Microscopic Imaging Modalities Research Articles

Super-Resolution Single Molecule FISH at the Drosophila Neuromuscular Junction.

The lack of an effective, simple, and highly sensitive protocol for fluorescent in situ hybridization (FISH) at the Drosophila larval neuromuscular junction (NMJ) has hampered the study of mRNA biology. Here, we describe our modified single molecule FISH (smFISH) methods that work well in whole mount Drosophila NMJ preparations to quantify primary transcription and count individual cytoplasmic mRNA molecules in specimens while maintaining ultrastructural preservation. The smFISH method is suitable for high-throughput sample processing and 3D image acquisition using any conventional microscopy imaging modality and is compatible with the use of antibody colabeling and transgenic fluorescent protein tags in axons, glia, synapses, and muscle cells. These attributes make the method particularly amenable to super-resolution imaging. With 3D Structured Illumination Microscopy (3D-SIM), which increases spatial resolution by a factor of 2 in X, Y, and Z, we acquire super-resolution information about the distribution of single molecules of mRNA in relation to covisualized synaptic and cellular structures. Finally, we demonstrate the use of commercial and open source software for the quality control of single transcript expression analysis, 3D-SIM data acquisition and reconstruction as well as image archiving management and presentation. Our methods now allow the detailed mechanistic and functional analysis of sparse as well as abundant mRNAs at the NMJ in their appropriate cellular context.

Multi-pass transmission electron microscopy

Feynman once asked physicists to build better electron microscopes to be able to watch biology at work. While electron microscopes can now provide atomic resolution, electron beam induced specimen damage precludes high resolution imaging of sensitive materials, such as single proteins or polymers. Here, we use simulations to show that an electron microscope based on a multi-pass measurement protocol enables imaging of single proteins, without averaging structures over multiple images. While we demonstrate the method for particular imaging targets, the approach is broadly applicable and is expected to improve resolution and sensitivity for a range of electron microscopy imaging modalities, including, for example, scanning and spectroscopic techniques. The approach implements a quantum mechanically optimal strategy which under idealized conditions can be considered interaction-free.

Remote focusing multifocal plane microscopy for the imaging of 3D single molecule dynamics with cellular context.

Three-dimensional (3D) single molecule fluorescence microscopy affords the ability to investigate subcellular traffcking at the level of individual molecules. An imaged single molecule trajectory, however, often reveals only limited information about the underlying biological process when insuffcient information is available about the organelles and other cellular structures with which the molecule interacts. A new 3D fluorescence microscopy imaging modality is described here that enables the simultaneous imaging of the trajectories of fast-moving molecules and the associated cellular context. The new modality is called remote focusing multifocal plane microscopy (rMUM), as it extends multifocal plane microscopy (MUM) with a remote focusing module. MUM is a modality that uses multiple detectors to image distinct focal planes within the specimen at the same time, and it has been demonstrated to allow the determination of 3D single molecule trajectories with high accuracy. Remote focusing is a method that makes use of two additional objective lenses to enable the acquisition of a z-stack of the specimen without having to move the microscope's objective lens or sample stage, components which are required by MUM to be fixed in place. rMUM's remote focusing module thus allows the cellular context to be imaged in the form of z-stacks as the trajectories of molecules or other objects of interest are imaged by MUM. In addition to a description of the modality, a discussion of rMUM data analysis and an example of data acquired using an rMUM setup are provided in this paper.

