Confined activation and subdiffractive localization enables whole-cell PALM with genetically expressed probes

We demonstrate three-dimensional (3D) super-resolution microscopy in whole fixed cells using photoactivated localization microscopy (PALM). The use of the bright, genetically expressed fluorescent marker photoactivatable monomeric (m)Cherry (PA-mCherry1) in combination with near diffraction-limited confinement of photoactivation using two-photon illumination and 3D localization methods allowed us to investigate a variety of cellular structures at <50 nm lateral and <100 nm axial resolution. Compared to existing methods, we have substantially reduced excitation and bleaching of unlocalized markers, which allows us to use 3D PALM imaging with high localization density in thick structures. Our 3D localization algorithms, which are based on cross-correlation, do not rely on idealized noise models or specific optical configurations. This allows instrument design to be flexible. By generating appropriate fusion constructs and expressing them in Cos7 cells, we could image invaginations of the nuclear membrane, vimentin fibrils, the mitochondrial network and the endoplasmic reticulum at depths of greater than 8 μm.     

多尺度的光声显微镜用于癌细胞及实体瘤成像

多尺度显微成像系统(M-PAM)被发展,并被用于成像从癌细胞到实体肿瘤的多尺度生物结构.该装置由二维运动平台,扫描振镜,物镜,聚焦超声换能器组成,其横向分辨率达到3 μm.结果显示该系统可以对体外培养黑色素瘤细胞与体内的黑色素瘤进行无标记成像.基于具有靶向性的探针,M-PAM系统可以对体外培养的U87-MG肿瘤细胞以及体内U87-MG实体肿瘤进行成像.综上所述,M-PAM系统将是研究肿瘤的有力工具.     

Multiplex FISH and three-dimensional DNA imaging with near infrared femtosecond laser pulses

We report on a novel technology for multicolor gene and chromosome detection as well as for three-dimensional (3D) DNA imaging by multiphoton excitation of multiple FISH fluorophores and DNA stains. Near infrared femtosecond laser pulses at 770 nm were used to simultaneously excite the visible fluorescence of a wide range of FISH fluorophores, such as FITC, DAC, Cy3, Cy5, Cy5.5, rhodamine, spectrum aqua, spectrum green, spectrum orange, Jenfluor, and Texas red as well as of DNA/chromosome stains, for example Hoechst 33342, DAPI, SYBR green, propidium iodide, ethidium homodimer, and Giemsa. In addition to the advantage of using only one excitation wavelength for a variety of fluorophores, multiphoton excitation provided the intrinsic possibility of 3D fluorescence imaging. The technology has been used in human genetics for the diagnosis of numerical chromosome aberrations and microdeletions. In particular, multicolor 3D images of the intranuclear localization of FISH-labeled chromosome territories in interphase nuclei of amniotic fluid cells have been obtained. Using the high light penetration depth at 770 nm, optical sectioning of Hoechst 33342-labeled DNA within living culture cells and within tissue of living tumor-bearing mice was performed.     

Neuroanatomical phenotyping of the mouse brain with three-dimensional autofluorescence imaging

The structural organization of the brain is important for normal brain function and is critical to understand in order to evaluate changes that occur during disease processes. Three-dimensional (3D) imaging of the mouse brain is necessary to appreciate the spatial context of structures within the brain. In addition, the small scale of many brain structures necessitates resolution at the ～10 μm scale. 3D optical imaging techniques, such as optical projection tomography (OPT), have the ability to image intact large specimens (1 cm(3)) with ～5 μm resolution. In this work we assessed the potential of autofluorescence optical imaging methods, and specifically OPT, for phenotyping the mouse brain. We found that both specimen size and fixation methods affected the quality of the OPT image. Based on these findings we developed a specimen preparation method to improve the images. Using this method we assessed the potential of optical imaging for phenotyping. Phenotypic differences between wild-type male and female mice were quantified using computer-automated methods. We found that optical imaging of the endogenous autofluorescence in the mouse brain allows for 3D characterization of neuroanatomy and detailed analysis of brain phenotypes. This will be a powerful tool for understanding mouse models of disease and development and is a technology that fits easily within the workflow of biology and neuroscience labs.     

