We applied phase-shifting digital holography to microscopy in this paper. At first lensless microscopy is proposed, in which no optical adjustment is necessary. Then, the method is applied to relax the limitation of focal depth in traditional optical microscopy. A theory for image formation and experimental verification using a few specimens are described.

keywords: microscopy, digital holography, phase shifting

Due to the finite focal depth of an imaging lens, a limitation to normal optical microscopy-is that, only the 2-dimensional (2-D) information of an object can be obtained at one time. Besides, it is not convenient for quantitative analysis the observed image. Optical sectioning microscopy (OSM) and scanning confocal microscopy (SCM) which use opto-electronic detection have been proposed for quantitative analysis of a 3-D object. However, the former requires critical mechanical adjustment, while the latter uses timeconsuming mechanical 3-D scanning. Holographic microscopy can solve these problems because it can record 3-D information at one time. But, the chemical processing of holograms and the mechanical focusing at the reconstructed images cause more or less trouble. A 3-D imaging technique without use of photographic recording called optical scanning holography has recently been reported. However, there are also some trouble owing to the twin-image noise.

A multiport-readout, frame-transfer charge coupled device (CCD) digital imaging system has been successfully constructed and tested for intermediate-high voltage electron microscopy (IVEM) applications up to 400 keV. The system employs a back-thinned CCD, made by MIT Lincoln laboratories, that comprises 2560 × 1960 pixels and a pixel size of 24 μm × 24 μm. With a frame transfer design, the imager fills nearly the entire usable area of a 100 mm-diameter silicon wafer (FIG. 1). In the current implementation, four of the eight on-chip readout ports are utilized in parallel each operating at a pixel rate of 1 or 2 MHz so that the entire CCD array can be read out in as short as ∼0.6 seconds. The frame-transfer readout functions as an electronic shutter which permits the rapid transfer of charges in the active pixels to four light-shielded buffers (FIG. 1) where the charges are readout and digitized while the active area of the CCD is integrating the next frame. At 2 MHz, charge transfer of 980 CCD rows will be completed in under 0.5 ms, which is much shorter than a typical exposure time of a few seconds. The camera head (FIG.2) and control electronics for CCD sensor were packaged by Photometries (Tuscon, Arizona). Two MaxVideo 200 image processing boards from Datacube (Peabody, MA) are used for high speed online imaging processing. The CCD sensor is electronically cooled to ∼ −40°C during operation.

For many years, we have been viewing stereo images created from a wide variety of microscopic sources. These images need to be easily viewed, shared and presented for stereo imagery to become more mainstream. We have been working for several years now to create affordable high resolution stereo display systems using the modern digital computers’ display capabilities. In the last couple of years, two methods have come into widespread use in our laboratory. One using shuttered goggles and the other using an active polarizing screen in front of the monitor. Both are synchronized with the display at refresh rates that allow for flicker free viewing. We use standard video display cards and shuttered glasses that cost under $300 (complete).

Unfortunately, the software and video cards turn over at such a fast rate that we have found our hardware “orphaned” several times.

One of the problems with remote imaging which purports to represent the real world (Tele-presence) is that the images are two-dimensional and the “real” world is three dimensional. When viewing many objects, the lack of depth perceptiorican be a serious deficiency. For example, one would want a micro-surgeon to have a good idea how deep to cut into tissue during a tele-presence operation. There is a wealth of three dimensional information in light, SEM, AFM, confocal, and computer reconstructed or simulated objects. To transfer that information faithfully over the networks using broadcast quality high definition video transmission technologies such as MPEG2 may require as much as 20 Mbps per channel. Furthermore, stereo imaging is particularly sensitive to any phase delays and jitter between the two eye channels. Control of these parameters requires advance quality of service features that current internet does not provide, but that may be available on high-performance intranets. All this may present a problem for routine use of the stereo facility .

The next generation of Internet, however, will be able to routinely provision much higher bandwidth and other quality of service, in general through protocols that will allow for not only full screen full resolution television but also the use of two channels (left and right) that can stay in fairly close synchrony.

A stacked CMOS active pixel image-sensor (APS) has been developed for detecting various kinds of charged particles and its noise performance has been measured and analyzed. The sensitivity for ions and electrons with keV energy level utilizes for ion microscopy such-as SIMS and electron microscopy, respectively.