Imaging of Three-Dimensional Single Molecule Dynamics in their Cellular Context

Single molecule microscopy has provided a wealth of information regarding the dynamics of receptors on the plasma membrane. In contrast, due to lack of appropriate imaging approaches, very little is known about three-dimensional (3D) single molecule trafficking in subcellular compartments. There are two major technical problems in imaging 3D subcellular single molecule dynamics. First, the dynamics of the single molecules are not confined to one focal plane of the microscope and second, having only the trajectories available of single molecules is typically not meaningful unless the cellular context of the single molecule dynamics is also imaged. For example, only by the simultaneous imaging of the single molecule dynamics and the cellular context can, for example, the diffusion of a receptor be analyzed on a non-stationary endosome. Here we introduce a novel microscopy imaging modality that is designed to simultaneously image both the dynamics of single molecules in three dimensions and the cellular context which may labeled with several fluorophores. Imaging of the single molecule dynamics is carried out using multi-focal plane microscopy (MUM), a previously demonstrated and powerful tool for this task. The cellular context is imaged using a remote focusing approach that is highly compatible with MUM imaging. The efficacy of this new imaging modality, called rMUM for remote focusing multifocal plane microscopy, is demonstrated by an investigation of the trafficking pathways of antibodies against the prostate-specific membrane antigen (PSMA) in prostate cancer cells. We demonstrate that novel experiments such as a single molecule diffusion study on sorting endosomes are possible with the new microscope, and we completely characterize the pathway of PSMA-specific antibodies, starting with diffusion towards the plasma membrane, followed by endocytosis, transport to sorting endosomes, and subsequent diffusion on sorting endosomes, and ending with either recycling or maturation into a multivesicular body.

High-definition optical coherence tomography imaging for noninvasive examination of heritage works

Cultural heritage works, such as ancient murals and historical paintings, are examined routinely for the purpose of conservation. Previous works have applied optical coherence tomography (OCT), which is a three-dimensional (3D) microscopic imaging modality in the field of heritage works conservation. The data acquired by OCT provides both 3D surface information of the object and structure information underneath the surface. Therefore, cross-sectional information on the object can be utilized to study layer structure of the painting and brush stroke techniques used by the artist. However, as demonstrated in previous studies, OCT has limited capability in high-definition (HD) examination of paintings or murals that are in macroscopic scale. HD examination of heritage works needs to scan large areas and process huge amounts of data, while OCT imaging has a limited field of view and processing power. To further advance the application of OCT in the conservation of heritage works, we demonstrate what we believe is a novel high-speed, large field-of-view (FOV) OCT imaging platform. Our results suggest that this OCT platform has the potential to become a nondestructive alternative for the analysis and conservation of paintings and murals.

Probing the light harvesting and charge rectification of bismuth nanoparticles behind the promoted photoreactivity onto Bi/BiOCl catalyst by (in-situ) electron microscopy

State-of-the-art electron microscopy has enabled us to investigate microstructural details down to sub-subångström and milli-electron-volt resolution level. The enhanced photoreactivity over bismuth hybridized BiOCl catalyst (Bi/BiOCl) has been reported recently, however, the mechanistic understandings of this improved photoreactivity especially the optical behavior of bismuth nanoparticles (Bi NPs) are still obscured and in debate. The optical absorption features of Bi NPs and the charge transfer characteristic between bismuth and BiOCl have been considered as the major physicochemical origin for the promoted photoreactivity. Based on the advanced (in-situ) electron microscopy of monochromated electron energy loss spectroscopy in scanning transmission electron microscopy imaging mode (Mono-STEM-EELS) along with related theoretical investigations, in this work, we for the first time distinguished and explained the optical absorption originated from the localized surface plasmon resonances (LSPR) effect and direct band gap transition in an individual bismuth nanoparticle as well as transportation of photogenerated carriers at the interface of Bi/BiOCl. These findings could provide better understandings about the origin of the improved photoreactivity of various bismuth-hybridized photocatalysts.

Simplified three-dimensional tissue clearing and incorporation of colorimetric phenotyping.

Tissue clearing methods promise to provide exquisite three-dimensional imaging information; however, there is a need for simplified methods for lower resource settings and for non-fluorescence based phenotyping to enable light microscopic imaging modalities. Here we describe the simplified CLARITY method (SCM) for tissue clearing that preserves epitopes of interest. We imaged the resulting tissues using light sheet microscopy to generate rapid 3D reconstructions of entire tissues and organs. In addition, to enable clearing and 3D tissue imaging with light microscopy methods, we developed a colorimetric, non-fluorescent method for specifically labeling cleared tissues based on horseradish peroxidase conversion of diaminobenzidine to a colored insoluble product. The methods we describe here are portable and can be accomplished at low cost, and can allow light microscopic imaging of cleared tissues, thus enabling tissue clearing and imaging in a wide variety of settings.

