Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. ^1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. ⁷ LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of <1 μm x <1 μm x <3 μm in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm³, and can be particularly useful for lab-on-a-chip platforms.     

Multimodal Holographic Microscopy: Distinction between Apoptosis and Oncosis

Identification of specific cell death is of a great value for many scientists. Predominant types of cell death can be detected by flow-cytometry (FCM). Nevertheless, the absence of cellular morphology analysis leads to the misclassification of cell death type due to underestimated oncosis. However, the definition of the oncosis is important because of its potential reversibility. Therefore, FCM analysis of cell death using annexin V/propidium iodide assay was compared with holographic microscopy coupled with fluorescence detection - “Multimodal holographic microscopy (MHM)”. The aim was to highlight FCM limitations and to point out MHM advantages. It was shown that the annexin V+/PI− phenotype is not specific of early apoptotic cells, as previously believed, and that morphological criteria have to be necessarily combined with annexin V/PI for the cell death type to be ascertained precisely. MHM makes it possible to distinguish oncosis clearly from apoptosis and to stratify the progression of oncosis.     

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.     

Stiffness Tomography by Atomic Force Microscopy

The atomic force microscope is a convenient tool to probe living samples at the nanometric scale. Among its numerous capabilities, the instrument can be operated as a nano-indenter to gather information about the mechanical properties of the sample. In this operating mode, the deformation of the cantilever is displayed as a function of the indentation depth of the tip into the sample. Fitting this curve with different theoretical models permits us to estimate the Young's modulus of the sample at the indentation spot. We describe what to our knowledge is a new technique to process these curves to distinguish structures of different stiffness buried into the bulk of the sample. The working principle of this new imaging technique has been verified by finite element models and successfully applied to living cells.     

A Fast Color Image Encryption Algorithm Using 4-Pixel Feistel Structure

Algorithms using 4-pixel Feistel structure and chaotic systems have been shown to resolve security problems caused by large data capacity and high correlation among pixels for color image encryption. In this paper, a fast color image encryption algorithm based on the modified 4-pixel Feistel structure and multiple chaotic maps is proposed to improve the efficiency of this type of algorithm. Two methods are used. First, a simple round function based on a piecewise linear function and tent map are used to reduce computational cost during each iteration. Second, the 4-pixel Feistel structure reduces round number by changing twist direction securely to help the algorithm proceed efficiently. While a large number of simulation experiments prove its security performance, additional special analysis and a corresponding speed simulation show that these two methods increase the speed of the proposed algorithm (0.15s for a 256*256 color image) to twice that of an algorithm with a similar structure (0.37s for the same size image). Additionally, the method is also faster than other recently proposed algorithms.     

Accuracy of Image Guidance Using Free-Breathing Cone-Beam Computed Tomography for Stereotactic Lung Radiotherapy

Movement of the target object during cone-beam computed tomography (CBCT) leads to motion blurring artifacts. The accuracy of manual image matching in image-guided radiotherapy depends on the image quality. We aimed to assess the accuracy of target position localization using free-breathing CBCT during stereotactic lung radiotherapy. The Vero4DRT linear accelerator device was used for the examinations. Reference point discrepancies between the MV X-ray beam and the CBCT system were calculated using a phantom device with a centrally mounted steel ball. The precision of manual image matching between the CBCT and the averaged intensity (AI) images restructured from four-dimensional CT (4DCT) was estimated with a respiratory motion phantom, as determined in evaluations by five independent operators. Reference point discrepancies between the MV X-ray beam and the CBCT image-guidance systems, categorized as left-right (LR), anterior-posterior (AP), and superior-inferior (SI), were 0.33 ± 0.09, 0.16 ± 0.07, and 0.05 ± 0.04 mm, respectively. The LR, AP, and SI values for residual errors from manual image matching were -0.03 ± 0.22, 0.07 ± 0.25, and -0.79 ± 0.68 mm, respectively. The accuracy of target position localization using the Vero4DRT system in our center was 1.07 ± 1.23 mm (2 SD). This study experimentally demonstrated the sufficient level of geometric accuracy using the free-breathing CBCT and the image-guidance system mounted on the Vero4DRT. However, the inter-observer variation and systematic localization error of image matching substantially affected the overall geometric accuracy. Therefore, when using the free-breathing CBCT images, careful consideration of image matching is especially important.     