Charge particles such as ions and electrons with kinetic energy of keV order are useful probes for surface analysis of material. A measurement system which yields two-dimensional image of charge particles is highly demanded. The conventional two-dimensional detection system is composed of a micro channel plate, a florescent plate which receives multiplied secondary electrons and generates a visible image, and a visible image sensor. However, its limited dynamic range and non-linearity in the ion-electron-to-photon conversion process make a quantitative measurement difficult. The proposed system using a stacked CMOS APS has several advantages over the conventional system such as high spatial resolution, no insensitive time, high S/N, wide dynamic range, nondestructive readout capability, high robustness, and low power consumption.

Many of the repetitive measurements required in the analysis of images in the semiconductor field can be replaced by partially or fully automated measurements. Automated measurements provide a number of benefits, including speed, repeatability, and removal of operator bias. The question of accuracy and precision in metrology applications is important to understand.

Recently we implemented a tool for automated analysis of resist bar images that was designed for use in acceptance testing-of lithographic stepper machines. The steppers are required to meet a defined set of criteria over the entire area of the wafer and through the focus range of the lens. Prior to the development of this package, the measurements were all done with a ruler and protractor on film.

The software identifies the boundary lines of the resist bar by locating a series of points along each edge and then fitting a line to those points. Edge identification is complicated by coating artifacts and by the variety in the images being analyzed. After identifying edge points along the base, side, and top of the resist bar, the software automatically generates information about the width, height, side wall angles, and various other measurements of the resist bar and stores the data in spread sheet format.

The development of high and intermediate voltage electron microscopes provides the ability to investigate the ultrastructure of thick biological samples. This produces a unique view of the 3-D interrelationships of cells and organelles. However, the analysis and display of this 3-D information has presented challenges, particularly to quantitative biologists. Major advances in adapting computed tomography (CT) to electron microscopy have provided the major means of 3-D analysis of cellular ultrastructure (1,2). In particular, tremendous gains in determining the structure of macromolecules have occurred with advances in electron microscopy CT (3). However, CT remains an arduous undertaking and one not well suited to analysis of the structural alterations within large populations of cells requiring many reconstructions. We have adapted an alternative approach, Tuned Aperture Computer Tomography (TACT®), for use with high and intermediate voltage electron microscopes. Although with EM specimens TACT® does not usually produce as high Z axis resolution as CT, the simplicity of the TACT® algorithms lend themselves to the rapid analysis of multiple samples. TACT® is already gaining acceptance in radiology (4, 5). Like CT, TACT® uses a series of images of the sample taken from different angular perspectives. However, unlike CT, for TACT® only a few projections are required and thus good reconstructions of beam sensitive material are possible. The quality of the reconstruction is determined by the quality of the input images, the total angular spread, and the individual tilt increments between images. Better images, a greater angular spread, and smaller angles between images will produce more accurate results.

Molecular microscopy is one of the most important structural approaches in cell biological investigations and can provide insight into complex biological questions that no other technique can provide. Currently, the technique typically requires the acquisition of very large numbers of transmission electron micrographs from frozen hydrated specimens using low dose techniques. The field is constrained by manual data acquisition methods that are slow, labor-intensive and result in a very low percentage of suitable images. We have developed a system, called Leginon, for automatically acquiring images from a transmission electron microscope. Our first prototype of this system demonstrated that we could acquire 1000 high magnification images per day from negatively stained catalase crystals. We have now extended this system to acquire low dose images of specimens embedded in vitreous ice.

Methods: Specimens were prepared on Quantifoil grids using techniques which have been described previously. The Leginon system uses a Philips CM200 TEM and a Gatan MSC CCD camera and is controlled by the emScope software library. The overall acquisition protocol requires (i) obtaining a low magnification image [660x] of a grid square from a Quantifoil grid (fig. 1a); (ii) automatically identifying holes containing ice of suitable thickness; (iii) acquiring an intermediate magnification image [6600x] of the identified hole (fig. 1b); (iv) identifying features of interest within the hole; (v) focusing at high magnification [38,000x] and finally (vi) acquiring a high magnification image (fig 1c,d).