Genetic toxicity assessment of engineered nanoparticles using a 3D in vitro skin model (EpiDerm™).

BackgroundThe rapid production and incorporation of engineered nanomaterials into consumer products alongside research suggesting nanomaterials can cause cell death and DNA damage (genotoxicity) makes in vitro assays desirable for nanosafety screening. However, conflicting outcomes are often observed when in vitro and in vivo study results are compared, suggesting more physiologically representative in vitro models are required to minimise reliance on animal testing.MethodBASF Levasil® silica nanoparticles (16 and 85 nm) were used to adapt the 3D reconstructed skin micronucleus (RSMN) assay for nanomaterials administered topically or into the growth medium. 3D dose-responses were compared to a 2D micronucleus assay using monocultured human B cells (TK6) after standardising dose between 2D / 3D assays by total nanoparticle mass to cell number. Cryogenic vitrification, scanning electron microscopy and dynamic light scattering techniques were applied to characterise in-medium and air-liquid interface exposures. Advanced transmission electron microscopy imaging modes (high angle annular dark field) and X-ray spectrometry were used to define nanoparticle penetration / cellular uptake in the intact 3D models and 2D monocultured cells.ResultsFor all 2D exposures, significant (p < 0.002) increases in genotoxicity were observed (≥100 μg/mL) alongside cell viability decreases (p < 0.015) at doses ≥200 μg/mL (16 nm-SiO2) and ≥100 μg/mL (85 nm-SiO2). In contrast, 2D-equivalent exposures to the 3D models (≤300 μg/mL) caused no significant DNA damage or impact on cell viability. Further increasing dose to the 3D models led to probable air-liquid interface suffocation. Nanoparticle penetration / cell uptake analysis revealed no exposure to the live cells of the 3D model occurred due to the protective nature of the skin model’s 3D cellular microarchitecture (topical exposures) and confounding barrier effects of the collagen cell attachment layer (in-medium exposures). 2D monocultured cells meanwhile showed extensive internalisation of both silica particles causing (geno)toxicity.ConclusionsThe results establish the importance of tissue microarchitecture in defining nanomaterial exposure, and suggest 3D in vitro models could play a role in bridging the gap between in vitro and in vivo outcomes in nanotoxicology. Robust exposure characterisation and uptake assessment methods (as demonstrated) are essential to interpret nano(geno)toxicity studies successfully.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-016-0161-5) contains supplementary material, which is available to authorized users.

Scanning Hard X-ray Microscopy Imaging Modalities for Geobiological Samples

Modern scanning X-ray microscopy can help to unravel the spatial context between biotic and abiotic compounds of geobiological assemblies with the aim to finally link chemical pathways to biological activities at the nanometre scale. This work presents some multi-modal imaging techniques provided by hard X-ray microscopes at synchrotron radiation sources to address analytical needs in geobiological research. Using the examples of 1) a calcified basal skeleton of the demosponge Astrosclera willeyana, 2) an anaerobic methane-oxidizing microbial mat and 3) a bacterial sulfur-oxidizing consortium, we illustrate the potential of scanning X-ray fluorescence and scanning transmission X-ray microscopy, and a novel quantitative approach of ptychographic imaging at single cell level.