Restored interlaced volumetric imaging increases image quality and scanning speed during intravital imaging in living mice

Dynamic intravital imaging is essential for revealing ongoing biological phenomena within living organisms and is influenced primarily by several factors: motion artifacts, optical properties and spatial resolution. Conventional imaging quality within a volume, however, is degraded by involuntary movements and trades off between the imaged volume, imaging speed and quality. To balance such trade‐offs incurred by two‐photon excitation microscopy during intravital imaging, we developed a unique combination of interlaced scanning and a simple image restoration algorithm based on biological signal sparsity and a graph Laplacian matrix. This method increases the scanning speed by a factor of four for a field size of 212 μm × 106 μm × 130 μm, and significantly improves the quality of four‐dimensional dynamic volumetric data by preventing irregular artifacts due to the movement observed with conventional methods. Our data suggest this method is robust enough to be applied to multiple types of soft tissue.     

Synchrotron radiation micro-CT at the micrometer scale for the analysis of the three-dimensional morphology of microcracks in human trabecular bone

Bone quality is an important concept to explain bone fragility in addition to bone mass. Among bone quality factors, microdamage which appears in daily life is thought to have a marked impact on bone strength and plays a major role in the repair process. The starting point for all studies designed to further our understanding of how bone microdamage initiate or dissipate energy, or to investigate the impact of age, gender or disease, remains reliable observation and measurement of microdamage. In this study, 3D Synchrotron Radiation (SR) micro-CT at the micrometric scale was coupled to image analysis for the three-dimensional characterization of bone microdamage in human trabecular bone specimens taken from femoral heads. Specimens were imaged by 3D SR micro-CT with a voxel size of 1.4 μm. A new tailored 3D image analysis technique was developed to segment and quantify microcracks. Microcracks from human trabecular bone were observed in different tomographic sections as well as from 3D renderings. New 3D quantitative measurements on the microcrack density and morphology are reported on five specimens. The 3D microcrack density was found between 3.1 and 9.4/mm3 corresponding to a 2D density between 0.55 and 0.76 /mm2. The microcrack length and width measured in 3D on five selected microcrack ranged respectively from 164 μm to 209 μm and 100 μm to 120 μm. This is the first time that various microcracks in unloaded human trabecular bone--from the simplest linear crack to more complex cross-hatch cracks--have been examined and quantified by 3D imaging at this scale. The suspected complex morphology of microcracks is here considerably more evident than in the 2D observations. In conclusion, this technique opens new perspective for the 3D investigation of microcracks and the impact of age, disease or treatment.     

Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy

We demonstrate three-dimensional (3D) super-resolution in live multicellular organisms using structured illumination microscopy (SIM). Sparse multifocal illumination patterns generated by a digital micromirror device (DMD) allowed us to physically reject out-of-focus light, enabling 3D subdiffractive imaging in samples eightfold thicker than had been previously imaged with SIM. We imaged samples at one 2D image per second, at resolutions as low as 145 nm laterally and 400 nm axially. In addition to dual-labeled, whole fixed cells, we imaged GFP-labeled microtubules in live transgenic zebrafish embryos at depths >45 μm. We captured dynamic changes in the zebrafish lateral line primordium and observed interactions between myosin IIA and F-actin in cells encapsulated in collagen gels, obtaining two-color 4D super-resolution data sets spanning tens of time points and minutes without apparent phototoxicity. Our method uses commercially available parts and open-source software and is simpler than existing SIM implementations, allowing easy integration with wide-field microscopes.     

Mineralized scale patterns on the cell periphery of the chrysophyte Mallomonas determined by comparative 3D Cryo-FIB SEM data processing

Unicellular protists can biomineralize spatially complex and functional shells. A typical cell of the photosynthetic synurophyte Mallomonas is covered by about 60–100 silica scales. Their geometric arrangement, the so-called scale case, mainly depends on the species and on the cell cycle. In this study, the scale case of the synurophyte Mallomonas was preserved in aqueous suspension using high-pressure freezing (HPF). From this specimen, a three-dimensional (3D) data set spanning a volume of about 25.6 μm × 19.2 μm × 4.2 μm with a voxel size of 12.5 nm × 12.5 nm × 25.0 nm was collected by Cryo-FIB SEM in 3 h and 24 min. SEM imaging using In-lens SE detection allowed to clearly differentiate between mineralized, curved scales of less than 0.2 μm thickness and organic cellular ultrastructure or vitrified ice. The three-dimensional spatial orientations and shapes of a minimum set of scales (N = 13) were identified by visual inspection, and manually segmented. Manual and automated segmentation approaches were comparatively applied to one arbitrarily selected reference scale using the differences in grey level between scales and other constituents. Computational automated routines and principal component analysis of the experimentally extracted data created a realistic mathematical model based on the Fibonacci pattern theory. A complete in silico scale case of Mallomonas was reconstructed showing an optimized scale coverage on the cell surface, similarly as it was observed experimentally. The minimum time requirements from harvesting the living cells to the final scale case determination by Cryo-FIB SEM and computational image processing are discussed.     