Treatment of human congenital malformations and for loss of tissue or organ function is a serious clinical concern, frequently supported by harvesting autogenous tissue for reconstructive surgery. This paper summarizes an alternate approach through novel tissue engineering methods and describes cell-polymer composites that could be fundamental in developing advances for digit and joint replacement. The work utilizes cells from different sources transplanted on biodegradable polymer matrices to form structures with defined shape, morphological features and composition resembling those of human phalanges and intact joints.

Polyglycolic acids (PGA) fibers (Albany International, Mansfield, MA) 15 μm in diameter were used as a mesh support for cell attachment and matrix formation. For cartilage and tendon cells, PGA alone served as a flat surface without specific shape. For bone cells, PGA was copolymerized with poly-L-lactic acid (PLLA) (Polysciences, Warrington, PA) and shaped after a human phalanx molded from cadaveric distal and middle phalangeal bones. Polymers were sterilized with ethylene oxide before use.

Study of the microscopic structure of bone dates back to the earliest use of the optical microscope. Understanding the complexities of the structure of this material has roughly paralleled advances in microscopic technique. Relatively recently a new powerful microscopic technique, atomic force microscopy (AFM), has been invented. So it should not be surprising to expect a new step in our understanding of bone. There have been several recent publications applying AFM to bone and its constituents. None of these studies have examined the shape of naturally occurring mineralites in bone. The issue we addsgss in this work is the elucidation of the three dimensional structure of the apatite mineralites in bone. Chiefly, two methods have been used previously to ascertain this structure: transmission electron microscopy (TEM) and X-ray diffraction (XRD,). The major difficulty with TEM is that it is a projection technique. Each image only yields information about geometry in a single plane. Landis et al. showed how to circumvent this problem using tomographic TEM. AFM provides a corroborative and perhaps more gentle method of directly measuring the structure of very small bone mineralites. It is possible that the interaction energy of the electron beam with very small mineralites is sufficient to vaporize them. The problem with XRD is that the line broadening used can have contributions from sources not directly related to mineralite size. In particular, if a single mineralite contains more than one crystalline domain, then the line broadening yields a measure of the domain size, not the overall mineralite size.

In order to understand the systematic fabrication of hierarchical structures found in biominerals, the process nature uses in the biosynthesis of systems with complex morphology and precise internal molecular organization must be mimicked. The multi-component organic supramolecular system reported here is a kinetically controlled model that demonstrates the three stages of biomineralization: supramolecular pre-organization, interfacial molecular recognition, and cellular processing.

Three molecular components are used in this system: acetic acid (HOAc), benzene, and Kemp's Triacid (l,3,5-Trimethylcyclohexane-l,3,5-tricarboxylic acid or KTA). KTA and HOAc cocrystallize as hydrogen bonded aggregates of KTA dimers capped by four HOAc molecules. KTA and benzene co-crystallize with benzene acting as spacers between one dimensional hydrogen bonded rod-like structures of KTA. Growth of microcrystalline KTA/benzene was initiated by immersing single crystals of KTA/HOAc in 1 mL of benzene for 10-120 sec. The product was isolated on filter paper and mounted with carbon tape on an aluminum stub. The samples were coated with ∼10 nm of 60/40 Au/Pd prior to imaging at 15 kV in a DS-130/LaB6 SEM or at 5kV in a DS-130FE SEM.

The need to optimize adhesion between the femoral stem and the surrounding tissue has led to the development of plasma sprayed HA-coated implants as attachment veichles for hip prostheses. The strength of attachment of bone to the HA surface cannot be fully explained by mechanical bonding at the macroscopic or microscopic level; thus, molecular interactions must contribute significantly to bone bonding, facilitating adhesion at the nanometer level. Observations of apatite crystallites and cell adhesion proteins in the direct vicinity of coated implants indicates that these play a role in modifying the surface for osteoblast attachment and subsequent collagen deposition.

An understanding of the mechanism of “bone bonding” could lead to improvements in the design of HA coatings, leading to enhanced performance of orthopedic implants. Transmission electron microscopy (TEM) has been used to elucidate a sequence of early bone bonding events at the interface of PSHA-coated titanium implants.

The deposition of biological apatite and subsequent formation of a bone/HA bond seem to depend on the dissolution of the coating and the reprecipitation of carbonated apatite. Previous investigations in vitro have shown that the degree of dissolution and reprecipitation decreases as the coating crystallinity increases. Thus, coatings which had undergone heat treatment were used to look at the effect of solubility of the coating on mechanism of bone bonding.