Acoustic resolution photoacoustic microscopy

Even if conventional optical imaging systems such as multiphoton microscopy (MPM), confocal microscopy (CM), fluorescence microscopy (FM), and optical coherence tomography (OCT) are regarded as revolutionary microscopic imaging modalities to reveal the inner information of biological tissues with very high spatial resolution, it is inherently restricted to image deep tissues due to strong optical scatting in biological tissues. Photoacoustic imaging (PAI) is a hybrid imaging modality to combine strong optical contrast and high ultrasonic resolution in deep tissues. In a microscopic imaging perspective, photoaocustic microscopy (PAM) can be implemented in two forms: optical-resolution (OR) and acoustic-resolution (AR) PAM. In OR-PAM, the lateral spatial resolution is determined by tight optical focusing, but the penetration depth is limited to one optical transport mean free path. In AR-PAM, the lateral spatial resolution is determined by loose acoustic focusing, but the penetration depth can be much enhanced and reach to several centimeters. Therefore, AR-PAM gains great attention for both preclinical and clinical applications. This review explains the principle, implementation, and applications of AR-PAM.

Choosing a Fluorescence Microscopy Imaging Modality for Live Quantitative Experiments

Journal Article Choosing a Fluorescence Microscopy Imaging Modality for Live Quantitative Experiments Get access Talley J Lambert, Talley J Lambert Harvard Medical School/Department of Cell Biology, Boston, MA USA Search for other works by this author on: Oxford Academic Google Scholar Jennifer C Waters Jennifer C Waters Harvard Medical School/Department of Cell Biology, Boston, MA USA Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 20, Issue S3, 1 August 2014, Pages 2122–2123, https://doi.org/10.1017/S1431927614012343 Published: 27 August 2014

Multispectral Fluorescence Ultramicroscopy: Three-Dimensional Visualization and Automatic Quantification of Tumor Morphology, Drug Penetration, and Antiangiogenic Treatment Response

Classic histology still represents the gold standard in tumor tissue analytics. However, two-dimensional analysis of single tissue slides does not provide a representative overview of the inhomogeneous tumor physiology, and a detailed analysis of complex three-dimensional structures is not feasible with this technique. To overcome this problem, we applied multispectral fluorescence ultramicroscopy (UM) to the field of tumor analysis. Optical sectioning of cleared tumor specimen provides the possibility to three-dimensionally acquire relevant tumor parameters on a cellular resolution. To analyze the virtual UM tumor data sets, we created a novel set of algorithms enabling the fully automatic segmentation and quantification of multiple tumor parameters. This new postmortem imaging technique was applied to determine the therapeutic treatment effect of bevacizumab on the vessel architecture of orthotopic KPLA breast cancer xenografts at different time points. A significant reduction of the vessel volume, number of vessel segments, and branching points in the tumor periphery was already detectable 1 day after initiation of treatment. These parameters remained virtually unchanged in the center of the tumor. Furthermore, bevacizumab-induced vessel normalization and reduction in vascular permeability diminished the penetration behavior of trastuzumab-Alexa 750 into tumor tissue. Our results demonstrated that this new imaging method enables the three-dimensional visualization and fully automatic quantification of multiple tumor parameters and drug penetration on a cellular level. Therefore, UM is a valuable tool for cancer research and drug development. It bridges the gap between common macroscopic and microscopic imaging modalities and opens up new three-dimensional (3D) insights in tumor biology.

Shading correction for whole slide image using low rank and sparse decomposition.

Many microscopic imaging modalities suffer from the problem of intensity inhomogeneity due to uneven illumination or camera nonlinearity, known as shading artifacts. A typical example of this is the unwanted seam when stitching images to obtain a whole slide image (WSI). Elimination of shading plays an essential role for subsequent image processing such as segmentation, registration, or tracking. In this paper, we propose two new retrospective shading correction algorithms for WSI targeted to two common forms of WSI: multiple image tiles before mosaicking and an already-stitched image. Both methods leverage on recent achievements in matrix rank minimization and sparse signal recovery. We show how the classic shading problem in microscopy can be reformulated as a decomposition problem of low-rank and sparse components, which seeks an optimal separation of the foreground objects of interest and the background illumination field. Additionally, a sparse constraint is introduced in the Fourier domain to ensure the smoothness of the recovered background. Extensive qualitative and quantitative validation on both synthetic and real microscopy images demonstrates superior performance of the proposed methods in shading removal in comparison with a well-established method in ImageJ.