Two-photon excitation chlorophyll fluorescence lifetime imaging: a rapid and noninvasive method for in vivo assessment of cadmium toxicity in a marine diatom Thalassiosira weissflogii

We demonstrate that a two-photon excitation fluorescence lifetime imaging technology can rapidly and noninvasively assess the cadmium (Cd)-induced toxic effects in a marine diatom Thalassiosira weissflogii. The chlorophyll, an intrinsic fluorophore, was used as a contrast agent for imaging of cellular structures and for assessment of cell toxicity. The assessment is based on an imaging-guided statistical analysis of chlorophyll fluorescence decay. This novel label-free imaging method is physically based and free of tedious preparation and preprocessing of algal samples. We first studied the chlorophyll fluorescence quenching induced by the infrared two-photon excitation laser and found that the quenching effects on the assessment of Cd toxicity could be well controlled and calibrated. In the toxicity study, chlorophyll fluorescence lifetime images were collected from the diatom samples after exposure to different concentrations of Cd. The alteration of chloroplast structure at higher Cd concentration was clearly identified. The decay of chlorophyll fluorescence extracted from recorded pixels of high signal-to-noise ratio in the fluorescence lifetime image was analyzed. The increase of average chlorophyll fluorescence lifetime following Cd treatment was observed, indicating the Cd inhibition effect on the electron transport chain in photosynthesis system. The findings of this study show that the temporal characteristics of chlorophyll fluorescence can potentially be utilized as a biomarker for indicating Cd toxicity noninvasively in algal cells.     

Imaging neuronal structure dynamics using 2‐photon super‐resolution patterned excitation reconstruction microscopy

Visualizing fine neuronal structures deep inside strongly light‐scattering brain tissue remains a challenge in neuroscience. Recent nanoscopy techniques have reached the necessary resolution but often suffer from limited imaging depth, long imaging time or high light fluence requirements. Here, we present two‐photon super‐resolution patterned excitation reconstruction (2P‐SuPER) microscopy for 3‐dimensional imaging of dendritic spine dynamics at a maximum demonstrated imaging depth of 130 μm in living brain tissue with approximately 100 nm spatial resolution. We confirmed 2P‐SuPER resolution using fluorescence nanoparticle and quantum dot phantoms and imaged spiny neurons in acute brain slices. We induced hippocampal plasticity and showed that 2P‐SuPER can resolve increases in dendritic spine head sizes on CA1 pyramidal neurons following theta‐burst stimulation of Schaffer collateral axons. 2P‐SuPER further revealed nanoscopic increases in dendritic spine neck widths, a feature of synaptic plasticity that has not been thoroughly investigated due to the combined limit of resolution and penetration depth in existing imaging technologies.      

Noninvasive reconstruction of the three-dimensional ventricular activation sequence during pacing and ventricular tachycardia in the canine heart

Single-beat imaging of myocardial activation promises to aid in both cardiovascular research and clinical medicine. In the present study we validate a three-dimensional (3D) cardiac electrical imaging (3DCEI) technique with the aid of simultaneous 3D intracardiac mapping to assess its capability to localize endocardial and epicardial initiation sites and image global activation sequences during pacing and ventricular tachycardia (VT) in the canine heart. Body surface potentials were measured simultaneously with bipolar electrical recordings in a closed-chest condition in healthy canines. Computed tomography images were obtained after the mapping study to construct realistic geometry models. Data analysis was performed on paced rhythms and VTs induced by norepinephrine (NE). The noninvasively reconstructed activation sequence was in good agreement with the simultaneous measurements from 3D cardiac mapping with a correlation coefficient of 0.74 ± 0.06, a relative error of 0.29 ± 0.05, and a root mean square error of 9 ± 3 ms averaged over 460 paced beats and 96 ectopic beats including premature ventricular complexes, couplets, and nonsustained monomorphic VTs and polymorphic VTs. Endocardial and epicardial origins of paced beats were successfully predicted in 72% and 86% of cases, respectively, during left ventricular pacing. The NE-induced ectopic beats initiated in the subendocardium by a focal mechanism. Sites of initial activation were estimated to be ～7 mm from the measured initiation sites for both the paced beats and ectopic beats. For the polymorphic VTs, beat-to-beat dynamic shifts of initiation site and activation pattern were characterized by the reconstruction. The present results suggest that 3DCEI can noninvasively image the 3D activation sequence and localize the origin of activation of paced beats and NE-induced VTs in the canine heart with good accuracy. This 3DCEI technique offers the potential to aid interventional therapeutic procedures for treating ventricular arrhythmias arising from epicardial or endocardial sites and to noninvasively assess the mechanisms of these arrhythmias.     

