Three dimensional image restoration in fluorescence lifetime imaging microscopy

A microscope set-up and numerical methods are described which enable the measurement and reconstruction of three-dimensional nanosecond fluorescence lifetime images in every voxel. The frequency domain fluorescence lifetime imaging microscope (FLIM) utilizes phase detection of high-frequency modulated light by homodyne mixing on a microchannel plate image intensifier. The output signal at the image intensifier's phosphor screen is integrated on a charge coupled device camera. A scanning stage is employed to obtain a series of phase-dependent intensity images at equally separated depths in a specimen. The Fourier transform of phase-dependent data gives three-dimensional (3D) images of the Fourier coefficients. These images are deblurred using an Iterative Constrained Tikhonov–Miller (ICTM) algorithm in conjunction with a measured point spread function. The 3D reconstruction of fluorescence lifetimes are calculated from the deblurred images of the Fourier coefficients. An improved spatial and temporal resolution of fluorescence lifetimes was obtained using this approach to the reconstruction of simulated 3D FLIM data. The technique was applied to restore 3D FLIM data of a live cell specimen expressing two green fluorescent protein fusion constructs having distinct fluorescence lifetimes which localized to separate cellular compartments.     

High-resolution wave field reconstruction using a through-focus series of images taken under limited electron dose

The three-dimensional Fourier filtering method and Schiske's Wiener filtering method are compared with the aim of high-resolution wave field reconstruction of an unstained deoxyribonucleic acid (DNA) molecular fiber using a through-focus series of images taken under a limited electron dose. There were some definite differences between the two reconstructed images, although the two kinds of processing are essentially equivalent except for the dimension and the filter used for processing. Through theoretical analyses together with computer simulations, the differences were proved to be primarily due to specimen drift during the experiment. Although the observed structure of the DNA molecular fiber was heavily damaged by electron beam irradiation, reconstructed images by the three-dimensional Fourier filtering method provided higher resolution information on the molecular structure even when relatively large specimen drift was included in the through-focus series. In contrast, in Schiske's Wiener filtering method, the detailed information of the structure was lost because of the drift, although the reconstructed image showed a higher signal-to-noise ratio. The three dimensional Fourier filtering method seems to be more applicable for observing radiation-sensitive materials under an extremely low electron dose, because specimen drift cannot be completely avoided.     

Computed tomography of cryogenic biological specimens based on X-ray microscopic images

Soft X-ray microscopy employs the photoelectric absorption contrast between water and protein in the 2.34-4.38 nm wavelength region to visualize protein structures down to 30 nm size without any staining methods. Due to the large depth of focus of the Fresnel zone plates used as X-ray objectives, computed tomography based on the X-ray microscopic images can be used to reconstruct the local linear absorption coefficient inside the three-dimensional specimen volume. High-resolution X-ray images require a high specimen radiation dose, and a series of images taken at different viewing angles is needed for computed tomography. Therefore, cryo microscopy is necessary to preserve the structural integrity of hydrated biological specimens during image acquisition. The cryo transmission X-ray microscope at the electron storage ring BESSY I (Berlin) was used to obtain a tilt series of images of the frozen-hydrated green alga Chlamydomonas reinhardtii. The living specimens were inserted into borosilicate glass capillaries and, in this first experiment, rapidly cooled by plunging into liquid nitrogen. The capillary specimen holders allow image acquisition over the full angular range of 180 degrees. The reconstruction shows for the first time details down to 60 nm size inside a frozen-hydrated biological specimen and conveys a clear impression of the internal structures. This technique is expected to be applicable to a wide range of biological specimens, such as the cell nucleus. It offers the possibility of imaging the three-dimensional structure of hydrated biological specimens close to their natural living state.     