A New Color Image Encryption Scheme Using CML and a Fractional-Order Chaotic System

The chaos-based image cryptosystems have been widely investigated in recent years to provide real-time encryption and transmission. In this paper, a novel color image encryption algorithm by using coupled-map lattices (CML) and a fractional-order chaotic system is proposed to enhance the security and robustness of the encryption algorithms with a permutation-diffusion structure. To make the encryption procedure more confusing and complex, an image division-shuffling process is put forward, where the plain-image is first divided into four sub-images, and then the position of the pixels in the whole image is shuffled. In order to generate initial conditions and parameters of two chaotic systems, a 280-bit long external secret key is employed. The key space analysis, various statistical analysis, information entropy analysis, differential analysis and key sensitivity analysis are introduced to test the security of the new image encryption algorithm. The cryptosystem speed is analyzed and tested as well. Experimental results confirm that, in comparison to other image encryption schemes, the new algorithm has higher security and is fast for practical image encryption. Moreover, an extensive tolerance analysis of some common image processing operations such as noise adding, cropping, JPEG compression, rotation, brightening and darkening, has been performed on the proposed image encryption technique. Corresponding results reveal that the proposed image encryption method has good robustness against some image processing operations and geometric attacks.     

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated ^1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated ^4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot ^8-10. We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.     

Practical Image Restoration of Thick Biological Specimens Using Multiple Focus Levels in Transmission Electron Microscopy

Three-dimensional electron tomographic studies of thick specimens such as cellular organelles or supramolecular structures require accurate interpretations of transmission electron micrograph intensities. In addition to microscope lens aberrations, thick specimen imaging is complicated by additional distortions resulting from multiple elastic and inelastic scattering. Extensive analysis of the mechanism of image formation using electron energy-loss spectroscopy and imaging as well as exit wavefront reconstruction demonstrated that multiple scattering does not contribute to the coherent component of the exit wave (Hanet al.,1996, 1995). Although exit wavefront restored images showed enhanced contrast and resolution, that technique, which requires the collection of more than 30 images at different focus levels, is not practical for routine data collection in 3D electron tomography, where usually over 100 projection views are required for each reconstruction. Using a 0.7-μm-thick specimen imaged at 200 keV, the accuracy of reconstructions using small numbers of defocused images and a simple linear filter (Schiske, 1968) was assessed by comparison to the complete exit wave restoration. We demonstrate that only four optimal focus levels are required to effectively restore the coherent component (deviation 5.1%). By contrast, the optimal single image (zero defocus) shows a 25.5% deviation to the exit wave restoration. Two pairs of under- and over-defocus images should be taken: one pair at quite high defocus (>10 μm) to differentiate the coherent (single elastic scattering) from the incoherent (multiple elastic and inelastic scattering) components, and the second pair to optimize information content at the highest desired resolution (e.g., 5 μm for (2.5 nm)⁻¹resolution). We also propose a new interpretation of the restored amplitude and phase components where the specimen mass-density is proportional to the logarithm of the amplitude component and linearly related to the phase component. This approach should greatly facilitate the collection of high resolution tomographic data from thick samples.     

A New Least Squares Based Calibration for an Ultrasound Tomography Scanner Using Radial Image Processing

In the context of the development of an ultrasound scanner where a probe turns around the part of the body to be studied, a new tomographic technique has been developed: the Ultrasound Reflection-mode Tomography Using Radial Image Processing (URTURIP Technique). This technique is used when a unique B-scan image is insufficient to correctly describe a cross-section. It utilises B-scan images obtained under different angles of view in the same plane to reconstruct a better cross-sectional image. Before using the URTURIP Technique, a calibration step is required to accurately determine the centre of rotation of the probe. To solve the calibration problem, a reference method has been developed, but it is very time-consuming. This paper presents a faster method based on a least squares fitting and is compared to the reference method in terms of accuracy and time-consuming.     

