ABSTRACT
Intravital two-photon microscopy enables deep-tissue imaging at high temporospatial resolution in live animals. However, the endosteal bone compartment and underlying bone marrow pose unique challenges to optical imaging as light is absorbed, scattered and dispersed by thick mineralized bone matrix and the adipose-rich bone marrow. Early bone intravital imaging methods exploited gaps in the cranial sutures to bypass the need to penetrate through cortical bone. More recently, investigators have developed invasive methods to thin the cortical bone or implant imaging windows to image cellular dynamics in weight-bearing long bones. Here, we provide a step-by-step procedure for the preparation of animals for minimally invasive, nondestructive, longitudinal intravital imaging of the murine tibia. This method involves the use of mixed bone marrow radiation chimeras to unambiguously double-label osteoclasts and osteomorphs. The tibia is exposed by a simple skin incision and an imaging chamber constructed using thermoconductive T-putty. Imaging sessions up to 12 h long can be repeated over multiple timepoints to provide a longitudinal time window into the endosteal and marrow niches. The approach can be used to investigate cellular dynamics in bone remodeling, cancer cell life cycle and hematopoiesis, as well as long-lived humoral and cellular immunity. The procedure requires an hour to complete and is suitable for users with minimal prior expertise in small animal surgery.
Subject(s)
Bone and Bones , Intravital Microscopy , Mice , Animals , Bone and Bones/diagnostic imaging , Intravital Microscopy/methods , Optical ImagingABSTRACT
Osteoclasts are large multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage-derived precursors that are thought to undergo apoptosis once resorption is complete. Here, by intravital imaging, we reveal that RANKL-stimulated osteoclasts have an alternative cell fate in which they fission into daughter cells called osteomorphs. Inhibiting RANKL blocked this cellular recycling and resulted in osteomorph accumulation. Single-cell RNA sequencing showed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and express a number of non-canonical osteoclast genes that are associated with structural and functional bone phenotypes when deleted in mice. Furthermore, genetic variation in human orthologs of osteomorph genes causes monogenic skeletal disorders and associates with bone mineral density, a polygenetic skeletal trait. Thus, osteoclasts recycle via osteomorphs, a cell type involved in the regulation of bone resorption that may be targeted for the treatment of skeletal diseases.
Subject(s)
Bone Resorption/pathology , Osteoclasts/pathology , RANK Ligand/metabolism , Animals , Apoptosis , Bone Resorption/metabolism , Cell Fusion , Cells, Cultured , Humans , Macrophages/cytology , Mice , Osteochondrodysplasias/drug therapy , Osteochondrodysplasias/genetics , Osteochondrodysplasias/metabolism , Osteochondrodysplasias/pathology , Osteoclasts/metabolism , Signal TransductionABSTRACT
Microfluidics in vitro assays recapitulate a blood vessel microenvironment using surface-immobilized agonists under biofluidic flows. However, these assays do not quantify intrathrombus mass and activities of adhesive platelets at the agonist margin and use fluorescence labeling, therefore limiting clinical translation potential. Here, we describe a label-free multimodal quantitative imaging flow assay that combines rotating optical coherent scattering microscopy and quantitative phase microscopy. The combined imaging platform enables real-time evaluation of membrane fluctuations of adhesive-only platelets and total intrathrombus mass under physiological flow rates in vitro. We call this multimodal quantitative imaging flow assay coherent optical scattering and phase interferometry (COSI). COSI records intrathrombus mass to picogram accuracy and shape changes to a platelet membrane with high spatial-temporal resolution (0.4 µm/s) under physiological and pathophysiological fluid shear stress (1800 and 7500 s-1). With COSI, we generate an axial slice of 4 µm from the coverslip surface, approximately equivalent to the thickness of a single platelet, which permits nanoscale quantification of membrane fluctuation (activity) of adhesive platelets during initial adhesion, spreading, and recruitment into a developing thrombus (mass). Under fluid shear, pretreatment with a broad range metalloproteinase inhibitor (250 µM GM6001) blocked shedding of platelet adhesion receptors that shown elevated adhesive platelet activity at average of 42.1 µm/s and minimal change in intrathrombus mass.
