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To determine the molecular and/or mechanical basis of cell migration using live cell imaging tools, it is necessary to correlate multiple 3D spatiotemporal events simultaneously. Fluorescence nanoscopy and label free nanoscale imaging can complement each other by providing both molecular specificity and structural dynamics of sub-cellular structure. In doing so, a combined imaging system would permit quantitative 3D spatial temporal detail of individual cellular components. In this paper, we empirically determined a series of optimal azimuthal scanning angles and rotating beam to achieve simultaneous and label-free nanoscale and fluorescence imaging. Label-free nanoscale imaging here refers to interferometric, brightfield (BF) and darkfield (DF) rotating coherence scattering (ROCS) microscopy, while fluorescence refers to high inclined Laminated Oblique (HiLO) and total internal reflection fluorescence (TIRF) imaging. The combined capabilities of interferometric, scattering and fluorescence imaging enables (1) the identification of molecular targets (substrate or organelle), (2) quantification of 3D cell morphodynamics, and (3) tracking of intracellular organelles in 3D. This combined imaging tool was then used to characterize migrating platelets and adherent endothelial cells, both critical to the process of infection and wound healing. The combined imaging results of over ∼1000 platelets, suggested that serum albumin (bovine) was necessary for platelets to migrate and scavenge fibrin/fibrinogen. Furthermore, we determine new asynchronous membrane fluctuations between the leading and rear edge of a migrating platelet. We further demonstrated that interferometric imaging permitted the quantification of mitochondria dynamics on lung microvascular cells (HMVEC). Our data suggests that axial displacement of mitochondria is minimized when it is closer to the nucleus or the leading edge of a cell membrane that exhibits retrograde motion. Taken together, this combined imaging platform has proven to quantify multiple spatial temporal events of a migrating cell, that will undoubtedly open ways to new quantitative correlative nanoscale live cell imaging.

Heterogenous fluid shear stress is known to provide mechanical cues for cell adhesion and translocation. To assemble 3D microstructures using current fabrication methods in a single channel and recapitulate in vivo heterogenous fluid flow would require hours of fabrication and specialized equipment. Inspired by the traditional art form of inside painting, we developed a technique for 3D fabrication of micro-patterned flow channels and mixed in vivo fluid flow in a matter of minutes. We termed this technique Multiphoton Inner Laser Lithography (MILL). We further showed that when combined with adaptive optics, MILL is compatible with both flat and curved channel shapes. MILL recapitulated in vivo tissue topology and 3D fluid flow within tissue stroma (low fluid shear, 0 - 3.5 dynes/cm2) and blood vessel (high fluid shear, 0 - 80 dynes/cm2). We demonstrate fibroblast cell and platelets adhere and translocate differently between laminar flow patterns that are homogenous versus heterogeneous in real time. Parallel strips of MILL channels were assembled for simultaneous platelet function test to quantify the efficacy of an antithrombotic GPVI Fab (~2000 microthrombi per test). The MILL technique can be readily reproduced in vivo fluid flow in minutes and benefit preclinical screening of drug pharmacokinetics.

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.

Background Collagen and fibrin engagement and activation of glycoprotein (GP) VI induces proteolytic cleavage of the GPVI ectodomain generating shed soluble GPVI (sGPVI). Collagen-mediated GPVI shedding requires intracellular signalling to release the sGPVI, mediated by A Disintegrin And Metalloproteinase 10 (ADAM10); however, the precise mechanism by which fibrin induces GPVI shedding remains elusive. Plasma sGPVI levels are elevated in patients with coagulopathies, sepsis, or inflammation and can predict onset of sepsis and sepsis-related mortality; therefore, it is clinically important to understand the mechanisms of GPVI shedding under conditions of minimal collagen exposure. Objectives Our aim was to characterize mechanisms by which fibrin-GPVI interactions trigger GPVI shedding. Methods Platelet aggregometry, sGPVI ELISA, and an ADAM10 fluorescence resonance energy transfer assay were used to measure fibrin-mediated platelet responses. Results Fibrin induced αIIbβ3-independent washed platelet aggregate formation, GPVI shedding, and increased ADAM10 activity, all of which were insensitive to pre-treatment with inhibitors of Src family kinases but were divalent cation- and metalloproteinase-dependent. In contrast, treatment of washed platelets with other GPVI ligands, collagen, and collagen-related peptide caused αIIbβ3-dependent platelet aggregation and GPVI release but did not increase constitutive ADAM10 activity. Conclusions Fibrin engages GPVI in a manner that differs from other GPVI ligands. Inclusion of polyanionic molecules disrupted fibrin-induced platelet aggregate formation and sGPVI release, suggesting that electrostatic charge may play a role in fibrin/GPVI engagement. It may be feasible to exploit this property and specifically disrupt GPVI/fibrin interactions whilst sparing GPVI/collagen engagement.Fibrin engages GPVI in a manner that differs from other GPVI ligands. Inclusion of polyanionic molecules disrupted fibrin-induced platelet aggregate formation and sGPVI release, suggesting that electrostatic charge may play a role in fibrin/GPVI engagement. It may be feasible to exploit this property and specifically disrupt GPVI/fibrin interactions whilst sparing GPVI/collagen engagement.