Quantitative Stain-Free and Continuous Multimodal Monitoring of Wound Healing In Vitro with Digital Holographic Microscopy

Impaired epithelial wound healing has significant pathophysiological implications in several conditions including gastrointestinal ulcers, anastomotic leakage and venous or diabetic skin ulcers. Promising drug candidates for accelerating wound closure are commonly evaluated in in vitro wound assays. However, staining procedures and discontinuous monitoring are major drawbacks hampering accurate assessment of wound assays. We therefore investigated digital holographic microscopy (DHM) to appropriately monitor wound healing in vitro and secondly, to provide multimodal quantitative information on morphological and functional cell alterations as well as on motility changes upon cytokine stimulation. Wound closure as reflected by proliferation and migration of Caco-2 cells in wound healing assays was studied and assessed in time-lapse series for 40 h in the presence of stimulating epidermal growth factor (EGF) and inhibiting mitomycin c. Therefore, digital holograms were recorded continuously every thirty minutes. Morphological changes including cell thickness, dry mass and tissue density were analyzed by data from quantitative digital holographic phase microscopy. Stimulation of Caco-2 cells with EGF or mitomycin c resulted in significant morphological changes during wound healing compared to control cells. In conclusion, DHM allows accurate, stain-free and continuous multimodal quantitative monitoring of wound healing in vitro and could be a promising new technique for assessment of wound healing.     

应用反向滤过及迭代重建方法评估标准放射剂量与低剂量颈椎CT图像质量的对比研究

目的：利用反向滤过重建（filtered back-projecfion,FBP）及迭代重建（iterative reconstruction,IR）方法评估标准剂量及低剂量对颈椎CT图像质量的影响。方法：40例受检对象行颈椎CT检查,将其随机分为两组：标准剂量组（SD,120kVp,275mAs）及低剂量组（LD,120kVp,150mAs）,随机选择管电流值,所有数据均行FBP及IR重建。测量C3C4及C6C7椎间盘水平椎间盘、脊神经、脊髓、韧带以及周围软组织的图像噪声值（Imagenoise,IN）,信噪比（signal—to—noise,SNR）及对比信噪比（contrast—to—noise,CNR）。结果：在测量的各椎间盘水平,迭代重建的信噪比及对比噪声比要明显高于反向滤过重建方法,并有效的降低了图像噪声。低剂量迭代重建图像与标准剂量反向滤过图像相比无明显统计学意义。排除剂量及扫描层面的影响,椎间盘、脊神经及韧带的图像质量,迭代重建评分要明显高于反向滤过重建,结果具有统计学差异;而低剂量迭代重建图像质量评分与标准剂量反向滤过重建相比无明显差异。软组织及椎体的图像质量,迭代重建图像质量评分要低于反向滤过重建方法,结果具有统计学差异;而低剂量迭代重建图像质量评分与标准剂量反向滤过重建相比无明显差异。整体病例图像质量评分,迭代重建方法要高于反向滤过重建方法,低剂量迭代重建方法要高于标准剂量反向滤过重建方法。结论：应用低剂量扫描方式以及迭代重建方法进行颈椎CT检查可以为I临床提供较好的图像质量,对于椎间盘、脊神经、脊髓显示较好,对于周围软组织以及椎体来说,图像质量相对较差,同时可以降低大约40％的放射剂量。     

应用反向滤过及迭代重建方法评估标准放射剂量与低剂量颈椎 CT图像质量的对比研究

目的：利用反向滤过重建（filtered back-projection,FBP）及迭代重建（iterative reconstruction,IR）方法评估标准剂量及低剂量 对颈椎CT 图像质量的影响。方法：40 例受检对象行颈椎CT 检查,将其随机分为两组：标准剂量组（SD,120 kVp, 275 mAs）及低 剂量组（LD,120 kVp,150 mAs）,随机选择管电流值,所有数据均行FBP 及IR 重建。测量C3 C4 及C6 C7 椎间盘水平椎间盘、脊 神经、脊髓、韧带以及周围软组织的图像噪声值（Image noise,IN）,信噪比（signal-to-noise,SNR）及对比信噪比（contrast-to-noise, CNR）。结果：在测量的各椎间盘水平,迭代重建的信噪比及对比噪声比要明显高于反向滤过重建方法,并有效的降低了图像噪 声。低剂量迭代重建图像与标准剂量反向滤过图像相比无明显统计学意义。排除剂量及扫描层面的影响,椎间盘、脊神经及韧带 的图像质量,迭代重建评分要明显高于反向滤过重建,结果具有统计学差异;而低剂量迭代重建图像质量评分与标准剂量反向滤 过重建相比无明显差异。软组织及椎体的图像质量,迭代重建图像质量评分要低于反向滤过重建方法,结果具有统计学差异;而 低剂量迭代重建图像质量评分与标准剂量反向滤过重建相比无明显差异。整体病例图像质量评分,迭代重建方法要高于反向滤 过重建方法,低剂量迭代重建方法要高于标准剂量反向滤过重建方法。结论：应用低剂量扫描方式以及迭代重建方法进行颈椎 CT 检查可以为临床提供较好的图像质量,对于椎间盘、脊神经、脊髓显示较好,对于周围软组织以及椎体来说,图像质量相对较 差,同时可以降低大约40%的放射剂量。     

