仪系统（SEM-CL）,研究电子源对有机荧光团及荧光探针标记细胞的激发特性,观测了有机物的CL信号的发射特性、强度衰减、成像方式及特点。结果 实验结果显示,在低能量（2.5～5 keV）和低束流（～10 pA）电子辐照下,有机荧光微珠发射出较强的荧光,CL像分辨率达到～30 nm。荧光微珠经过12 min辐照,信号强度衰减了25%,CL像仍保持了可接受的发光强度和足够的信噪比。此外,还获得了从细胞表面到内部一定深度内,荧光标记的亚细胞结构信息。结论 在SEM-CL系统中,可以同时获得由电子束激发产生的电子像和CL像,实现阴极荧光与电子显微镜关联（CCLEM）成像。本实验的研究结果为CCLEM技术应用于生物结构研究提供了数据及技术支持。     

Studies of Biominerals Relevant to the Search for Life on Mars

The evidence of the water erosion on Mars is particularly interesting since present climatic conditions are such that liquid water cannot exist at the surface. But, if water was present on the planet in the past, there may have been life, too. Since the discovery of carbonates on Mars also may have very important implications on the possibility that life developed there, we are studying minerals that can have biotic or abiotic origin: calcite (CaCO₃) and aragonite, a metastable state of calcite. We have analysed biomineral aragonite, in the form of recent sea shells, as well as crystals of mineral aragonite. Infrared spectroscopy in the 2–25 μm wavelength range reveals that, after thermal processing, the biotic samples have a different spectral behaviour from the abiotic ones. As a result, it is possible to distinguish abiotic mineral aragonite from aragonite of recent biological origin. Obviously, if life existed in the past on the Red Planet, we could expect to find “ancient” biotic carbonates, which should therefore be investigated, in order to search for a way of discriminating them from abiotic minerals. For this reason, at the beginning we have considered samples of crushed fossil shells of aragonite composition. Afterwards, in order to take into account that fossilization processes almost always produce a transformation of metastable form (aragonite) into more stable form (calcite), we also studied samples of mineral calcite and different types of fossils completely transformed into calcite. All these biotic fossil samples show the same spectral behaviour as the fresh biotic material after thermal annealing at 485°C. Instead, the calcite behaves like abiotic aragonite. Furthermore, it is known that seashells and other biominerals are formed through an intimate association of inorganic materials with organic macromolecules. The macromolecules control the nucleation, structure, morphology, crystal orientation and spatial confinement of the inorganic phase: this differentiates biominerals from minerals. Analysing the aragonite or calcite fossils with a Scanning Electron Microscope, we found that the fossilization process did not modify the structure of the biominerals which maintain their microscopic characteristics. Looking at the morphology of fossil biominerals, it is evident that the crystals are arranged in complex architectures compared with the compact structure of the mineral crystals. In conclusion, the properties and structure of the biominerals are different from those of the minerals. The rapid increase of the crystalline structure developed under biotic conditions makes these minerals less resistant to thermal treatments, compared with samples of abiotic origin. This result holds both for recent shells as well as all fossil samples. The spectroscopic behaviour of all analysed calcium carbonates of biotic origin is different from that of the abiotic one. Therefore, the infrared spectroscopy is a valid technique to discern the origin of the samples and a powerful tool for analysing in-situ and “sample-return” Mars missions specimens. Also Optical and Scanning Electron Microscopy can be useful to support this type of studies. ^*Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005     

A new technique for preparing biominerals for atomic-force microscopy

Summary We describe a methodology that enables the orienting and cleaving of biominerals to consistently yield low z-axis (height) surfaces suitable for high-resolution imaging by atomic-force microscopy. The methodology permits biominerals to be prepared without chemical fixation, resin embedding, or sectioning, and demonstrates a utility for a variety of silicified and calcified biominerals.     

Fingerprint‐to‐CH stretch continuously tunable high spectral resolution stimulated Raman scattering microscope

Stimulated Raman scattering (SRS) microscopy is a label‐free method generating images based on chemical contrast within samples, and has already shown its great potential for high‐sensitivity and fast imaging of biological specimens. The capability of SRS to collect molecular vibrational signatures in bio‐samples, coupled with the availability of powerful statistical analysis methods, allows quantitative chemical imaging of live cells with sub‐cellular resolution. This application has substantially driven the development of new SRS microscopy platforms. Indeed, in recent years, there has been a constant effort on devising configurations able to rapidly collect Raman spectra from samples over a wide vibrational spectral range, as needed for quantitative analysis by using chemometric methods. In this paper, an SRS microscope which exploits spectral shaping by a narrowband and rapidly tunable acousto‐optical tunable filter (AOTF) is presented. This microscope enables spectral scanning from the Raman fingerprint region to the Carbon‐Hydrogen (CH)‐stretch region without any modification of the optical setup. Moreover, it features also a high enough spectral resolution to allow resolving Raman peaks in the crowded fingerprint region. Finally, application of the developed SRS microscope to broadband hyperspectral imaging of biological samples over a large spectral range from 800 to 3600 cm^?1, is demonstrated.     