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.

vBack Focal Plane interferometry (BFPI) is a widely used technique in particle tracking, providing high-speed, highresolution measurements. We present Structured Back Focal Plane Interferometry (SBFPI), a modification to BFPI utilizing structured beams and structured detectors [1]. Utilising Conical Wavefront Gaussian Beams and Annular Quadrant Photodetectors, SBFPI significantly increases tracking range while maintaining sensitivity and robustness to aberrations. We studied the variation in detection range and sensitivity over our parameters. A maximum increase in axial and lateral tracking range of 4.25-fold and 1.83-fold was found respectively, concomitant with sensitivity decreases of 53% and 61%. We also found parameter combinations with smaller range improvements but which retain sensitivity, in particular a 2.31 fold axial range increase with a 22.4% reduction in sensitivity, and these can be tuned to each experiment. We also studied the effect of aberrations on signal integrity.

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 ∼ 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.

In this work, we propose a label-free COSI system to quantify morphological changes and platelet activity along non-patterned collagen fibers within millisecond in microfluidics channels under flow at sub-platelet imaging resolution.

In this work, we propose a label-free COSI system to investigating morphological changes and platelet-platelet interactions within a thrombus during embolism events to interrogate prothrombotic events within a microfluidics channel under flow.

Living cells adjust their cytoskeletal organization and mechanically change their overall shape by reacting to the changes of the microenvironment. The ability to quantify these dynamic events in micro and nanoscale in real-time at the same time contributes to our understanding of the functional response of living cells. The combination to achieve both microscale and nanoscale imaging simultaneous at volumetric speeds is challenging. Traditional TIRF microscopy has excelled in measuring surface interaction but yet limited in imaging depth and requires fluorescent labelling. Likewise, the ability to quantify the total volume and shape change of biological cells as they interact requires either confocal microscopy or lightsheet microscopy. In this paper, we propose an in toto label free approach through coherent optical interference to measure volumetric information and surface interaction at the same time to provide a full view of the cell during dynamic activities.

Commercial microscopy systems make use of tandem scanning i.e. either slow or fast scanning. We constructed, for the first time, an advanced control system capable of delivering a dynamic line scanning speed ranging from 2.7 kHz to 27 kHz and achieve variable frame rates from 5 Hz to 50 Hz (512 × 512). The dynamic scanning ability is digitally controlled by a new customized open-source software named PScan1.0. This permits manipulation of scanning rates either to gain higher fluorescence signal at slow frame rate without increasing laser power or increase frame rates to capture high speed events. By adjusting imaging speed from 40 Hz to 160 Hz, we capture a range of calcium waves and transient peaks from soma and dendrite of single fluorescence neuron (CAL-520AM). Motion artifacts arising from respiratory and cardiac motion in small animal imaging reduce quality of real-time images of single cells in-vivo. An image registration algorithm, integrated with PScan1.0, was shown to perform both real time and post-processed motion correction. The improvement is verified by quantification of blood flow rates. This work describes all the steps necessary to develop a high performance and flexible polygon-mirror based multiphoton microscope system for in-vivo biological 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 0.8 mm, with up to 167% recovery of fluorescence intensity 335 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 500 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.

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&minus, 1, and peak wall shears of 3700 to 29,000 s&minus, 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.

The application of advanced optical imaging technology for platelet biology is in its infancy. In this talk, we shall introduce the potential impact of optical imaging in understanding the roles of platelets in thrombus formation and in hemostasis. We will summarize how techniques in optical imaging has enabled exploration of morphological, molecular and fluidic parameters that determine platelet adhesion, activation and consequent roles in thrombus formation. Finally, we discuss the convergence of multiple imaging modalities towards a complete understanding of platelet roles in thrombus formation.

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.

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.

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.

