Optical Sectioning Microscopy Research Articles

Pico-projector-based optical sectioning microscopy for 3D chlorophyll fluorescence imaging of mesophyll cells

A pico-projector-based optical sectioning microscope (POSM) was constructed using a pico-projector to generate structured illumination patterns. A net rate of 5.8 × 106 pixel/s and sub-micron spatial resolution in three-dimensions (3D) were achieved. Based on the pico-projector’s flexibility in pattern generation, the characteristics of POSM with different modulation periods and at different imaging depths were measured and discussed. With the application of different modulation periods, 3D chlorophyll fluorescence imaging of mesophyll cells was carried out in freshly plucked leaves of four species without sectioning or staining. For each leaf, an average penetration depth of 120 μm was achieved. Increasing the modulation period along with the increment of imaging depth, optical sectioning images can be obtained with a compromise between the axial resolution and signal-to-noise ratio. After ∼30 min imaging on the same area, photodamage was hardly observed. Taking the advantages of high speed and low damages of POSM, the investigation of the dynamic fluorescence responses to temperature changes was performed under three different treatment temperatures. The three embedded blue, green and red light-emitting diode light sources were applied to observe the responses of the leaves with different wavelength excitation.

Design and Development of Nonlinear Optical Microscope System: Simple Implementation with epi-Illumination Platform

During the research using fluorescence-tagged or auto-fluorescence molecules, meaningful information is often buried deep inside the tissue, not its surface. Therefore, especially in the field of biomedical imaging, acquiring optically sectioned images from deep inside the tissue is very important. As well know already, confocal laser scanning microscopy (the most well-known optical sectioning microscopy) gives axially-resolved fluorescence information using the physical background blocking component called pinhole. However, the axial range of imaging is practically limited due to such optical phenomena as the light scattered and absorbed in the tissue. However, nonlinear optical microscopy (e.g. Multiphoton microscopy, harmonic generation microscopy, coherent anti-Stokes Raman spectroscopy) realized by the development of ultrafast light sources has been used for visualizing various tissues, especially in vivo, because of their low sensitivity to the limitation caused by the scattering and the absorption of light. Although nonlinear optical microscopy gives deep tissue image, it is not easy for many researcher to build customized nonlinear system. Here, we introduce an easy and simple way designing and developing such nonlinear optical microscope with upright or inverted epi-illumination platform using commercial optical components only.

Three-dimensional super-resolution structured illumination microscopy with maximum a posteriori probability image estimation.

We introduce and demonstrate a new high performance image reconstruction method for super-resolution structured illumination microscopy based on maximum a posteriori probability estimation (MAP-SIM). Imaging performance is demonstrated on a variety of fluorescent samples of different thickness, labeling density and noise levels. The method provides good suppression of out of focus light, improves spatial resolution, and allows reconstruction of both 2D and 3D images of cells even in the case of weak signals. The method can be used to process both optical sectioning and super-resolution structured illumination microscopy data to create high quality super-resolution images.

Neonatal Tolerance: Donor B Cells Home to Host Lymphoid Organs, Interact With Apoptotic Cells, Produce IL-10 and Prolong Heart Allograft Survival.