Three-dimensional distributions of elements in biological samples by energy-filtered electron tomography

By combining electron tomography with energy-filtered electron microscopy, we have shown the feasibility of determining the three-dimensional distributions of phosphorus in biological specimens. Thin sections of the nematode, Caenorhabditis elegans were prepared by high-pressure freezing, freeze-substitution and plastic embedding. Images were recorded at energy losses above and below the phosphorus L2,3 edge using a post-column imaging filter operating at a beam energy of 120 keV. The unstained specimens exhibited minimal contrast in bright-field images. After it was determined that the specimen was sufficiently thin to allow two-window ratio imaging of phosphorus, pairs of pre-edge and post-edge images were acquired in series over a tilt range of +/-55 degrees at 5 degrees increments for two orthogonal tilt axes. The projected phosphorus distributions were aligned using the pre-edge images that contained inelastic contrast from colloidal gold particles deposited on the specimen surface. A reconstruction and surface rendering of the phosphorus distribution clearly revealed features 15-20 nm in diameter, which were identified as ribosomes distributed along the stacked membranes of endoplasmic reticulum and in the cytoplasm. The sensitivity of the technique was estimated at < 35 phosphorus atoms per voxel based on the known total ribosomal phosphorus content of approximately 7000 atoms. Although a high electron dose of approximately 10(7)e/nm2 was required to record two-axis tilt series, specimens were sufficiently stable to allow image alignment and tomographic reconstruction.     

Techniques for obtaining and observing complementary images with a low-temperature field emission SEM and subsequent comparison of the identical cells in freeze-etch replicas viewed with a TEM

Initial evidence shows that low-temperature (LT) field emission scanning electron microscopy (FESEM) provides high-resolution complementary images of frozen, fractured biological tissues and that the coating or replicas from the tissues can be recovered and viewed in the TEM to compare the identical cellular structures. To observe frozen specimens, a Hitachi S-4000 FESEM was equipped with an Oxford CT 1500 Cryotrans System. The standard Oxford specimen carrier was modified to accommodate a Denton complementary freeze-etch cap that would hold up to 6 hinged 24K gold specimen holders. This combination allowed observation of complementary images of sputter-coated, freeze-etched biological specimens at magnifications up to X30,000. Resolution of sputter-coated images was also compared with that from evaporative coatings. Use of high-vacuum evaporation of Pt/C in conjunction with LT-FESEM provided useful resolutio up to X100,000. In addition, after these specimens were observed in the LT- FESEM, their coating, which consisted of a freeze-etch replica, could be recovered from the frozen tissues and subsequently observed in the TEM at even higher resolutions. Consequently, complementary images of frozen, fractured, fully hydrated cells could be observed in the LT-FESEM and then compared to the complementary images of the identical cells that were present in the replicas, which were recovered from the frozen specimens, and subsequently observed in the TEM. Being able to evaluate, compare, and contrast data from these two different EM imaging techniques as well as from complementary surfaces, not only provides additional information about the ultrastructure of a specimen, but also helps to assess the resolution of coating films, the presence of contaminants and the three-dimensional distortion in replicas.     

A nondestructive method for three-dimensional reconstruction of luminescence materials: Principles,data acquisition,image processing

In the study presented here we have tried to state the principles and calculate and visualize models of three-dimensional (3-D)-cathodoluminescence reconstruction of luminescence structures by scanning electron microscopy (SEM). The new technique does not destroy the specimen and uses the variable energy of the electron beam to penetrate to different depths in the specimen volume. The SEM in color cathodoluminescence mode (CCL-SEM) detects integrated panchromatic CL-images for different energies of the electron beam. The use of electron scattering theory in solids and theories of cathodoluminescence and color allow the production of problem-oriented software for the routine processing of primary images. Processed images represent the CCL-SEM displays of separated layers (without CL information from other layers) up to the maximum depth penetrated by the beam. The 3-D reconstruction is carried out through algorithms developed using a personal computer, software, and a set of processed two-dimensional (2-D) images. The first experimental work was accomplished using a multilayer SiC mesastructure. The final reconstructed image of SiC material demonstrates separated epitaxial layers of different SiC polytypes and Z sections (YOZ and XOZ sections). The 3-D image represents the space distribution of CL-spectral data in color CL interpretation.     