Imaging brain tissue slices with terahertz near-field microscopy

Photoconductive antenna microprobe (PCAM)-based terahertz (THz) near-field imaging technique is promising for biomedical detection due to its excellent biocompatibility and high resolution; yet it is limited by its imaging speed and the difficulty in the control of the PCAM tip-sample separation. In this work, we successfully realized imaging of mouse brain tissue slices using an improved home-built PCAM-based THz near-field microscope. In this system, the imaging speed was enhanced by designing and applying a voice coil motor-based delay-line. The tip-sample separation control was implemented by developing an image analysis-based technique. Compared with conventional PCAM-based THz near-field systems, our improved system is 100 times faster in imaging speed and the tip-sample separation can be controlled to a few micrometers (e.g., 3 μm), satisfying the requirements of THz near-field imaging of biological samples. It took about ~30 min (not the tens of hours it took to acquire the same kind of image previously) to collect a THz near-field image of brain tissue slices of BALb/c mice (500 μm × 500 μm) with pixel size of 20 μm × 20 μm. The results show that the mouse brain slices can be properly imaged and different regions in the slices (i.e., the corpus callosum region and the cerebrum region) can be identified unambiguously. Evidently, the work demonstrated here provides not only a convincing example but a useful technique for imaging biological samples with THz near-field microscopy. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2741, 2019.     

A biopsy‐needle compatible varifocal multiphoton rigid probe for depth‐resolved optical biopsy

In this work, we report a biopsy‐needle compatible rigid probe, capable of performing three‐dimensional (3D) two‐photon optical biopsy. The probe has a small outer diameter of 1.75 mm and fits inside a gauge‐14 biopsy needle to reach internal organs. A carefully designed focus scanning mechanism has been implemented in the rigid probe, which, along with a rapid two‐dimensional MEMS scanner, enables 3D imaging. Fast image acquisition up to 10 frames per second is possible, dramatically reducing motion artifacts during in vivo imaging. Equipped with a high‐numerical aperture micro‐objective, the miniature rigid probe offers a high two‐photon resolution (0.833 × 6.11 μm, lateral × axial), a lateral field of view of 120 μm, and an axial focus tuning range of 200 μm. In addition to imaging of mouse internal organs and subcutaneous tumor in vivo, first‐of‐its‐kind depth‐resolved two‐photon optical biopsy of an internal organ has been successfully demonstrated on mouse kidney in vivo and in situ.      

Noninvasive cineangiography by magnetic resonance global coherent free precession

Cardiovascular disease is primarily diagnosed using invasive X-ray cineangiography. Here we introduce a new concept in magnetic resonance imaging (MRI) that, for the first time, produces similar images noninvasively and without a contrast agent. Protons in moving blood are 'tagged' every few milliseconds as they travel through an arbitrary region in space. Simultaneous with ongoing tagging of new blood, previously tagged blood is maintained in a state of global coherent free precession (GCFP), which allows acquisition of consecutive movie frames as the heart pushes blood through the vascular bed. Body tissue surrounding the moving blood is never excited and therefore remains invisible. In 18 subjects, pulsating blood could be seen flowing through three-dimensional (3D) space for distances of up to 16 cm outside the stationary excitation region. These data underscore that our approach noninvasively characterizes both anatomy and blood flow in a manner directly analogous to invasive procedures.     

Visualizing the “sandwich” structure of osteocytes in their native environment deep in bone in vivo

Osteocytes are the most abundant cells in bone and always the focus of bone research. They are embedded in the highly scattering mineralized bone matrix. Consequently, visualizing osteocytes deep in bone with subcellular resolution poses a major challenge for in vivo bone research. Here we overcome this challenge by demonstrating 3‐photon imaging of osteocytes through the intact mouse skull in vivo. Through broadband transmittance characterization, we establish that the excitation at the 1700‐nm window enables the highest optical transmittance through the skull. Using label‐free third‐harmonic generation (THG) imaging excited at this window, we visualize osteocytes through the whole 140‐μm mouse skull and 155 μm into the brain in vivo. By developing selective labeling technique for the interstitial space, we visualize the “sandwich” structure of osteocytes in their native environment. Our work provides novel imaging methodology for bone research in vivo.      