Subject(s)
Platelet Adhesiveness , Thrombosis , Blood Platelets , Humans , Microfluidics , Stress, MechanicalABSTRACT
Intensity shot noise in digital holograms distorts the quality of the phase images after phase retrieval, limiting the usefulness of quantitative phase microscopy (QPM) systems in long term live cell imaging. In this paper, we devise a hologram-to-hologram neural network, Holo-UNet, that restores high quality digital holograms under high shot noise conditions (sub-mW/cm2 intensities) at high acquisition rates (sub-milliseconds). In comparison to current phase recovery methods, Holo-UNet denoises the recorded hologram, and so prevents shot noise from propagating through the phase retrieval step that in turn adversely affects phase and intensity images. Holo-UNet was tested on 2 independent QPM systems without any adjustment to the hardware setting. In both cases, Holo-UNet outperformed existing phase recovery and block-matching techniques by â¼ 1.8 folds in phase fidelity as measured by SSIM. Holo-UNet is immediately applicable to a wide range of other high-speed interferometric phase imaging techniques. The network paves the way towards the expansion of high-speed low light QPM biological imaging with minimal dependence on hardware constraints.
ABSTRACT
Moldable, transparent polydimethylsiloxane (PDMS) elastomer microdevices enable a broad range of complex studies of three-dimensional cellular networks in their microenvironment in vitro. However, the uneven distribution of refractive index change, external to PDMS devices and internally in the sample chamber, creates a significant optical path difference (OPD) that distorts the light sheet beam and so restricts diffraction limited performance. We experimentally showed that an OPD of 120 µm results in the broadening of the lateral point spread function by over 4-fold. In this paper, we demonstrate steps to adapt a commercial inverted selective plane illumination microscope (iSPIM) and remove the OPD so as to achieve sub-micrometer imaging ranging from 0.6 ± 0.04 µm to 0.91 ± 0.03 µm of a fluorescence biological sample suspended in regular saline (RI ≈1.34) enclosed in 1.2 to 2 mm thick micromolded PDMS microdevices. We have proven that the removal of the OPD from the external PDMS layer by refractive index (RI) matching with a readily accessible, inexpensive sucrose solution is critical to achieve a >3-fold imaging resolution improvement. To monitor the RI matching process, a single-mode fiber (SMF) illuminator was integrated into the iSPIM. To remove the OPD inside the PDMS channel, we used an electrically tunable lens (ETL) that par-focuses the light sheet beam with the detection objective lens and so minimised axial distortions to attain sub-micrometer imaging resolution. We termed this new light sheet imaging protocol as modified inverted selective plane illumination microscopy (m-iSPIM). Using the high spatial-temporal 3D imaging of m-iSPIM, we experimentally captured single platelet (≈2 µm) recruitment to a platelet aggregate (22.5 µm × 22.5 µm × 6 µm) under flow at a 150 µm depth within a microfluidic channel. m-iSPIM paves the way for the application of light sheet imaging to a wide range of 3D biological models in microfluidic devices which recapitulate features of the physiological microenvironment and elucidate subcellular responses.
Subject(s)
Lenses , Microscopy , Dimethylpolysiloxanes , Lab-On-A-Chip Devices , LightingABSTRACT
Removal of complex aberrations at millisecond time scales over millimeters in distance in multiphoton laser scanning microscopy limits the total spatiotemporal imaging throughput for deep tissue imaging. Using a single low resolution deformable mirror and time multiplexing (TM) adaptive optics, we demonstrate video rate aberration correction (5â ms update rate for a single wavefront mask) for a complex heterogeneous distribution of refractive index differences through a depth of up to 1.1â mm and an extended imaging FOV of up to 0.8â mm, with up to 167% recovery of fluorescence intensity 335â µm from the center of the FOV. The proposed approach, termed raster adaptive optics (RAO), integrates image-based aberration retrieval and video rate removal of arbitrarily defined regions of dominant, spatially varied wavefronts. The extended FOV was achieved by demonstrating rapid recovery of up to 50 distinct wavefront masks at 500â ms update rates that increased imaging throughput by 2.3-fold. Because RAO only requires a single deformable mirror with image-based aberration retrieval, it can be directly implemented on a standard laser scanning multiphoton microscope.