Introduction: Newborn mice injected with a mixture of semi-allogeneic (F1) bone marrow (BMC) and spleen cells (SC) accept donor-type transplants as adults. B cells are abundant in both BMC and SC and have been reported to tolerize CD8+ T cells. To determine what role, if any, B cells in the tolerizing inoculum play in this classic tolerance model, we studied the effect of purified B cells injected into newborn mice. Methods: F1-B cells and allogeneic-B cells (allo-B) were purified from spleen using StemCell Technologies EasySep® CD19 positive selection kits. C3H/He (C3H; H-2k) neonates were injected within 24 hours of birth with 106 purified B cells, GFP-expressing (C57BL/6 [B6; H-2b] or C3H X B6 [H-2k/b]) or CFSE-labelled (BALB/c [BALB; H-2d] or C3H X BALB [H-2k/d]). Injected cells were tracked by whole body/organ imaging; their interactions and fates were studied with high-resolution optical-sectioning microscopy. Impact on heart allograft survival was also determined. Results: By whole organ imaging (day 3), purified F1-B cells were detected in primary and secondary lymphoid organs (spleen, lymph node and bone). At day 6, injected B cells in spleen were positioned in follicular regions. F1- and allo-B cells both interacted with host CD8+ T cells; F1-B cells remained intact while allo-B cells fragmented and were taken up by host DC. Survival of F1-B cells suggested they induced tolerance in host CD8+ T cells. Consistent with that, F1-B cells also interacted with host apoptotic cells in spleen. Apoptotic cells have been reported to induce B cells to produce IL-10 and in turn convert T cells into Tr1-type regulatory cells. IL-10-producing F1-B cells were observed interacting with host T cells in day 3 spleens. To determine if other mechanisms played a role in prolonging F1-B cell survival we examined host Foxp3 regulatory and apoptotic T cells; in both cases events were few. Purified F1-B cells prolonged survival of donor-type hearts to 100 days in 6 of 7 transplanted mice. Beat scores were low, however, and one day-105 beating heart showed infiltrating host macrophages and T cells including CD8+ T cells. Conclusion: Neonatally-injected purified F1-B cells actively regulate the neonatal immune system and prolong survival of donor-type heart grafts transplanted as adults. By themselves, however, F1-B cells do not induce a robust tolerance suggesting additional F1 cell types are required for the process.

Modulated-alignment dual-axis (MAD) confocal microscopy for deep optical sectioning in tissues.

A strategy is presented to enable optical-sectioning microscopy with improved contrast and imaging depth using low-power (0.5 - 1 mW) diode laser illumination. This technology combines the inherent strengths of focal-modulation microscopy and dual-axis confocal (DAC) microscopy for rejecting out-of-focus and multiply scattered background light in tissues. The DAC architecture is unique in that it utilizes an intersecting pair of illumination and collection beams to improve the spatial-filtering and optical-sectioning performance of confocal microscopy while focal modulation selectively 'labels' in-focus signals via amplitude modulation. Simulations indicate that modulating the spatial alignment of dual-axis beams at a frequency f generates signals from the focal volume of the microscope that are modulated at 2f with minimal modulation of background signals, thus providing nearly an order-of-magnitude improvement in optical-sectioning contrast compared to DAC microscopy alone. Experiments show that 2f lock-in detection enhances contrast and imaging depth within scattering phantoms and fresh tissues.

Aggregates of nisin with various bactoprenol-containing cell wall precursors differ in size and membrane permeation capacity

Many lantibiotics use the membrane bound cell wall precursor Lipid II as a specific target for killing Gram-positive bacteria. Binding of Lipid II usually impedes cell wall biosynthesis, however, some elongated lantibiotics such as nisin, use Lipid II also as a docking molecule for pore formation in bacterial membranes. Although the unique nisin pore formation can be analyzed in Lipid II-doped vesicles, mechanistic details remain elusive. We used optical sectioning microscopy to directly visualize the interaction of fluorescently labeled nisin with membranes of giant unilamellar vesicles containing Lipid II and its various bactoprenol precursors. We quantitatively analyzed the binding and permeation capacity of nisin when applied at nanomolar concentrations. Specific interactions with Lipid I, Lipid II and bactoprenol-diphosphate (C55-PP), but not bactoprenol-phosphate (C55-P), resulted in the formation of large molecular aggregates. For Lipid II, we demonstrated the presence of both nisin and Lipid II in these aggregates. Membrane permeation induced by nisin was observed in the presence of Lipid I and Lipid II, but not in the presence of C55-PP. Notably, the size of the C55-PP–nisin aggregates was significantly smaller than that of the aggregates formed with Lipid I and Lipid II. We conclude that the membrane permeation capacity of nisin is determined by the size of the bactoprenol-containing aggregates in the membrane. Notably, transmitted light images indicated that the formation of large aggregates led to a pinch-off of small vesicles, a mechanism, which probably limits the growth of aggregates and induces membrane leakage.