In this work, we have demonstrated that Fourier Ptychographic microscopy (FPM) is able to recover spatially varying off-axis aberrations generated from lenses fabricated using moldless methods.

For the first time, a polygon-mirror laser scanning microscope is engineered to provide rapid changes in the field of view to achieve vector scanning and also ultrafine pixel resolution (50 nm) without increasing the overall acquisition rate.

Multiphoton laser scanning microscopes exhibit highly localized nonlinear optical excitation and are powerful instruments for in-vivo deep tissue imaging. Customized multiphoton microscopy has a significantly superior performance for in-vivo imaging because of precise control over the scanning and detection system. To date, there have been several flexible software platforms catered to custom built microscopy systems i.e. ScanImage, HelioScan, MicroManager, that perform at imaging speeds of 30-100fps. In this paper, we describe a flexible software framework for high speed imaging systems capable of operating from 5 fps to 1600 fps. The software is based on the MATLAB image processing toolbox. It has the capability to communicate directly with a high performing imaging card (Matrox Solios eA/XA), thus retaining high speed acquisition. The program is also designed to communicate with LabVIEW and Fiji for instrument control and image processing. Pscan 1.0 can handle high imaging rates and contains sufficient flexibility for users to adapt to their high speed imaging systems.

Here we propose a region-recognition approach with iterative thresholding, which is adaptively tailored to extract the appropriate region or shape of spatial frequency. In order to justify the method, we tested it with different samples and imaging conditions (different objectives). We demonstrate that our method provides a useful method for rapid imaging of cellular dynamics in microfluidic and cell cultures.

Wearable computing devices, e.g. Google Glass, Smart watch, embodies the new human design frontier, where technology interfaces seamlessly with human gestures. During examination of any subject in the field (clinic, surgery, agriculture, field survey, water collection), our sensory peripherals (touch and vision) often go hand-in-hand. The sensitivity and maneuverability of the human fingers are guided with tight distribution of biological nerve cells, which perform fine motor manipulation over a range of complex surfaces that is often out of sight. Our sight (or naked vision), on the other hand, is generally restricted to line of sight that is ill-suited to view around corner. Hence, conventional imaging methods are often resort to complex light guide designs (periscope, endoscopes etc) to navigate over obstructed surfaces. Using modular design strategies, we constructed a prototype miniature microscope system that is incorporated onto a wearable fixture (thimble). This unique platform allows users to maneuver around a sample and take high resolution microscopic images. In this paper, we provide an exposition of methods to achieve a thimble microscopy; microscope lens fabrication, thimble design, integration of miniature camera and liquid crystal display.

Digital holographic microscopy (DHM) is a powerful in-vitro biological imaging tool. In this paper, we report a fully automated off-axis digital holographic microscopy system completed with a graphical user interface in the Matlab © environment. The interface primarily includes Fourier domain processing, phase reconstruction, aberration compensation and autofocusing. A variety of imaging operations such as region of interest selection, de-noising mode (filtering and averaging), low frame rate imaging for immediate reconstruction and high frame rate imaging routine (~ 27 fps) are implemented to facilitate ease of use.

Intravital multiphoton microscopy has emerged as a powerful technique to visualize cellular processes in-vivo. Real time processes revealed through live imaging provided many opportunities to capture cellular activities in living animals. The typical parameters that determine the performance of multiphoton microscopy are speed, field of view, 3D imaging and imaging depth; many of these are important to achieving data from in-vivo. Here, we provide a full exposition of the flexible polygon mirror based high speed laser scanning multiphoton imaging system, PCI-6110 card (National Instruments) and high speed analog frame grabber card (Matrox Solios eA/XA), which allows for rapid adjustments between frame rates i.e. 5 Hz to 50 Hz with 512 × 512 pixels. Furthermore, a motion correction algorithm is also used to mitigate motion artifacts. A customized control software called Pscan 1.0 is developed for the system. This is then followed by calibration of the imaging performance of the system and a series of quantitative in-vitro and in-vivo imaging in neuronal tissues and mice.

