Minimally invasive longitudinal intravital imaging of cellular dynamics in intact long bone.

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.

Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption.

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.

Label-free multimodal quantitative imaging flow assay for intrathrombus formation in vitro.

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.

Holo-UNet: hologram-to-hologram neural network restoration for high fidelity low light quantitative phase imaging of live cells.

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/cm 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.

Modified inverted selective plane illumination microscopy for sub-micrometer imaging resolution in polydimethylsiloxane soft lithography devices.

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.

Raster adaptive optics for video rate aberration correction and large FOV multiphoton imaging.

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 (5 ms update rate for a single wavefront mask) for a complex heterogeneous distribution of refractive index differences through a depth of up to 1.1 mm and an extended imaging FOV of up to 0.

Structured Back Focal Plane Interferometry (SBFPI).

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).

Novel Stenotic Microchannels to Study Thrombus Formation in Shear Gradients: Influence of Shear Forces and Human Platelet-Related Factors.

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.

Soluble GPVI is elevated in injured patients: shedding is mediated by fibrin activation of GPVI.

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.

In situ retrieval and correction of aberrations in moldless lenses using Fourier ptychography.

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.

Compact flexible multi-pass rotary delay line using spinning micro-machined mirrors.

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.

Design and fabrication of a passive droplet dispenser for portable high resolution imaging system.

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.

Dynamic axial control over optically levitating particles in air with an electrically-tunable variable-focus lens.

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.

Intravital microscopic interrogation of peripheral taste sensation.

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.

Shaping self-imaging bottle beams with modified quasi-Bessel beams.

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.

Resolving stable axial trapping points of nanowires in an optical tweezers using photoluminescence mapping.

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.

Fabrication and operation of GRIN probes for in vivo fluorescence cellular imaging of internal organs in small animals.

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.

Effect of pulse temporal shape on optical trapping and impulse transfer using ultrashort pulsed lasers.

We investigate the effects of pulse duration on optical trapping with high repetition rate ultrashort pulsed lasers, through Lorentz-Mie theory, numerical simulation, and experiment. Optical trapping experiments use a 12 femtosecond duration infrared pulsed laser, with the trapping microscope's temporal dispersive effects measured and corrected using the Multiphoton Intrapulse Interference Phase Scan method. We apply pulse shaping to reproducibly stretch pulse duration by 1.

Construction and calibration of an optical trap on a fluorescence optical microscope.

The application of optical traps has come to the fore in the last three decades. They provide a powerful, sterile and noninvasive tool for the manipulation of cells, single biological macromolecules, colloidal microparticles and nanoparticles. An optically trapped microsphere may act as a force transducer that is used to measure forces in the piconewton regime.

Experimental study of holographic generation of fractional Bessel beams.

We demonstrate the experimental generation of a fractional Bessel beam by holographic means. Such fractional modes of Bessel beams possess an intrinsic opening gap across concentric intensity rings on propagation. We also show that the opening gaps within the fractional modes are diffraction free for a working distance while a fractional helical wave front is maintained.

Optical tweezers with multiple optical forces using double-hologram interference.

In earlier work, we introduced new ways of generating a series of interference patterns formed from Laguerre-Gaussian (LG) beams, which are being used as advanced optical tweezers in creating and manipulating three-dimensional structures. In this work, we have succeeded in demonstrating, for the first time to our knowledge, double LG and LG beams with a Gaussian-beam interference using a Michelson interferometer. We have been able to observe LG interference of unequal azimuthal charge by using just two holograms.