ABSTRACT
Thrombus formation in hemostasis or thrombotic disease is initiated by the rapid adhesion, activation, and aggregation of circulating platelets in flowing blood. At arterial or pathological shear rates, for example due to vascular stenosis or circulatory support devices, platelets may be exposed to highly pulsatile blood flow, while even under constant flow platelets are exposed to pulsation due to thrombus growth or changes in vessel geometry. The aim of this study is to investigate platelet thrombus formation dynamics within flow conditions consisting of either constant or variable shear. Human platelets in anticoagulated whole blood were exposed ex vivo to collagen type I-coated microchannels subjected to constant shear in straight channels or variable shear gradients using different stenosis geometries (50%, 70%, and 90% by area). Base wall shears between 1800 and 6600 s-1, and peak wall shears of 3700 to 29,000 s-1 within stenoses were investigated, representing arterial-pathological shear conditions. Computational flow-field simulations and stenosis platelet thrombi total volume, average volume, and surface coverage were analysed. Interestingly, shear gradients dramatically changed platelet thrombi formation compared to constant base shear alone. Such shear gradients extended the range of shear at which thrombi were formed, that is, platelets became hyperthrombotic within shear gradients. Furthermore, individual healthy donors displayed quantifiable differences in extent/formation of thrombi within shear gradients, with implications for future development and testing of antiplatelet agents. In conclusion, here, we demonstrate a specific contribution of blood flow shear gradients to thrombus formation, and provide a novel platform for platelet functional testing under shear conditions.
Subject(s)
Biomechanical Phenomena , Shear Strength , Thrombosis/etiology , Algorithms , Blood Coagulation , Blood Platelets/metabolism , Constriction, Pathologic , Humans , Models, Biological , Platelet Adhesiveness , Platelet Aggregation , Thrombosis/metabolism , Thrombosis/pathologyABSTRACT
Back focal plane interferometry (BFPI) is one of the most straightforward and powerful methods for achieving sub-nanometer particle tracking precision at high speed (MHz). BFPI faces technical challenges that prohibit tunable expansion of linear detection range with minimal loss to sensitivity, while maintaining robustness against optical aberrations. In this paper, we devise a tunable BFPI combining a structured beam (conical wavefront) and structured detection (annular quadrant photodiode). This technique, which we termed Structured Back Focal Plane Interferometry (SBFPI), possesses three key novelties namely: extended tracking range, low loss in sensitivity, and resilience to spatial aberrations. Most importantly, the conical wavefront beam preserves the axial Gouy phase shift and lateral beam waist that can then be harnessed in a conventional BFPI system. Through a series of experimental results, we were able to tune detection sensitivity and detection range over the SBFPI parameter space. We also identified a figure of merit based on the experimental optimum that allows us to identify optimal SBPFI configurations that balance both range and sensitivity. In addition, we also studied the resilience of SBFPI against asymmetric spatial aberrations (astigmatism of up to 0.8 λ) along the lateral directions. The simplicity and elegance of SBFPI will accelerate its dissemination to many associated fields in optical detection, interferometry and force spectroscopy.
ABSTRACT
In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2-fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub-diffraction limited pixels (60 nm per pixels over the FOV of 512 µm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 µm) within fluorescent giant unilamellar vesicles and micro-vascular dynamics after laser-induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological-imaging capacity of any polygon scanning microscope system.
Subject(s)
Microscopy, Fluorescence, Multiphoton , Signal-To-Noise Ratio , Animals , Lasers/adverse effects , Mice , Vascular System Injuries/diagnostic imagingABSTRACT
Soluble glycoprotein VI (sGPVI) is shed from the platelet surface and is a marker of platelet activation in thrombotic conditions. We assessed sGPVI levels together with patient and clinical parameters in acute and chronic inflammatory conditions, including patients with thermal injury and inflammatory bowel disease and patients admitted to the intensive care unit (ICU) for elective cardiac surgery, trauma, acute brain injury, or prolonged ventilation. Plasma sGPVI was measured by enzyme-linked immunosorbent assay and was elevated on day 14 after thermal injury, and was higher in patients who developed sepsis. sGPVI levels were associated with sepsis, and the value for predicting sepsis was increased in combination with platelet count and Abbreviated Burn Severity Index. sGPVI levels positively correlated with levels of D-dimer (a fibrin degradation product) in ICU patients and patients with thermal injury. sGPVI levels in ICU patients at admission were significantly associated with 28- and 90-day mortality independent of platelet count. sGPVI levels in patients with thermal injury were associated with 28-day mortality at days 1, 14, and 21 when adjusting for platelet count. In both cohorts, sGPVI associations with mortality were stronger than D-dimer levels. Mechanistically, release of GPVI was triggered by exposure of platelets to polymerized fibrin, but not by engagement of G protein-coupled receptors by thrombin, adenosine 5'-diphosphate, or thromboxane mimetics. Enhanced fibrin production in these patients may therefore contribute to the observed elevated sGPVI levels. sGPVI is an important platelet-specific marker for platelet activation that predicts sepsis progression and mortality in injured patients.