Electron backscatter diffraction (EBSD) analysis of maniraptoran eggshells with important implications for microstructural and taphonomic interpretations

Electron backscatter diffraction (EBSD) is a useful tool for gathering crystallographic information from carbonate biominerals because it maps out the orientation of crystal grains very precisely. EBSD has become popular in invertebrate palaeontology but its application in vertebrate palaeontology remains limited. However, the study of fossil eggshells is a field where EBSD has wide potential applicability and provides a quantitative approach to fossil eggshell research as well as new qualitative data. Here we analyse fossil and extant maniraptoran dinosaur eggshells using EBSD analysis emphasizing four different aspects. The mapping imaging clarifies previously ambiguous characters such as squamatic ultrastructure and allows a more objective evaluation of avian and non‐avian maniraptoran eggshell. In particular, our results imply that the presence of an external zone in the manirpatoran eggshell is not diagnostic of avian eggshell. EBSD analysis can be also used for differentiating true pore canals from cracks in the eggshell radial section, thereby determining the biological genuineness or otherwise of a pore‐like structure. Finally, the misorientation angle distribution of the material shows a clear dichotomy that may reflect reproductive brooding strategy, although further studies on contact incubation of palaeognaths and neognaths are needed to confirm this.     

The role of secondary ion mass spectrometry (SIMS) in biological microanalysis: technique comparisons and prospects.

The virtues and limitations of SIMS ion microscopy are compared with other spectroscopic techniques applicable to biological microanalysis, with a special emphasis on techniques for elemental localization in biological tissue (electron, X-ray, laser, nuclear, ion microprobes). Principal advantages of SIMS include high detection sensitivity, high depth resolution, isotope specificity, and possibilities for three-dimensional imaging. Current limitations, especially in comparison to X-ray microanalysis, center on lateral spatial resolution and quantification. Recent SIMS instrumentation advances involving field emission liquid metal ion sources and laser post-ionization will help to minimize these limitations in the future. The molecular surface analysis capabilities of static SIMS, especially with the new developments in commercial time-of-flight spectrometers, are promising for application to biomimetic, biomaterials, and biological tissue or cell surfaces. However, the direct microchemical imaging of biomolecules in tissue samples using SIMS will be hindered by limited concentrations, small analytical volumes, and the inefficiencies of converting surface molecules to structurally significant gas phase ions. Indirect detection using elemental or isotopically tagged molecules, however, shows considerable promise for molecular imaging studies using SIMS ion microscopy.     

Quasi-cubic magnetite/silica core-shell nanoparticles as enhanced MRI contrast agents for cancer imaging

Development of magnetic resonance imaging (MRI) contrast agents that can be readily applied for imaging of biological tissues under clinical settings is a challenging task. This is predominantly due to the expectation of an ideal MR agent being able to be synthesized in large quantities, possessing longer shelf life, reasonable biocompatibility, tolerance against its aggregation in biological fluids, and high relaxivity, resulting in better contrast during biological imaging. Although a repertoire of reports address various aforementioned issues, the previously reported results are far from optimal, which necessitates further efforts in this area. In this study, we demonstrate facile large-scale synthesis of sub-100 nm quasi-cubic magnetite and magnetite/silica core-shell (Mag@SiO2) nanoparticles and their applicability as a biocompatible T2 contrast agent for MRI of biological tissues. Our study suggests that silica-coated magnetite nanoparticles reported in this study can potentially act as improved MR contrast agents by addressing a number of aforementioned issues, including longer shelf life and stability in biological fluids. Additionally, our in vitro and in vivo studies clearly demonstrate the importance of silica coating towards improved applicability of T2 contrast agents for cancer imaging.     

New magnetic resonance contrast agents as biochemical reporters

Magnetic resonance imaging is a noninvasive, volume rendering diagnostic technique that uses lanthanide complexes to enhance proton relaxation. Magnetic resonance imaging is not limited by light scattering as optical microscopic techniques are, and allows imaging of whole animals. Clinical contrast agents are nonspecific and report solely on anatomy, whereas contrast agents that can be activated can be tailored to report on the physiological status or metabolic activity of biological systems. These new classes of magnetic resonance contrast agents represent a substantial leap in the type of information that can be derived from imaging experiments, and are the focus of this review.     