Characterization of a three-dimensional double-helix point-spread function for fluorescence microscopy in the presence of spherical aberration

We characterize the three-dimensional (3-D) double-helix (DH) point-spread function (PSF) for depth-variant fluorescence microscopy imaging motivated by our interest to integrate the DH-PSF in computational optical sectioning microscopy (COSM) imaging. Physical parameters, such as refractive index and thickness variability of imaging layers encountered in 3-D microscopy give rise to depth-induced spherical aberration (SA) that change the shape of the PSF at different focusing depths and render computational approaches less practical. Theoretical and experimental studies performed to characterize the DH-PSF under varying imaging conditions are presented. Results show reasonable agreement between theoretical and experimental DH-PSFs suggesting that our model can predict the main features of the data. The depth-variability of the DH-PSF due to SA, quantified using a normalized mean square error, shows that the DH-PSF is more robust to SA than the conventional PSF. This result is also supported by the frequency analysis of the DH-PSF shown. Our studies suggest that further investigation of the DH-PSF's use in COSM is warranted, and that particle localization accuracy using the DH-PSF calibration curve in the presence of SA can be improved by accounting for the axial shift due to SA.

DMD-based LED-illumination Super-resolution and optical sectioning microscopy

Super-resolution three-dimensional (3D) optical microscopy has incomparable advantages over other high-resolution microscopic technologies, such as electron microscopy and atomic force microscopy, in the study of biological molecules, pathways and events in live cells and tissues. We present a novel approach of structured illumination microscopy (SIM) by using a digital micromirror device (DMD) for fringe projection and a low-coherence LED light for illumination. The lateral resolution of 90 nm and the optical sectioning depth of 120 μm were achieved. The maximum acquisition speed for 3D imaging in the optical sectioning mode was 1.6×107 pixels/second, which was mainly limited by the sensitivity and speed of the CCD camera. In contrast to other SIM techniques, the DMD-based LED-illumination SIM is cost-effective, ease of multi-wavelength switchable and speckle-noise-free. The 2D super-resolution and 3D optical sectioning modalities can be easily switched and applied to either fluorescent or non-fluorescent specimens.

Microscopic Delineation of Medulloblastoma Margins in a Transgenic Mouse Model Using a Topically Applied VEGFR-1 Probe

The unambiguous demarcation of tumor margins is critical at the final stages in the surgical treatment of brain tumors because patient outcomes have been shown to correlate with the extent of resection. Real-time high-resolution imaging with the aid of a tumor-targeting fluorescent contrast agent has the potential to enable intraoperative differentiation of tumor versus normal tissues with accuracy approaching the current gold standard of histopathology. In this study, a monoclonal antibody targeting the vascular endothelial growth factor receptor 1 (VEGFR-1) was conjugated to fluorophores and evaluated as a tumor contrast agent in a transgenic mouse model of medulloblastoma. The probe was administered topically, and its efficacy as an imaging agent was evaluated in vitro using flow cytometry, as well as ex vivo on fixed and fresh tissues through immunohistochemistry and dual-axis confocal microscopy, respectively. Results show a preferential binding to tumor versus normal tissue, suggesting that a topically applied VEGFR-1 probe can potentially be used with real-time intraoperative optical sectioning microscopy to guide brain tumor resections.