Feature-based image reconstruction in three dimensions for electron microscopy Part 2: Image reconstruction

The three-dimensional reconstruction of an electron microscopy specimen from a feature-based representation of the images taken from it is described. The method involves the assembling of the features, their correlation, and the measurement of the disparities between the original images.     

Improvement in the resolution of three-dimensional data sets collected using optical serial sectioning

In this paper an approach for improving the quality of 3-D microscopic images obtained through optical serial sectioning is described and implemented. A serially sectioned image is composed of a sequence of 2-D images obtained by incrementing the focusing plane of the microscope through the specimen of interest; ideally, the image obtained at each focusing plane should be in focus, and should contain information lying only within that plane. In practice, however, the images obtained contain redundant information from neighbouring focusing planes and are blurred by a three-dimensional low-pass distortion. These degradations are a consequence of the limited aperture of any optical system; using principles of geometric optics and allowing for the passage of light through the specimen, we are able to demonstrate that the microscope distortion can be described as a linear system, if the absorption of the specimen is assumed to be linear and non-diffractive. The transfer function of the microscope is found to zero a biconic region of 3-D spatial frequencies orientated along the optical axis; a closed-form expression is derived for the low-pass transfer function of the microscope outside the region of missing frequencies. The planar resolution of the serial sections can be greatly improved by convolving the image obtained with the inverse of the low-pass distortion function, although the missing cone of frequencies is not recoverable. The reconstruction technique is demonstrated using both simulated images, to demonstrate more clearly the effects of the distortion and the accuracy of the subsequent reconstruction, and actual experiments with a pollen grain and a stained preparation of human cerebellum tissue.     

Automated three-dimensional X-ray analysis using a dual-beam FIB

We present a fully automated method for three-dimensional (3D) elemental analysis demonstrated using a ceramic sample of chemistry (Ca)MgTiO(x). The specimen is serially sectioned by a focused ion beam (FIB) microscope, and energy-dispersive X-ray spectrometry (EDXS) is used for elemental analysis of each cross-section created. A 3D elemental model is reconstructed from the stack of two-dimensional (2D) data. This work concentrates on issues arising from process automation, the large sample volume of approximately 17 x 17 x 10 microm(3), and the insulating nature of the specimen. A new routine for post-acquisition data correction of different drift effects is demonstrated. Furthermore, it is shown that EDXS data may be erroneous for specimens containing voids, and that back-scattered electron images have to be used to correct for these errors.     

Seventh Report of the Nomenclature Committee

By using standard cine-micrography apparatus in conjunction with a simple turntable under the microscope it is possible to locate and orient serial sections for sequential photography on cine film. The location of the sections is sufficiently accurate to enable the film to be projected at normal speeds. The effect is to produce a steady image giving the viewer the impression of travelling through the specimen. This can assist in the interpretation of serial sections and in the three-dimensional reconstruction of microscopic structures. The film loops thus obtained can also be used directly for teaching purposes, allowing students to obtain quickly the mental images required for a three-dimensional understanding of microstructure. The method can be used in interpreting any series of sections without special preparation. It is particularly valuable for embryological studies and for investigating elongated sub-structures such as blood vessels.     