A novel spectrofluorometric technique for specific biocompatibility testing of implantable materials by cell culture. Report on use for multiparameter analysis of human osteoblasts cultured on commercially pure titanium and hydroxyapatite

The authors describe a novel spectrofluorometric technique based on double-labelled fluorescence imaging using immunoconjugates labelled with fluorochromes. Following isolation and characterization, cells are seeded on the surface of disks of the material(s) to be tested. After application of a primary antibody and an antibody bearing a fluorochrome, the signal emitted by the molecules in the extracellular matrix on the surface of the test disks is measured by spectrofluorimetry. Measurement is thus independent of the surface characteristics of the test material. Measured values are compared with pre-established standard curves. This technique facilitates determination of the characteristic molecules expressed by a given cell type,thus allowing accurate evaluation of the response of pertinent biological samples to implantable biomaterials. This revised version was published online in July 2006 with corrections to the Cover Date.     

Atomic force microscopy: from cell imaging to molecular manipulation

The atomic force microscope (AFM) allows to explore the surface of biological samples bathed in physiological solutions, with vertical and horizontal resolutions ranging from nanometers to angstr?ms. Complex biological structures as well as single molecules can be observed and recent examples of the possibilities offered by the AFM in the imaging of intact cells, isolated membranes, membrane model systems and single molecules are discussed in this review. Applications where the AFM tip is used as a nanotool to manipulate biomolecules and to determine intra and intermolecular forces from single molecules are also presented.     

Tuning of the Zernike phase-plate for visualization of detailed ultrastructure in complex biological specimens

In order to acquire phase-contrast images with adequate contrast, conventional TEM requires large amount of defocus. Increasing the defocus improves the low-frequency components but attenuates the high-frequency ones. On the other hand, Zernike phase-contrast TEM (ZPC-TEM) can recover low-frequency components without losing the high-frequency ones under in-focus conditions. ZPC-TEM however, has another problem, especially in imaging of complex biological specimens such as cells and tissues; strong halos appear around specimen structures, and these halos hinder the interpretation of images. Due to this problem, the application of ZPC-TEM has been restricted to imaging of smaller particles. In order to improve the halo appearance, we fabricated a new quarter-wave thin film phase-plate with a smaller central hole and tested it on vitreous biological specimens. ZPC-TEM with the new plate could successfully visualize, in in-focus images, the intracellular fine features of cultured cells and brain tissues. This result indicates that reduction of the central hole diameter makes ZPC-TEM applicable on size scales ranging from protein particles to tissue sections. The application of ZPC-TEM to vitreous biological specimens will be a powerful method to advance the new field of imaging science for ultrastructures in close-to-physiological state.     

Computer-integrated polarisation (CIP) in the analysis of fossils: a case of study in a Palaeozoic coral (Sinopora,Syringoporicae, Carboniferous)

Computer-integrated polarisation (CIP) method has been applied satisfactorily in the study of fossils skeletons of Sinopora (tabulate coral, Auloporida and Carboniferous). A previous characterisation of sample by scanning electron microscopy, atomic force microscopy and cathodoluminescence (CL) with the purpose of distinguishing the diagenetical alteration was done. Subsequently, a crystallographic comparison between CIP and electron-backscattering diffraction has been made getting a very good correlation between both methods. The CIP method allows obtaining c-axis orientation images, pole figures, and measure and mapping the misorientation of uniaxial biominerals in recent and fossil skeletons. This technique can only be used in uniaxial biominerals (calcite, quartz and hydroxylapatite), limiting its use for biaxial or bimineralic and polimineralic biominerals. CIP method has good spatial resolution (limited by camera); in our example 90 nm. The main advantage of this technique, versus other with similar properties, is the fast acquisition of data in low and high magnifications. This method does not require special treatment of samples and can be very useful for the analysis of microstructures in thin and ultra-thin sections. CIP method detects diagenetic alterations in fossil skeletons by modifications in the inner arrangement of biominerals, which combined with CL offers valuable geochemical and crystallographic information.     

Characterization of biominerals in species of Canna (Cannaceae)

Plant biominerals are not always well characterized, although this information is important for plant physiology and can be useful for taxonomic purposes. In this work, fresh plant material of seven wild neotropical species of genus Canna, C. ascendens, C. coccinea, C. indica, C. glauca, C. plurituberosa, C. variegatifolia and C. fuchsina sp. ined., taken from different habitats, were studied to characterize the biominerals in their internal tissues. For the first time, samples from primary and secondary veins of leaves were investigated by means of infrared spectroscopy, complemented with X-ray powder diffractometry and scanning electron microscopy. The spectroscopic results, supported by X-ray powder diffractometry, suggest that the calcium oxalate is present in the form of whewellite (CaC2O4 x H2O) in all the investigated samples. It is interesting to emphasize that all IR spectra obtained were strongly similar in all species studied, thus indicating an identical chemical composition in terms of the biominerals found. In this sense, the results suggest that the species of Canna show similar ability to produce biogenic silica and produce an identical type of calcium oxalate within their tissues. These results can be an additional trait to support the relationship among the families of Zingiberales.     

Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy

In this protocol, we describe a 3D imaging technique known as 'volume electron microscopy' or 'focused ion beam scanning electron microscopy (FIB/SEM)' applied to biological tissues. A scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and a backscattered electron (BSE) detector, used to image the milled surfaces, generates a large series of images that can be combined into a 3D rendered image of stained and embedded biological tissue. Structural information over volumes of tens of thousands of cubic micrometers is possible, revealing complex microanatomy with subcellular resolution. Methods are presented for tissue processing, for the enhancement of contrast with osmium tetroxide/potassium ferricyanide, for BSE imaging, for the preparation and platinum deposition over a selected site in the embedded tissue block, and for sequential data collection with ion beam milling; all this takes approximately 90 h. The imaging conditions, procedures for alternate milling and data acquisition and techniques for processing and partitioning the 3D data set are also described; these processes take approxiamtely 30 h. The protocol is illustrated by application to developing chick cornea, in which cells organize collagen fibril bundles into complex, multilamellar structures essential for transparency in the mature connective tissue matrix. The techniques described could have wide application in a range of fields, including pathology, developmental biology, microstructural anatomy and regenerative medicine.     

Scanning tunneling microscopy with applications to biological surfaces

Each major advance in the field of microscopy has eventually been translated into major advances in the biological and medical sciences. The scanning tunneling microscope (STM) offers exciting new ways of imaging biological surfaces with resolution to the sub-molecular scale. Rigid, conductive surfaces can readily be imaged with the STM with atomic resolution. Unfortunately, few biological surfaces are sufficiently conductive or rigid enough to be examined directly with the STM. At present, non-conductive surfaces can be examined in two ways: 1) Sufficiently thin molecular layers attached to conductive substrates so that tunneling can occur through the molecules; or 2) coating or replicating non-conductive surfaces with metal layers so as to make them conductive, then imaging with the STM. We present images of biological and organic molecules obtained with these techniques that demonstrate the possibilities and limitations of each. Future advances leading to atomic resolution STM of biological surfaces depend on significant progress in the art and science of making biomaterials compatible with the restrictions of the instrument.     

Chemical contrast for imaging living systems: molecular vibrations drive CARS microscopy

Cellular biomolecules contain unique molecular vibrations that can be visualized by coherent anti-Stokes Raman scattering (CARS) microscopy without the need for labels. Here we review the application of CARS microscopy for label-free imaging of cells and tissues using the natural vibrational contrast that arises from biomolecules like lipids as well as for imaging of exogenously added probes or drugs. High-resolution CARS microscopy combined with multimodal imaging has allowed for dynamic monitoring of cellular processes such as lipid metabolism and storage, the movement of organelles, adipogenesis and host-pathogen interactions and can also be used to track molecules within cells and tissues. The CARS imaging modality provides a unique tool for biological chemists to elucidate the state of a cellular environment without perturbing it and to perceive the functional effects of added molecules.     

Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging.

Multiphoton excitation fluorescence imaging generates an optical section of sample by restricting fluorophore excitation to the plane of focus. High photon densities, achieved only in the focal volume of the objective, are sufficient to excite the fluorescent probe molecules by density-dependent, multiphoton excitation processes. We present comparisons of confocal with multiphoton excitation imaging of identical optical sections within a sample. These side-by-side comparisons of imaging modes demonstrate a significant advantage of multiphoton imaging; data can be obtained from deeper within biological specimens. Observations on a variety of biological samples showed that in all cases there was at least a twofold improvement in the imaging penetration depth obtained with multiphoton excitation relative to confocal imaging. The more pronounced degradation in image contrast deep within a confocally imaged sample is primarily due to scattered emission photons, which reduce the signal and increase the local background as measurements of point spread functions indicated that resolution does not significantly change with increasing depth for either mode of microscopy. Multiphoton imaging does not suffer from degradation of signal-to-background to nearly the same extent as confocal imaging because this method is insensitive to scatter of the emitted signal. Direct detection of emitted photons using an external photodetector mounted close to the objective (possible only in a multiphoton imaging system) improves system sensitivity and the utilization of scattered emission photons for imaging. We demonstrate that this technique provides yet further improvements in the capability of multiphoton excitation imaging to produce good quality images from deeper within tissue relative to confocal imaging.