Multi-color miniature dual-axis confocal microscope for point-of-care pathology

We present a miniature microelectromechanical systems-based dual-axis confocal microscope capable of spatially coregistered fluorescence and reflectance imaging at multiple wavelengths. This device has a 10 mm diameter scan head with a 2 mm diameter tip for convenient use during surgery to guide tumor resection. The microscope has an adjustable focal depth of 20-200 micrometers and is capable of imaging with an axial resolution of 9 micrometers and in-plane resolution of 4 micrometers over a field of view of 450×450 micrometers. Simultaneous two-color imaging of individual optical sections is achieved by using a pair of grating-prism assemblies to compensate for chromatic dispersion in the 2 mm diameter gradient index relay lens at the distal tip of the device. Experimental measurements of the axial response of the microscope, as well as two-color images of a reflective bar target and fresh mouse brain tissues, demonstrate the performance of our device and its potential for multicolor in vivo optical sectioning microscopy.

Three-Dimensional Imaging and Modeling of Anatomic Structures, Sectional and Radiological Anatomy, and Staining Techniques

The first three-dimensional (3D) reconstruction was performed by Born (1883) and His (1885) using serial sections. Since then, a variety of reconstruction techniques have been developed, and the construction of physical models has become important in anatomy. The first computer-aided 3D reconstruction was achieved by Glaser and Van der Loos in 1965. With the improvements in both computer hardware and software tools, computerized modelling of anatomical structures has become very useful for visualizing complex 3D forms. Three-dimensional visualization of various microanatomic structures using special preparation and staining methods is important. Devices used for this purpose such as MicroCT, microMRI, SEM, and Confocal microscopy and so forth. are development parallel to the technological advances and give excellent 3D images, and it allows a better understanding of gross and microanatomic structures. On the other hand, first studies on cross-sectional anatomy date back to the 16th century. The widespread use of cross-sectional imaging techniques such as CT and MRI has gained an increasing importance in sectional anatomy. The most known cadaveric cross-sectional study, the visible human project, is an outgrowth of the National Library Medicine. In this special issue, we reviewed and edited seven articles on three-dimensional imaging and modeling of anatomic structures, sectional and radiological anatomy, and staining techniques. K. K. Schmid (University of Nebraska, USA) Marx and Samal (University of Nebraska-Lincoln, USA) discussed three-dimensional regression, a technique that can be used for mapping images and shapes that are represented by sets of three-dimensional landmark coordinates, for comparing and mapping 3D anatomical structures in their research paper. Y. Hoshino and coworkers (University of Pittsburgh, USA) reviewed previous research about image analysis of the anterior cruciate ligament (ACL) anatomy and its application to ACL reconstruction surgery. They concluded that three-dimensional image analysis of the ACL anatomy and its application to the navigation system is becoming more prevalent and reliable for advancing the anatomic studies related to the native ACL and the ACL reconstruction procedure. A. Arencibia et al. (Veterinary Faculty, University of Las Palmas de Gran Canaria) investigated imaging features of the temporomandibular joint (TMJ) using computed tomography and magnetic resonance imaging in two normal camels. They stated in conclusion that CT is an excellent method for the detailed assessment of the bony structures, and MRI is a valid imaging modality for the evaluation of the soft tissues. Processing of CT and MR images allowes to appreciate different bone structures and soft tissues of the TMJ, assisting in the interpretation of the images. J. A. N. Buytaert and colleagues (University of Antwerp and Ghent University, Belgium) discussed the first implementation of (laser) light-sheet-based fluorescence microscopy (LSFM) to image biomedical tissue in three-dimensional orthogonal plane fluorescence optical sectioning microscopy (OPFOS). They have shown with several applications that the OPFOS (and derived) methods are a valuable addition for sectional imaging and three-dimensional modeling of anatomic structures. The review paper entitled “Ultrahigh voltage electron microscopy (UHVEM) links neuroanatomy and neuroscience/neuroendocrinology” by H. Sakamoto (Okayama University, Japan) and M. Kawata (Kyoto Prefectural University of Medicine, Japan) stressed that UHVEM and light microscopy are useful and powerful tools for studying molecular and/or chemical neuroanatomy at the ultrastructural level. D. R. Marker et al. (The Johns Hopkins Hospital and University School of Medicine, Weill Cornell Medical College, USA) discussed strategic improvements for gross anatomy web-based teaching in their research paper. To better meet the expectations of gross anatomy students, electronic radiology teaching files for first-year coursework were organized into a web site. The web site was custom designed to provide material that directly correlated to the gross anatomy dissection and lectures. Quick links provided sets of images grouped by anatomic location. Their findings suggest that a well-organized web portal can provide a user-friendly, valuable educational resource for medical students who are studying gross anatomy. Finally, Herber and coworkers (University of California, USA) in their study entitled “Imaging an adapted dentoalveolar complex” used micro-X-ray computed tomography combined with 3D modeling using image processing, scanning electron microscopy, fluorochrome labeling, conventional histology (H&E, TRAP), and immunohistochemistry (RANKL, OPN) to elucidate the dynamic nature of bone, the periodontal ligamentspace, and cementum in the rat periodontium. They concluded that studies focusing on a functional rat dentoalveolar complex should acknowledge the baseline phenomena of bone-PDL-cementum adaptation, especially in regards to the physiological distal drift, before additional experimental variables are imposed. We hope that this special issue will contribute as a stimulus for further research into three-dimensional imaging and modeling of anatomic structures, sectional and radiological anatomy, and staining techniques. We are very grateful to the contributing authors for their scientific contributions to this special issue. We also thank the reviewers who spend their valuable time and thoughts on each paper. We appreciate the Hindawi Publishing Corporation for their help in the guidance of the procedure. Tuncay Peker Nadir Gulekon Ilkan Tatar Levent Sarikcioglu David Kachlik