In silico optimization of radioluminescence microscopy

Radioluminescence microscopy (RLM) is a high‐resolution method for imaging radionuclide uptake in live cells within a fluorescence microscopy environment. Although RLM currently provides sufficient spatial resolution and sensitivity for cell imaging, it has not been systematically optimized. This study seeks to optimize the parameters of the system by computational simulation using a combination of numerical models for the system's various components: Monte‐Carlo simulation for radiation transport, 3D optical point‐spread function for the microscope, and stochastic photosensor model for the electron multiplying charge coupled device (EMCCD) camera. The relationship between key parameters and performance metrics relevant to image quality is examined. Results show that Lu₂O₃:Eu yields the best performance among 5 different scintillator materials, and a thickness: 8 μm can best balance spatial resolution and sensitivity. For this configuration, a spatial resolution of ~20 μm and sensitivity of 40% can be achieved for all 3 magnifications investigated, provided that the user adjusts pixel binning and electron multiplying (EM) gain accordingly. Hence the primary consideration for selecting the magnification should be the desired field of view and magnification for concurrent optical microscopy studies. In conclusion, this study estimates the optimal imaging performance achievable with RLM and promotes further development for more robust imaging of cellular processes using radiotracers.      

Optimization of cell morphology measurement via single-molecule tracking PALM

In neurons, the shape of dendritic spines relates to synapse function, which is rapidly altered during experience-dependent neural plasticity. The small size of spines makes detailed measurement of their morphology in living cells best suited to super-resolution imaging techniques. The distribution of molecular positions mapped via live-cell Photoactivated Localization Microscopy (PALM) is a powerful approach, but molecular motion complicates this analysis and can degrade overall resolution of the morphological reconstruction. Nevertheless, the motion is of additional interest because tracking single molecules provides diffusion coefficients, bound fraction, and other key functional parameters. We used Monte Carlo simulations to examine features of single-molecule tracking of practical utility for the simultaneous determination of cell morphology. We find that the accuracy of determining both distance and angle of motion depend heavily on the precision with which molecules are localized. Strikingly, diffusion within a bounded region resulted in an inward bias of localizations away from the edges, inaccurately reflecting the region structure. This inward bias additionally resulted in a counterintuitive reduction of measured diffusion coefficient for fast-moving molecules; this effect was accentuated by the long camera exposures typically used in single-molecule tracking. Thus, accurate determination of cell morphology from rapidly moving molecules requires the use of short integration times within each image to minimize artifacts caused by motion during image acquisition. Sequential imaging of neuronal processes using excitation pulses of either 2 ms or 10 ms within imaging frames confirmed this: processes appeared erroneously thinner when imaged using the longer excitation pulse. Using this pulsed excitation approach, we show that PALM can be used to image spine and spine neck morphology in living neurons. These results clarify a number of issues involved in interpretation of single-molecule data in living cells and provide a method to minimize artifacts in single-molecule experiments.     

Fast, three-dimensional super-resolution imaging of live cells

We report super-resolution fluorescence imaging of live cells with high spatiotemporal resolution using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and 3D super-resolution images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast-switching probes enabled us to achieve 2D imaging at spatial resolutions of ～25 nm and temporal resolutions as fast as 0.5 s. We also demonstrated live-cell 3D super-resolution imaging. We obtained 3D spatial resolution of ～30 nm in the lateral direction and ～50 nm in the axial direction at time resolutions as fast as 1-2 s with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we also demonstrated two-color 3D super-resolution imaging in live cells. These imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level.     

An efficient way of high-contrast, quasi-3D cellular imaging: off-axis illumination

An imaging system enabling a convenient visualisation of cells and other small objects is presented. It represents an adaptation of the optical microscope condenser, accommodating a built-in edge (relief) diaphragm brought close to the condenser iris diaphragm and enabling high-contrast pseudo-relief (quasi-3D) imaging. The device broadens the family of available apparatus based on the off-axis (or anaxial, asymmetric, inclined, oblique, schlieren-type, sideband) illumination. The simplicity of the design makes the condenser a user-friendly, dedicated device delivering high-contrast quasi-3D images of phase objects. Those are nearly invisible under the ordinary (axial) illumination. The phase contrast microscopy commonly used in visualisation of phase objects does not deliver the quasi-3D effect and introduces a disturbing 'halo' effect around the edges. The performance of the device presented here is demonstrated on living cells and tissue replicas. High-contrast quasi-3D images of cell-free preparations of biological origin (paper fibres and microcrystals) are shown as well.