Subject(s)
Fibrin/physiology , Inflammation/blood , Platelet Activation , Platelet Membrane Glycoproteins/analysis , Predictive Value of Tests , Biomarkers/blood , Burns/blood , Burns/mortality , Burns/pathology , Disease Progression , Fibrin Fibrinogen Degradation Products/analysis , Humans , Inflammation/mortality , Inflammatory Bowel Diseases/blood , Inflammatory Bowel Diseases/mortality , Inflammatory Bowel Diseases/pathology , Mortality , Platelet Count , Platelet Membrane Glycoproteins/metabolism , Sepsis/blood , Sepsis/mortality , Sepsis/pathology , SolubilityABSTRACT
Liquid droplets cured at low temperatures or using ultraviolet light are primary approaches for fabricating refractive lenses without molds. Until now the performance of moldless lens fabrication process relied heavily on this step to precisely control the shape of each liquid droplet. Hence, a major hurdle in lenses fabricated from liquid droplets is the large variability of droplet shapes because they are sensitive to small amounts of interfacial forces. The shape of the final droplet critically affects the imaging performance of the lenses and cannot be reversed easily. Here, we aim to overcome this hurdle by performing in situ aberration correction using Fourier ptychography techniques. We demonstrate, for the first time, that computational optics can reverse high amounts of optical aberrations in moldless lenses and achieve high resolution imaging. In terms of imaging resolution, we successfully increased the resolving power of low powered moldless elastomer lenses by almost three-fold, from a numerical aperture of 0.035 to 0.099. The computational approach directly elucidates the spatially varying wavefront aberrations from each lens using the same imaging system. This provides direct feedback of droplet lens fabrication techniques without the need for advanced wavefront correction methods. The application of computational imaging onto moldless lenses, using consumer digital imaging systems, lends itself to the global efforts in decentralising high resolution image intensive scientific tools to the wider community.
ABSTRACT
We propose a new method to extend the path length tunability of rotary delay-lines. This method was shown to achieve a duty cycle of >80% and repetition rates of over 40 kHz. The new method relies on a new multi-segmented micro-machined mirror and serial injection of a single reflection onto separate segments of this mirror. The tunability is provided by the relative positioning of each reflective point on the mirror segments. There are two distinct modes of operation: synchronous and asynchronous. By simply manipulating the spatial position of the returning paths over the respective mirror segments, we can switch between increasing the repetition rate (asynchronous mode) or the total delay path (synchronous mode). We experimentally demonstrated up to 8 m/s scans with repetition rates of up to 42.7 kHz. Furthermore, we present numerical simulations of 18 reflection points to illustrate possibility of achieving a scan speed of up to 80 m/s. Through intermediate combinations of synchronous and asynchronous operation modes with 4 or more passes, we also show that the system can simultaneously increase both repetition rate and scan depth.
ABSTRACT
Moldless lens manufacturing techniques using standard droplet dispensing technology often require precise control over pressure to initiate fluid flow and control droplet formation. We have determined a series of interfacial fluid parameters optimised using standard 3D printed tools to extract, dispense and capture a single silicone droplet that is then cured to obtain high quality lenses. The dispensing process relies on the recapitulation of liquid dripping action (Rayleigh-Plateau instability) and the capturing method uses the interplay of gravitational force, capillary forces and liquid pinning to control the droplet shape. The key advantage of the passive lens fabrication approach is rapid scale-up using 3D printing by avoiding complex dispensing tools. We characterise the quality of the lenses fabricated using the passive approach by measuring wavefront aberration and high resolution imaging. The fabricated lenses are then integrated into a portable imaging system; a wearable thimble imaging device with a detachable camera housing, that is constructed for field imaging. This paper provides the full exposition of steps, from lens fabrication to imaging platform, necessary to construct a standalone high resolution imaging system. The simplicity of our methodology can be implemented using a regular desktop 3D printer and commercially available digital imaging systems.
ABSTRACT
Automated label-free quantitative imaging of biological samples can greatly benefit high throughput diseases diagnosis. Digital holographic microscopy (DHM) is a powerful quantitative label-free imaging tool that retrieves structural details of cellular samples non-invasively. In off-axis DHM, a proper spatial filtering window in Fourier space is crucial to the quality of reconstructed phase image. Here we describe a region-recognition approach that combines shape recognition with an iterative thresholding method to extracts the optimal shape of frequency components. The region recognition technique offers fully automated adaptive filtering that can operate with a variety of samples and imaging conditions. When imaging through optically scattering biological hydrogel matrix, the technique surpasses previous histogram thresholding techniques without requiring any manual intervention. Finally, we automate the extraction of the statistical difference of optical height between malaria parasite infected and uninfected red blood cells. The method described here paves way to greater autonomy in automated DHM imaging for imaging live cell in thick cell cultures.