Bacterial cell identification in differential interference contrast microscopy images.

BackgroundMicroscopy image segmentation lays the foundation for shape analysis, motion tracking, and classification of biological objects. Despite its importance, automated segmentation remains challenging for several widely used non-fluorescence, interference-based microscopy imaging modalities. For example in differential interference contrast microscopy which plays an important role in modern bacterial cell biology. Therefore, new revolutions in the field require the development of tools, technologies and work-flows to extract and exploit information from interference-based imaging data so as to achieve new fundamental biological insights and understanding.ResultsWe have developed and evaluated a high-throughput image analysis and processing approach to detect and characterize bacterial cells and chemotaxis proteins. Its performance was evaluated using differential interference contrast and fluorescence microscopy images of Rhodobacter sphaeroides.ConclusionsResults demonstrate that the proposed approach provides a fast and robust method for detection and analysis of spatial relationship between bacterial cells and their chemotaxis proteins.

Evaluations of auto-focusing methods under a microscopic imaging modality for metaphase chromosome image analysis.

Background: Auto-focusing is an important operation in high throughput imaging scanning. Although many auto-focusing methods have been developed and tested for a variety of imaging modalities, few investigations have been performed on the selection of an optimal auto-focusing method that is suitable for the pathological metaphase chromosome analysis under a high resolution scanning microscopic system.Objective: The purpose of this study is to investigate and identify an optimal auto-focusing method for the pathological metaphase chromosome analysis.Methods: In this study, five auto-focusing methods were applied and tested using metaphase chromosome images acquired from bone marrow and blood specimens. These methods were assessed by measuring a number of indices including execution time, accuracy, number of false maxima, and full width at half maximum (FWHM).Results: For the specific condition investigated in this study, the results showed that the Brenner gradient and threshold pixel counting methods were the optimal methods for acquiring high quality metaphase chromosome images from the bone marrow and blood specimens, respectively.Conclusions: Selecting an optimal auto-focusing method depends on the specific clinical tasks. This study also provides useful information for the design and implementation of the high throughput microscopic image scanning systems in the future digital pathology.

Comment on “Modified full-field optical coherence tomography: A novel tool for rapid histology of fresh tissues”

Optical coherence tomography (OCT) is one of several three-dimensional microscopy techniques that may revolutionize pathology practice.[1] These techniques essentially create histology-like images without glass microscope slides. This type of imaging can even be performed in vivo,[2] and represents a tremendous opportunity for pathology as a medical specialty. Not only can we pathologists revolutionize our workflow to drastically decrease turn-around time; we can also begin to project our substantial microscopic diagnostic expertise into realms that were previously off limits (i.e., in vivo diagnostics). Mukherjee et al.[1] present an excellent report of their experience with a novel “full-field” OCT-based imaging platform. They demonstrate high quality images that resemble low-to-medium magnification histopathology photomicrographs. This is extremely exciting, and one can imagine potential applications for such images: enhanced specimen grossing; reduced need for frozen sections during intraoperative consultation; perhaps even sign-out of specific surgical pathology tests such as postpartum tubal ligation specimens. This last may seem controversial, but existing OCT images may already be of high enough quality to permit assessment for the presence of complete cross sections of fallopian tube tissue.[3] It is likely that image quality will continue to increase, perhaps to the extent that it may rival what is possible with traditional microscopy (i.e., “routine” diagnostic work). Maturation of 3D histopathology may push OCT and related techniques far beyond glass-based methods, especially with computer-assisted diagnostic tools.[4] It is very possible that diagnostic-quality in vivo images may even make traditional biopsy obsolete, especially in certain situations (i.e., surveillance of Barrett's esophagus). Mukherjee's experience is also interesting because they are reporting on a system that it is commercially available, designed for use with histopathology specimens, and produces DICOM-compatible images that by definition can be incorporated into existing image management infrastructure such as radiology PACS systems. The rise of three-dimensional microscopic imaging modalities represents a fabulous opportunity for Pathology, but it also may foreshadow the end of traditional microscope-based practice. The stakes are very high, and we may have to compete with other specialties for “turf”. We should do so vigorously, leveraging our strong advantages related to existing microscopic morphology expertise, our culture of providing definitive diagnosis (vs. “impressions”), and possibly even cost. If we do not seize this opportunity we may be left in a passive role as these technologies develop around us, perhaps even rendering our old microscopic techniques obsolete. Mukherjee et al have provided us with a tantalizing glimpse of the future, and it is bright.