The enamel of human teeth is composed of parallel rods (bundles) of long, closely-packed, ribbonlike hydroxyapatite (HAP) Ca10(PO4)6(OH)2 crystallites oriented normal to the tooth surface. Both the unique structure of the crystallites and the higher order arrangement of the crystallites are believed to be a consequence of interactions between proteins secreted by the enamel forming tissues and the growing crystallites. A better understanding of the precise molecular mechanism that controls enamel crystallite formation could potentially lead to both novel enamel repair strategies and crystal growth control systems. It has been previously reported that the unidirectional flow of calcium ions across a membrane can be used to form ribbon-like structures of a related form of calcium phosphate. We have created a similar model system which utilizes the unidirectional flow of calcium ions to generate ribbon-like HAP crystallites. Ultimately, this system will be modified so that enamel proteins can be introduced into the crystallization milieu to better characterize their role in enamel formation.

SEM and TEM samples were prepared using the experimental setup shown in Fig. 1. Several tubes containing a 50mM HAP solution dissolved in 0.1M HC1 were placed upside down into a 50mM MOPS pH buffering solution at pH 7.0.

Neural Tissue Engineering involves the use of three-dimensional biomaterial scaffolds for transplantation of neural tissue or for induction of regeneration of severed peripheral or central nerves. There is a paucity of information regarding the optimization parameters for the design of 3D constructs from the mechanical, morphological, or charge perspective such that neurite extension is maximized. In addition, cytoskeletal organization and response of neurons in 3D matrices to various stimuli is not well understood. Our laboratory has been using a combination of electron, force, light, video and confocal microscopy to address these issues. Custom surface rendering image reconstruction techniques were used to reconstruct the three-dimensional structure of neurons extending processes in 3D as visualized by confocal microscopy. We have previously reported that low concentration gels of hydroxyethylated agarose are capable of supported neurite extension from a variety of neural cells in vitro.

Agarose hydrogels were fabricated in concentrations ranging from 0.75%-3.0% (w/v). These highly hydrated gels were prepared for electron microscopy using a variety of sample preparation protocols including freeze drying, and critical point drying and their morphological structure and pore radius was computed using scanning electron microscopy.

Near Field Scanning Optical Microscopy (NSOM) is the newest member of the Scanning Probe Microscopy (SPM) family, which includes Scanning Tunneling Microscopy (STM) and Atomic Force Microscopy (AFM). Like its predecessors, it uses a sharp tip and a very precise positioning system to scan the surface of samples. The important difference is that the NSOM tip is made of an optical fiber, with a sub-wavelength size aperture at its end, which is brought to near-field proximity to the sample. In the near-field regime, the resolution is only limited by the size of the light source (the aperture), and not by the wavelength of light, thus allowing NSOM to obtain images with subwavelength resolution. Applying NSOM to biological samples is very appealing, since biological research has developed a wide variety of fluorescent dyes which can be directed with high specificity to objects of interest. Moreover, biological research relies to a large extent on conventional fluorescence microscopy, which is diffraction limited. Application of NSOM to biological problems requires the ability to image rough and soft samples in liquid, a task that presents many challenges.

We have developed a NSOM which is capable of imaging cells in liquid with a resolution of ∼150 nm. This has enabled us to image the organization of fluorescently labeled proteins on the membrane of cells in liquid, and demonstrate for the first time that the proteins are clustered.

Transfection of mammalian cells can be enhanced by forming complexes between exogenous DNA and cationic peptides which facilitate DNA transport through cell membranes. Although complexes of plasmid DNA and cationic peptides have been characterized by microscopy, little is known about the complexes formed between oligonucleotides (ODN) and cationic peptides or how the ultrastructure of these complexes affects the intracellular delivery via the cell membrane. Because ODN have potential as therapeutic reagents in the treatment of many diseases, there is a need to develop improved methods for effective delivery of these molecules. By analyzing molecular interactions between cationic peptides and ODN and their effects on cell membranes, cell viability, and DNA delivery, improved delivery compounds can be designed. In this paper, we compare the interactions of a 28 base ODN with three cationic peptides: a novel compound, MNLEK (H-Met-(Nle-Lys4)7-Nle-NH2), an established transfection reagent, poly(L-lysine) (∼ 54 kDa), and a recently described cationic peptide which delivers both plasmid DNA and antisense ODN to cells, KALA (WEAKLAKALAKALAKHLAKALAKALKACEA).