Light emitting diodes: Light engines to simplify and economize advanced microscopy

THE old adage, ‘‘A picture is worth a thousand words,’’ is especially true in biological research, with microscopic images serving to mark the development of key concepts in biology since Hooke first described ‘‘cells’’ in thin slices of cork (1). More than 400 years later, microscopic imaging still serves as a primary data source for biological research, with increasingly advanced techniques that provide information on molecular scale interactions and dynamics. Such advanced molecular imaging techniques take advantage of ever more sophisticated light sources, optics, detectors, and image processing methods, many of which can be quite complex and expensive. At the same time, new technologies and production methods, many driven by the consumer market, have resulted in key components of imaging systems becoming smaller, simpler, and cheaper. The convergence of these effects is having profound effects on our options for light sources for microscopic imaging. The properties of the illumination source are critical to many advanced optical microscopy methods. Lasers play a key role in many of these owing to the monochromatic and coherent nature of their light, plus their ability to be pulsed and otherwise controlled. However, illumination with high-pressure mercury gas discharge or xenon lamps is still the standard for most imaging applications. These light sources provide bright, broad-spectrum excitation (300–900 nm) that can be easily selected by excitation filters for fluorescent imaging applications. However, there are quite a few disadvantages that limit the wide-spread of mercury or xenon lamps-based microscopy system in biological applications. First, sometimes these traditional light sources are too bright and harmful for the living organisms being studied. Excessive brightness can cause rapid photo-bleaching of the dyes used to stain the cells/ tissues. UV light especially, if not blocked, can have significant deleterious effects on live cells. Second, there are substantial costs involved in using mercury or xenon lamps because of their relatively short lifespan. The life of a mercury lamp is usually several hundred hours, and the intensity of these lamps decays progressively during this time. In addition, gas discharge lamps require some time to warm up before lamp intensity reaches steady state. Therefore, once mercury lamps are turned on, they have to be left on for hours to enable fluorescent measurements as needed without delay, thereby further shortening the lamp lifetime. Third, there are also some hazards issues of using mercury or xenon lamps. These lamps generate a significant amount of heat and therefore introduce complications when used in a confined space. When mercury lamps die, they can explode and damage the lenses or mirrors within the lamp housing. These factors combine to make gas discharge lamps inconvenient for lab-based applications and impractical for field applications, including use in portable, miniature, or low cost imaging devices. By contrast, light-emitting diodes (LEDs) show great pro