Analysis of fluorescence from algae fossils of the Neoproterozoic Doushantuo formation of China by confocal laser scanning microscope

Chinese algae fossils can provide unique information about the evolution of the early life. Thin sections of Neoproterozoic algae fossils, from Guizhou, China, were studied by confocal laser scanning microscopy, and algae fossils were fluorescenced at different wavelengths when excited by laser light of 488 nm, 476 nm, and 568 nm wavelength. When illuminated by 488 nm laser light, images of the algae fossils were sharper and better defined than when illuminated by 476 nm and 568 nm laser light. The algae fossils fluoresce at a wide range of emission wavelengths. The three-dimensional images of the fluorescent algae fossils were compared with the transmission images taken by light microscope. We found that the fluorescence image of the confocal laser scanning microscope in a single optical section could pass for the transmission image taken by a light microscope. We collected images at different sample depths and made a three-dimensional reconstruction of the algae fossils. And on the basis of the reconstruction of the three-dimensional fluorescent images, we conclude that the two algae fossils in our present study are red algae.     

Tomography of asymmetric bulk specimens imaged by scanning electron microscopy

The scanning electron microscope produces nanometer-resolution surface images of biological samples preserved in a life-like state. Extracting three-dimensional information from these two-dimensional images has been the subject of long and ongoing research. We present here a general method and theoretical basis for reconstructing the surfaces of SEM specimens imaged from multiple directions by back-projection. The resulting reconstructions are faithful representations of the original specimen geometry, even when the input images are blurred and have low signal-to-noise ratio.     

三维显微图像带约束的迭代解卷积复原算法

阐述显微镜透明厚样本成像原理和三维显微图像带约束的迭代解卷积复原算法。根据显微镜透明厚样本成像原理,对已知三维清晰图像进行退化处理,并且使用带约束的迭代解卷积算法去除退化图像中的散焦信息。试验结果表明,图像散焦信息的干扰得到有效的去除,清晰度和信噪比得到明显的改善,并且该算法可以恢复成像过程中丢失的部分频率成分,实现超分辨率复原。当迭代次数较大时,复原效果优于邻域法和线性方法。     

High-angle annular dark-field STEM observation of Xe nanocrystals embedded in Al

High-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) observation of Xe precipitates embedded in crystalline membranes has been made using electron probes of atomic dimensions and HAADF-STEM images of Xe precipitates qualitatively different from conventional TEM observation results have been obtained. Multislice-based HAADF-STEM simulation has been made and it has been revealed that the intensity of images of Xe atoms at positions displaced from Al matrix columns decreases rapidly as the thickness increases. Even in a thin specimen, the off-site Xe atoms of the precipitate at deep locations, were not observable. Therefore, different images are expected for specimens of different thicknesses or depths of these precipitates. These results indicate that the observation of precipitates in crystalline membranes requires some care.     

Atomic force microscopy of freeze-fracture replicas of rat atrial tissue

Atomic force microscopy (AFM) has provided three-dimensional (3-D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well-established technique of transmission electron microscopy (TEM) was performed. Freeze-fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large-scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3-D surface contour) and TEM (2-D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily available with TEM.     

Visualization of copper(II) in velvet mesquite (Prosopis Juliflora-Velutina) and soybeans (Glycine max) by earth field magnetic resonance imaging

The goal of this research was to visualize the uptake of copper by plants to promote phytoremediation. The hypothesis was that regions of the plant that contain more copper have enhanced nuclear magnetic resonance using copper as a paramagnetic contrast agent. Earth field magnetic resonance imaging (EFMRI) spectrometry uses a magnetic field 300,000 times weaker than a 600 MHz nuclear magnetic resonance spectrometer. As in conventional human MRI, this portable instrument is capable of recording one-, two-, and three-dimensional images, although the sample size is reduced. The enhancement of signal was first demonstrated recording the two dimensional images of different concentrations of aqueous copper sulfate. The study also focused on growing mesquite and soybean plants in hydroponic media containing 0.25 mM copper nitrate for four and three weeks, respectively, and recording their EFMRI images with plants grown without copper nitrate. The results showed that EFMRI allows the probing the plant absorption of copper without having to dissect the specimen and therefore requires less sample preparation and time than current techniques. This technique may be beneficial to phytoremediation by imaging plants to evaluate their copper accumulation.     