ABSTRACT
Efficient delivery of viruses, proteins and biological macromelecules into a micrometer-sized focal spot of an XFEL beam for coherent diffraction imaging inspired new development in touch-free particle injection methods in gaseous and vacuum environments. This paper lays out our ongoing effort in constructing an all-optical particle delivery approach that uses piconewton photophoretic and femtonewton light-pressure forces to control particle delivery into the XFEL beam. We combine a spatial light modulator (SLM) and an electrically tunable lens (ETL) to construct a variable-divergence vortex beam providing dynamic and stable positioning of levitated micrometer-size particles, under normal atmospheric pressure. A sensorless wavefront correction approach is used to reduce optical aberrations to generate a high quality vortex beam for particle manipulation. As a proof of concept, stable manipulation of optically-controlled axial motion of trapped particles is demonstrated with a response time of 100ms. In addition, modulation of trapping intensity provides a measure of the mass of a single, isolated particle. The driving signal of this oscillatory motion can potentially be phase-locked to an external timing signal enabling synchronization of particle delivery into the x-ray focus with XFEL pulse train.
ABSTRACT
Intravital microscopy is a powerful tool in neuroscience but has not been adapted to the taste sensory organ due to anatomical constraint. Here we developed an imaging window to facilitate microscopic access to the murine tongue in vivo. Real-time two-photon microscopy allowed the visualization of three-dimensional microanatomy of the intact tongue mucosa and functional activity of taste cells in response to topically administered tastants in live mice. Video microscopy also showed the calcium activity of taste cells elicited by small-sized tastants in the blood circulation. Molecular kinetic analysis suggested that intravascular taste sensation takes place at the microvilli on the apical side of taste cells after diffusion of the molecules through the pericellular capillaries and tight junctions in the taste bud. Our results demonstrate the capabilities and utilities of the new tool for taste research in vivo.
Subject(s)
Microscopy/methods , Taste Perception/physiology , Taste/physiology , Animals , Calcium/metabolism , Imaging, Three-Dimensional , Male , Mice, Inbred BALB C , Mucous Membrane/anatomy & histology , Taste Buds , Tongue/anatomy & histology , Tongue/blood supply , Tongue/physiologyABSTRACT
Coherent generated self-imaging bottle beams, typically formed by interfering two coherent quasi-Bessel beams, possess a periodic array of intensity maxima and minima along their axial direction. In practice, the overall quality of the self-repeating intensity patterns is prone to unresolved large intensity variations. In this Letter, we increased consistency of intensity of self-imaging bottle beams through a spatial frequency optimization routine. By doing so, we increased the effective length of self-imaging bottle beams by 74%. Further, we showed that this approach is applicable to higher-order self-imaging beams that display complex intensity structures. The enhancement in these modified self-imaging beams could play a significant role in optical trapping, imaging, and lithography.
ABSTRACT
Axially resolved microphotoluminescence mapping of semiconductor nanowires held in an optical tweezers reveals important new experimental information regarding equilibrium trapping points and trapping stability of high aspect ratio nanostructures. In this study, holographic optical tweezers are used to scan trapped InP nanowires along the beam direction with respect to a fixed excitation source and the luminescent properties are recorded. It is observed that nanowires with lengths on the range of 3-15 µm are stably trapped near the tip of the wire with the long segment positioned below the focus in an inverted trapping configuration. Through the use of trap multiplexing we investigate the possibility of improving the axial stability of the trapped nanowires. Our results have important implication for applications of optically assisted nanowire assembly and optical tweezers based scanning probes microscopy.
ABSTRACT
Intravital fluorescence microscopy has emerged as a powerful technique to visualize cellular processes in vivo. However, owing to their size, the objective lenses required have limited physical accessibility to various tissue sites in the internal organs of small animals. The use of small-diameter probes using graded-index (GRIN) lenses expands the capabilities of conventional intravital microscopes to minimally invasive imaging of internal organs. In this protocol, we describe the detailed steps for the fabrication of front- and side-view GRIN probes and the integration and operation of the probes in a confocal microscope to enable visualization of fluorescent cells and microvasculature in various mouse organs. Some experience in building an optical setup is required to complete the protocol. We also present longitudinal imaging of immune cells in renal allografts and tumor development in the colon. Fabrication and integration can be completed in 5-7 h, and a typical in vivo imaging session takes 1-2 h.