For high resolution scanning electron microscopy (HRSEM), DU145 human prostate cancer cells were grown overnight on poly-(D-lysine) coated, UV irradiated silicon chips.

Nano-mechanical properties of composite biomaterials are governed by the morphological features and chemical distribution of heterogeneous multiple subunits. However, very little is known about such structure-function correlation at the nanoscale. We have used wool fibers as a model of composite biomaterials for such study. Wool fibers provide an ideal two component cylindrical model for such study as their ultrastructure and chemical composition have been well characterized, they have a core cellular component surrounded by an outer cell layer. The core is 16-40 micron in diameter and rich in axially aligned keratin microfibrils. Outer cells have multiple laminar layers, 60-600 nm thick and are distinctly rich in disulfide bonds. In this study, we have used an atomic force microscope (AFM) to examine the micromechanical properties of the different structural components using complementary techniques of nano-indentation and force-volume imaging. AFM images of transverse sections of wool fibers were obtained in ambient environment (Figure 1).

The exo-cuticle showed a significantly higher mechanical stiffness than the internal cellular core region (Figure 2).

Gel networks that mimic the properties of naturally occurring mammalian elastin have been synthesized for the development of novel biomaterials. Hydrogels with controllable and predictable mechanical properties are pursued for use in drug encapsulation and delivery systems, as scaffolds for tissue engineering and for the development of prosthetic implantable devices. The techniques of genetic engineering and microbial expression have been used to produce a 90KD recombinant protein based starting material that was polymerized into a solvent swollen gel network by the introduction of covalent bifunctional crosslinks at regularly spaced lysyl residues. SEM methods have been used to describe solvent and temperature dependent structural features.

The polypeptide precursor to the elastin-mimetic gels, poly[(VPGVG)4VPGKG], was obtained through bacterial expression (pET system) of an oligomerized gene coding for tandem repeats of the monomer. Gel networks were formed by crosslinking the protein with a bifunctional NHS-ester in both water and anhydrous DMSO. For conventional below-lens SEM studies, samples were dehydrated in an ethanol gradient, critical point dried from CO2(l), mounted on stubs with carbon tape and sputter coated with a 4-6 nm layer of Au/Pd.

Adherent cells generally construct the immediate substrate upon which they reside. This may occur via synthesis and secretion of new materials and/or by rearrangement and modification of existing substrate. The response of adherent cell types to an existing substrate can be influenced by a number of factors which include both the chemical and physical nature of the substrate. Cell adhesion, proliferation, differentiation and death can all be substrate dependent. Much effort has been directed toward chemical modification of substrates to regulate one or more of the parameters noted above. A significant, but somewhat smaller, degree of attention has been paid to the effects of the topography and microtopography on the cell response to substrate materials. Studies to date strongly suggest the topography is a significant factor in cell-substrate interactions. As noted above, it is most probable that both the chemistry and the structure of a substrate simultaneously influence the cellular response. However we wished to determine, particularly for artificial substrates, the role which microtopography can play in cell-substrate interactions.

Every time we take a breath, we are inhaling the results of twentieth century combustion technology. Combustion processes generally produce a multitude of soot and other sub micron sized particulates. The human lungs, via the process of cilia movement expel most of these particles; others are broken down with the aid of macrophage agents. These macrophages absorb particles and incorporate the constitute elements into our bodies. These elements maybe expelled, or they may remain in the body and accumulate over time, as is the case with certain heavy metals. Limited prior research on ‘single-particle’ interaction with lung or bronchial tissue has been conducted. Related research has focused on the statistical significance of soot inhalation on the lung tissue of rodents and primates [1]. Using the methods of single particle examination, founded by previous research into single particle source allocation [2], the examination particles of from human lung and bronchial tissues was performed.

Research on the particle characterization shown here is based on the application of an innovative method developed at MIT, which utilized high resolution transmission electron microscopy (HRTEM), scanning transmission electron microscopy (STEM) coupled with electron energy loss spectroscopy (EELS) and energy dispersive X-ray analysis (EDX).