Processing of secondary ion microscope images: an example of application to the thyroid

Ion microscopy is a microanalytical method by which one can obtain distribution images of any chemical element with isotope discrimination even at very low local concentrations, in successive slices of the specimen. These images are obtained at the price of progressive erosion of the specimen, so that the analysis may not be replayed and it is necessary to record the maximum amount of information during specimen erosion. We present an improvement of this method using a highly sensitive camera connected to a video analog-digital converter. The images are acquired and digitized on line and may be processed by an image computer. We illustrate the technique described with an application of ion microscopy that is made possible by digital recording and processing of images. This application concerns the precise comparison of iodine isotopes and phosphorus distributions in sections of the thyroid gland of rats which were submitted to an iodine-deficient diet followed by an injection of ¹²⁹ I.     

Microtomography from limited projections in conventional TEM for 3D reconstruction of an intact cell

It is possible to generate three-dimensional reconstructions of whole, non-sectioned biological cells in conventional TEM using an 80 kV tungsten source. A TEM specimen stage was modified to accommodate a precise single-axis tilting mechanism controlled by a digital stepping motor interfaced to a computer. For image collection, a video camera was optically coupled to the TEM phosphorescent screen, and the video image was digitized by a frame buffer interfaced to a computer. Specimen tilt and projection image collection were fully computer-automated. This microtomography system design could be readily adapted for most TEMs. Image reconstruction was achieved through computation on projection images from limited tilts; typically less than thirty projection images were needed for a coarse 3D reconstruction. The iterative reconstruction algorithm used certain statistical assumptions about the distribution of image gray values. Since microtomography was performed on non-sectioned whole mount cells viewed under an 80 kV electron beam, methods of embedment-free specimen preparation with chemical fixation and extraction were employed. These methods were utilized successfully to permit good image formation of the entire cell mitotic nucleus a few micrometers in thickness. The 3D reconstruction of a single kidney cell mitotic nucleus was carried out and shown to produce a reasonable microtomogram of gross features like the condensed chromosomes.     

Methods for compensation of the light attenuation with depth of images captured by a confocal microscope

A confocal laser scanning microscope (CLSM) enables us to capture images from a biological specimen in different depths and obtain a series of precisely registered fluorescent images. However, images captured from deep layers of the specimen may be darker than images from the topmost layers because of light loss distortions. This effect causes difficulties in subsequent analysis of biological objects. We propose a solution using two approaches: either an online method working already during image acquisition or an offline method assisting as a postprocessing step. In the online method, the gain value of a photomultiplier tube of a CLSM is controlled according to the difference of mean image intensities between the reference and currently acquired image. The offline method consists of two stages. In the first stage, a standard histogram maintaining relative frequencies of gray levels and improving brightness and contrast is created from all images in the series. In the second stage, individual image histograms are warped according to this standard histogram. The methods were tested on real confocal image data captured from human placenta and rat skeletal muscle specimens. It was shown that both approaches diminish the light attenuation in images captured from deep layers of the specimen.     

Computer-simulated high-resolution transmission electron microscopy (HRTEM) in tourmaline

The contrast distributions observed in high-resolution transmission electron microscopy (HRTEM) images of tourmaline depend on the types and magnitudes of the exchange components present and on the degree of atom overlap along the direction of observation. Furthermore, the fractional atomic coordinates in tourmalines are valid only for the specific specimen refined. These properties make the interpretation of experimental HRTEM images of tourmaline using image simulation if not impossible at least extremely difficult. A correct interpretation of experimental HRTEM images of tourmaline is possible provided the structural refinement data on the same crystal are available. Nevertheless, it is possible to interpret the experimental HRTEM images of tourmaline if the composition of the structural model chosen during image simulations approximates the composition of the specimen studied by electron microscopy. A good control of the composition of the specimen studied and an appropriate choice of a structural model for image simulation are therefore as important as properly controlling specimen thickness, specimen tilt, beam tilt and objective lens defocus.