Poisson Noise Reduction in Deconvolution Microscopy

Computational optical sectioning microscopy is a powerful tool to reconstruct three-dimensional images from optical two-dimensional sections of a biological specimen acquired by means of a fluorescence microscope. Due to limiting factors in the imaging systems, the images are degraded by both the optical system and detection process. Each of the two-dimensional section of the three-dimensional data set are blurred by contributions of light from other out-of-focus planes. Besides, they are also corrupted by noise due to quantum fluctuations of light. In this work we present a method to perform the restoration of three-dimensional data obtained by fluorescence microscopy. The algorithm consists of the use of a noise reduction procedure based on the Anscombe transformation followed by the Richardson-Lucy deconvolution algorithm. Results showed an improvement on deconvolution performance when using phantoms and real cell images.

The contribution of apoptosis-inducing factor (AIF) to villous trophoblast differentiation

Apoptosis is postulated to be a delayed but important part of the differentiation of placental villous cytotrophoblasts (CT) into functional syncytiotrophoblast (ST). This hypothesis is based on the observation that the externalization of phosphatidylserine and the activation of caspase 8 are required for trophoblast differentiation. In contradiction to this hypothesis we have previously found that differentiation occurs in the presence of both broad spectrum and caspase 8 specific inhibitors. Apoptosis-inducing factor (AIF) is a mitochondria-associated protein which is known to translocate to the nucleus and induce caspase-independent nuclear condensation, phosphatidylserine externalization and cell death. Thus AIF nuclear translocation may result in the apoptotic-like features associated with trophoblast differentiation and may be an obligatory event for differentiation to proceed. AIF translocation was assessed in isolated primary trophoblasts by optical section microscopy of antibody stained cells. We found AIF to be strongly expressed in the villous trophoblast and that small amounts of AIF were localized to the nucleus of the cells. Significantly, inhibitors of AIF translocation (calpeptin and zFA-fmk) blocked translocation but not differentiation of the cells. We conclude that AIF translocation is not involved in CT differentiation in isolated cell culture.

The OPFOS Microscopy Family: High-Resolution Optical Sectioning of Biomedical Specimens

We report on the recently emerging (laser) light-sheet-based fluorescence microscopy field (LSFM). The techniques used in this field allow to study and visualize biomedical objects nondestructively in high resolution through virtual optical sectioning with sheets of laser light. Fluorescence originating in the cross-section of the sheet and sample is recorded orthogonally with a camera. In this paper, the first implementation of LSFM to image biomedical tissue in three dimensions—orthogonal-plane fluorescence optical sectioning microscopy (OPFOS)—is discussed. Since then many similar and derived methods have surfaced, (SPIM, ultramicroscopy, HR-OPFOS, mSPIM, DSLM, TSLIM, etc.) which we all briefly discuss. All these optical sectioning methods create images showing histological detail. We illustrate the applicability of LSFM on several specimen types with application in biomedical and life sciences.

Point-spread function engineering to reduce the impact of spherical aberration on 3D computational fluorescence microscopy imaging

Wavefront encoding (WFE) with different cubic phase mask designs was investigated in engineering 3D point-spread functions (PSF) to reduce their sensitivity to depth-induced spherical aberration (SA) which affects computational complexity in 3D microscopy imaging. The sensitivity of WFE-PSFs to defocus and to SA was evaluated as a function of phase mask parameters using mean-square-error metrics to facilitate the selection of mask designs for extended-depth-of-field (EDOF) microscopy and for computational optical sectioning microscopy (COSM). Further studies on pupil phase contribution and simulated WFE-microscope images evaluated the engineered PSFs and demonstrated SA insensitivity over sample depths of 30 μm. Despite its low sensitivity to SA, the successful WFE design for COSM maintains a high sensitivity to defocus as it is desired for optical sectioning.