A comparative study of high resolution microscopy imaging modalities using a three-dimensional resolution measure

From an acquired image, single molecule microscopy makes possible the determination of the distance separating two closely spaced biomolecules in three-dimensional (3D) space. Such distance information can be an important indicator of the nature of the biomolecular interaction. Distance determination, however, is especially difficult when, for example, the imaged point sources are very close to each other or are located near the focal plane of the imaging setup. In the context of such challenges, we compare the limits of the distance estimation accuracy for several high resolution 3D imaging modalities. The comparisons are made using a Cramer-Rao lower bound-based 3D resolution measure which predicts the best possible accuracy with which a given distance can be estimated. Modalities which separate the detection of individual point sources (e.g., using photoactivatable fluorophores) are shown to provide the best accuracy limits when the two point sources are very close to each other and/or are oriented near parallel to the optical axis. Meanwhile, modalities which implement the simultaneous imaging of the point sources from multiple focal planes perform best when given a near-focus point source pair. We also demonstrate that the maximum likelihood estimator is capable of attaining the limit of the accuracy predicted for each modality.

Laser-scanning optical-resolution photoacoustic microscopy

We have developed a laser-scanning optical-resolution photoacoustic microscopy method that can potentially fuse with existing optical microscopic imaging modalities. To acquire an image, the ultrasonic transducer is kept stationary during data acquisition, and only the laser light is raster scanned by an x-y galvanometer scanner. A lateral resolution of 7.8 microm and a circular field of view with a diameter of 6 mm were achieved in an optically clear medium. Using a laser system working at a pulse repetition rate of 1,024 Hz, the data acquisition time for an image consisting of 256 x 256 pixels was less than 2 min.

Prospects for 3D, nanometer-resolution imaging by confocal STEM

Confocal STEM is a new electron microscopy imaging mode. In a microscope with spherical aberration-corrected electron optics, it can produce three-dimensional (3D) images by optical sectioning. We have adapted the linear imaging theory of light confocal microscopy to confocal STEM and use it to suggest optimum imaging conditions for a confocal STEM limited by fifth-order spherical aberration. We predict that current or near-future microscopes will be able to produce 3D images with 1 nm vertical resolution and sub-Ångstrom lateral resolution. Multislice simulations show that we will need to be cautious in interpreting these images, however, as they can be complicated by dynamical electron scattering.

Structural Evidence for &amp;alpha;-Synuclein Fibrils Using &amp;lt;italic&amp;gt;in Situ&amp;lt;/italic&amp;gt; Atomic Force Microscopy

Human alpha-synuclein is a presynaptic terminal protein and can form insoluble fibrils that are believed to play an important role in the pathogenesis of several neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies and Lewy body variant of Alzheimer's disease. In this paper, in situ atomic force microscopy has been used to study the structural properties of alpha-synuclein fibrils in solution using two different atomic force microscopy imaging modes: tapping mode and contact mode. In the in situ contact mode atomic force microscopy experiments alpha-synuclein fibrils quickly broke into fragments, and a similar phenomenon was found using tapping mode atomic force microscopy in which alpha-synuclein fibrils were incubated with guanidine hydrochloride (0.6 M). The alpha-synuclein fibrils kept their original filamentous topography for over 1 h in the in situ tapping mode atomic force microscopy experiments. The present results provide indirect evidence on how beta-sheets assemble into alpha-synuclein fibrils on a nanometer scale.