Introduction Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired clonal haematopoietic stem cell disorder characterised by haemolytic anaemia, thrombosis and bone marrow failure. Thrombosis is considered a major cause for high morbidity and mortality in PNH patients (Hillmen P et al N Eng J Med 1995). However, the underlying mechanisms of thrombosis in PNH are still poorly understood. Various factors including defective interactions between the complement system, platelets and coagulation systems have been proposed (Peacock-Young B et al Haematologica 2017). Platelet function and clot formation in this disorder have not been comprehensively evaluated. Here we describe standard and novel techniques to evaluate platelet function in blood from PNH patients, to elucidate the underlying pro-thrombotic mechanisms. Methods Whole blood was collected from five PNH patients and compared to same-day healthy donor (HD) controls. The samples were collected at trough for those patients who were on eculizumab (Complement C5 inhibitor used in the treatment of PNH). Surface levels of platelet adhesion proteins and activation markers P-selectin and phosphatidylserine (PS) were assessed using flow cytometry. Plasma soluble GPVI (a platelet-specific activation marker) and cytokine levels were measured by ELISA. Clot formation was assessed by viscoelastic testing (ROTEM). Adhesion of platelets to collagen under flow in a whole blood assay was evaluated by Digital Holographic Microscopy (DHM) and thrombus height, surface area and volume quantified using custom-built MatLab-based software. Results Clinical characteristics of PNH patients were highly variable: two patients had history of thrombosis, three patients were on eculizumab, four patients were thrombocytopenic ( Conclusion Analysis of these PNH patients with highly variable clinical characteristics did not identify a unifying platelet lesion. DHM could detect and quantify parameters of small thrombi in real time, in PNH patient samples and these data were consistent with enhanced thrombogenic potential in PNH patients. Mechanisms beyond platelet activation that contribute to increased thrombosis in these patients and the impact of eculizumab therapy on thrombotic propensity need to be explored further. Disclosures D'Rozario: Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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.

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.

We present a novel, compact delay line demonstrating time domain OCT A-line scan rates of up to 40 kHz (theoretical maximum 1.4 MHz) with scan depths of 400 µm (theoretical maximum of 14 mm).

The authors received funding from the National Health and Medical Research Council of Australia and the Australian Research Council.

Optical micromanipulation of transparent microparticles such as cellular materials relies upon the application of optical forces that are crucially dependent on the refractive index contrast between the particle and the surrounding medium. We briefly review the application of optical forces for cell manipulation and sorting, highlighting some of the key experiments over the last twenty years. We then introduce a new technique for enhancing the dielectric contrast of mammalian cells, which is a result of cells naturally taking up microspheres from their environment. We explore how these intracellular dielectric tags can influence the scattering and gradient forces upon these cells from an externally applied optical field. We show that intracellular polymer microspheres can serve as highly directional optical scatterers and that scattering forces can enable sorting through axial guiding onto laminin-coated glass coverslips upon which the selected cells adhere. Such internal dielectric tagging presents a simple, inexpensive, sterile technique to enhance optical manipulation procedures for cellular material and may enable new sorting techniques within microfluidic systems.

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. By setting up a well-calibrated single-beam optical trap within a fluorescence microscope system, one can measure forces and collect fluorescence signals upon biological systems simultaneously. In this protocol, we aim to provide a clear exposition of the methodology of assembling and operating a single-beam gradient force trap (optical tweezers) on an inverted fluorescence microscope. A step-by-step guide is given for alignment and operation, with discussion of common pitfalls.

We review the emergent techniques of microfluidic sorting of colloidal and cellular samples using optical forces. We distinguish between what we term as passive and active forms of particle sorting where we can sort either with the use of a fluorescent marker (active) or based on physical attributes alone (passive). We then examine cell sorting with optical potential landscapes such as a Bessel light beam and a multibeam interference pattern. For both forms of optical potential energy landscape, we further present the possibility of enhancing the optical sorting process by tagging dielectric microspheres onto the cells. The results suggest that the methodology of tagging can enhance the sorting of cells as they subsequently respond more strongly to an applied optical field or potential energy landscape. This technique presents a simple method to enhance the sorting process.

We demonstrate an off-axis digital holographic microscopy system for imaging infected red blood cell and propose to combine that with flow channels to study adhesion bonds of infected blood cells at the cellular and molecular level.

The growing power of imaging and computing power of smartphones is creating the possibility of converting your smartphone into a high power pocket microscopy system. High quality miniature microscopy lenses attached to smartphone are typically made with glass or plastics that can only be produce at low cost with high volume. To revise the paradigm of microscope lenses, we devised a simple droplet lens fabrication technique that which produces low cost and high performance lens. Each lens is integrated into thin 3-D printed holder with complimentary light emitted diode (LEDs) that clips onto majority of smartphones. The integrated device converts a smartphone into a high power optical microscope/dermatoscope at around $2. This low cost device has wide application in a multitude of practical uses such as material inspection, dermascope and educational microscope.