Optical sectioning microscopy with planar or structured illumination

A key requirement for performing three-dimensional (3D) imaging using optical microscopes is that they be capable of optical sectioning by distinguishing in-focus signal from out-of-focus background. Common techniques for fluorescence optical sectioning are confocal laser scanning microscopy and two-photon microscopy. But there is increasing interest in alternative optical sectioning techniques, particularly for applications involving high speeds, large fields of view or long-term imaging. In this Review, I examine two such techniques, based on planar illumination or structured illumination. The goal is to describe the advantages and disadvantages of these techniques.

Realistic 3D Computer Model of the Gerbil Middle Ear, Featuring Accurate Morphology of Bone and Soft Tissue Structures

In order to improve realism in middle ear (ME) finite-element modeling (FEM), comprehensive and precise morphological data are needed. To date, micro-scale X-ray computed tomography (μCT) recordings have been used as geometric input data for FEM models of the ME ossicles. Previously, attempts were made to obtain these data on ME soft tissue structures as well. However, due to low X-ray absorption of soft tissue, quality of these images is limited. Another popular approach is using histological sections as data for 3D models, delivering high in-plane resolution for the sections, but the technique is destructive in nature and registration of the sections is difficult. We combine data from high-resolution μCT recordings with data from high-resolution orthogonal-plane fluorescence optical-sectioning microscopy (OPFOS), both obtained on the same gerbil specimen. State-of-the-art μCT delivers high-resolution data on the 3D shape of ossicles and other ME bony structures, while the OPFOS setup generates data of unprecedented quality both on bone and soft tissue ME structures. Each of these techniques is tomographic and non-destructive and delivers sets of automatically aligned virtual sections. The datasets coming from different techniques need to be registered with respect to each other. By combining both datasets, we obtain a complete high-resolution morphological model of all functional components in the gerbil ME. The resulting 3D model can be readily imported in FEM software and is made freely available to the research community. In this paper, we discuss the methods used, present the resulting merged model, and discuss the morphological properties of the soft tissue structures, such as muscles and ligaments.

Optical Microscopy – Preliminary Classification of Two R Chondrites

CML 215 and CML 392 are two unclassified meteorites that belong to the class of Rumuruti (or R) chondrites. Using optical thin section microscopy both specimens were preliminary classified as R3-5. Both meteorites contain hydrous phase amphibole, which indicate interaction of the parent body (or bodies) of these meteorites with OH- oxidant. Presence of this phase in CML 215 and 392 brings the number of amphibole-bearing R chondrites to three. At this point is uncertain whether CML 215 and 392 are paired.

Size-dependent endocytosis of gold nanoparticles studied by three-dimensional mapping of plasmonic scattering images

BackgroundUnderstanding the endocytosis process of gold nanoparticles (AuNPs) is important for the drug delivery and photodynamic therapy applications. The endocytosis in living cells is usually studied by fluorescent microscopy. The fluorescent labeling suffers from photobleaching. Besides, quantitative estimation of the cellular uptake is not easy. In this paper, the size-dependent endocytosis of AuNPs was investigated by using plasmonic scattering images without any labeling.ResultsThe scattering images of AuNPs and the vesicles were mapped by using an optical sectioning microscopy with dark-field illumination. AuNPs have large optical scatterings at 550-600 nm wavelengths due to localized surface plasmon resonances. Using an enhanced contrast between yellow and blue CCD images, AuNPs can be well distinguished from cellular organelles. The tracking of AuNPs coated with aptamers for surface mucin glycoprotein shows that AuNPs attached to extracellular matrix and moved towards center of the cell. Most 75-nm-AuNPs moved to the top of cells, while many 45-nm-AuNPs entered cells through endocytosis and accumulated in endocytic vesicles. The amounts of cellular uptake decreased with the increase of particle size.ConclusionsWe quantitatively studied the endocytosis of AuNPs with different sizes in various cancer cells. The plasmonic scattering images confirm the size-dependent endocytosis of AuNPs. The 45-nm-AuNP is better for drug delivery due to its higher uptake rate. On the other hand, large AuNPs are immobilized on the cell membrane. They can be used to reconstruct the cell morphology.