Digital Holographic Microscopy for Non-Invasive Monitoring of Cell Cycle Arrest in L929 Cells

Digital holographic microscopy (DHM) has emerged as a powerful non-invasive tool for cell analysis. It has the capacity to analyse multiple parameters simultaneously, such as cell- number, confluence and phase volume. This is done while cells are still adhered and growing in their culture flask. The aim of this study was to investigate whether DHM was able to monitor drug-induced cell cycle arrest in cultured cells and thus provide a non-disruptive alternative to flow cytometry. DHM parameters from G1 and G2/M cell cycle arrested L929 mouse fibroblast cells were collected. Cell cycle arrest was verified with flow cytometry. This study shows that DHM is able to monitor phase volume changes corresponding to either a G1 or G2/M cell cycle arrest. G1-phase arrest with staurosporine correlated with a decrease in the average cell phase volume and G2/M-phase arrest with colcemid and etoposide correlated with an increase in the average cell phase volume. Importantly, DHM analysis of average cell phase volume was of comparable accuracy to flow cytometric measurement of cell cycle phase distribution as recorded following dose-dependent treatment with etoposide. Average cell phase volume changes in response to treatment with cell cycle arresting compounds could therefore be used as a DHM marker for monitoring cell cycle arrest in cultured mammalian cells.     

Direct Visualization of Mucus Production by the Cold-Water Coral Lophelia pertusa with Digital Holographic Microscopy

Lophelia pertusa is the dominant reef-building organism of cold-water coral reefs, and is known to produce significant amounts of mucus, which could involve an important metabolic cost. Mucus is involved in particle removal and feeding processes, yet the triggers and dynamics of mucus production are currently still poorly described because the existing tools to study these processes are not appropriate. Using a novel microscopic technique—digital holographic microscopy (DHM)–we studied the mucus release of L. pertusa under various experimental conditions. DHM technology permits μm-scale observations and allows the visualization of transparent mucoid substances in real time without staining. Fragments of L. pertusa were first maintained in flow-through chambers without stressors and imaged with DHM, then exposed to various stressors (suspended particles, particulate food and air exposure) and re-imaged. Under non-stressed conditions no release of mucus was observed, whilst mucus strings and sheaths were produced in response to suspended particles (activated charcoal and drill cuttings sediment) i.e. in a stressed condition. Mucus strings and so-called ‘string balls’ were also observed in response to exposure to particulate food (brine shrimp Artemia salina). Upon air-exposure, mucus production was clearly visible once the fragments were returned to the flow chamber. Distinct optical properties such as optical path length difference (OPD) were measured with DHM in response to the various stimuli suggesting that different mucus types are produced by L. pertusa. Mucus produced to reject particles is similar in refractive index to the surrounding seawater, suggesting that the energy content of this mucus is low. In contrast, mucus produced in response to either food particle addition or air exposure had a higher refractive index, suggesting a higher metabolic investment in the production of these mucoid substances. This paper shows for the first time the potential of DHM technology for the detection, characterization and quantification of mucus production through OPD measurements in L. pertusa.     

Serotyping of Adenoviruses Using Immune Electron Microscopy

Immune electron microscopy may be used to determine the type specificity of several common human adenoviruses. The method described utilized equine antisera and was used to serotype adenoviruses within hours after typical cytopathic effect was observed in tissue culture. Adenovirus types 1, 2, 3, 5, 7a, 8, and 11 were isolated from clinical specimens and serotyped by serum neutralization and immune electron microscopy. Five of the seven types were identified consistently and identically by the two methods. Types 7a and 11 were indistinguishable by immune electron microscopy.     

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.     

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.