Computational cytometer based on magnetically modulated coherent imaging and deep learning.

Detecting rare cells within blood has numerous applications in disease diagnostics. Existing rare cell detection techniques are typically hindered by their high cost and low throughput. Here, we present a computational cytometer based on magnetically modulated lensless speckle imaging, which introduces oscillatory motion to the magnetic-bead-conjugated rare cells of interest through a periodic magnetic force and uses lensless time-resolved holographic speckle imaging to rapidly detect the target cells in three dimensions (3D). In addition to using cell-specific antibodies to magnetically label target cells, detection specificity is further enhanced through a deep-learning-based classifier that is based on a densely connected pseudo-3D convolutional neural network (P3D CNN), which automatically detects rare cells of interest based on their spatio-temporal features under a controlled magnetic force. To demonstrate the performance of this technique, we built a high-throughput, compact and cost-effective prototype for detecting MCF7 cancer cells spiked in whole blood samples. Through serial dilution experiments, we quantified the limit of detection (LoD) as 10 cells per millilitre of whole blood, which could be further improved through multiplexing parallel imaging channels within the same instrument. This compact, cost-effective and high-throughput computational cytometer can potentially be used for rare cell detection and quantification in bodily fluids for a variety of biomedical applications

Low-cost portable microscopy systems for biomedical imaging and healthcare applications

In recent years, the development of low-cost portable microscopes (LPMs) has opened new possibilities for disease detection and biomedical research, especially in resource-limited areas. Despite these advancements, the majority of existing LPMs are hampered by sophisticated optical and mechanical designs, require extensive post-data analysis, and are often tailored for specific biomedical applications, limiting their broader utility. Furthermore, creating an optical-sectioning microscope that is both compact and cost effective presents a significant challenge. Addressing these critical gaps, this PhD study aims to: (1) develop a universally applicable LPM featuring a simplified mechanical and optical design for real-time biomedical imaging analysis, and (2) design a novel, smartphone-based optical sectioning microscope that is both compact and affordable. These objectives are driven by the need to enhance accessibility to quality diagnostic tools in varied settings, promising a significant leap forward in the democratization of biomedical imaging technologies. With 3D printing, optimised optical design, and AI techniques, we can develop LPM’s real time analysis functionality. I conducted a literature review on LPMs and related applications in my study and implemented two low-cost prototype microscopes and one theoretical study. 1) The first project is a portable AI fluorescence microscope based on a webcam and the NVIDIA Jetson Nano (NJN) with real-time analysis functionality. The system was 3D printed, weighing ~250 grams with a size of 145mm × 172 mm × 144 mm (L×W×H) and costing ~$400. It achieves a physical magnification of ×5 and can resolve 228.1 lp/mm USAF features. The system can recognise and count fluorescent beads and human red blood cells (RBCs). 2) I developed a smartphone-based optical sectioning microscope using the HiLo technique. To our knowledge, it is the first smartphone-based HiLo microscope that offers low-cost optical-sectioned widefield imaging. It has a 571.5 μm telecentric scanning range and an 11.7 μm axial resolution. I successfully used it to realize optical sectioning imaging of fluorescent beads. For this system, I developed a new low-cost HiLo microscopy technique using microlens arrays (MLAs) with incoherent light-emitting diode (LED) light sources. I conducted a numerical simulation study assessing the integration of uncoherent LEDs and MLAs for a low-cost HiLo system. The MLA can generate structured illumination in HiLo. How the MLA’s geometry structure and physical parameters affect the image performance were discussed in detail. This PhD thesis explores the advancement of low-cost portable microscopes (LPMs) through the integration of 3D printing, optimized optical design, and artificial intelligence (AI) techniques to enhance their real-time analysis capabilities. The research involved a comprehensive literature review on LPMs and their applications, leading to the development of two innovative prototype LPMs, alongside a theoretical study. These works contribute significantly to the field by not only addressing the technical and financial barriers associated with advanced microscopy but also by laying the groundwork for future innovations in portable and accessible biomedical imaging. Through its focus on simplification, affordability, and practicality, the research holds promise for substantially expanding the reach and impact of diagnostic imaging technologies, especially in those resource-limited areas.In recent years, the development of low-cost portable microscopes (LPMs) has opened new possibilities for disease detection and biomedical research, especially in resource-limited areas. Despite these advancements, the majority of existing LPMs are hampered by sophisticated optical and mechanical designs, require extensive post-data analysis, and are often tailored for specific biomedical applications, limiting their broader utility. Furthermore, creating an optical-sectioning microscope that is both compact and cost effective presents a significant challenge. Addressing these critical gaps, this PhD study aims to: (1) develop a universally applicable LPM featuring a simplified mechanical and optical design for real-time biomedical imaging analysis, and (2) design a novel, smartphone-based optical sectioning microscope that is both compact and affordable. These objectives are driven by the need to enhance accessibility to quality diagnostic tools in varied settings, promising a significant leap forward in the democratization of biomedical imaging technologies. With 3D printing, optimised optical design, and AI techniques, we can develop LPM’s real time analysis functionality. I conducted a literature review on LPMs and related applications in my study and implemented two low-cost prototype microscopes and one theoretical study. 1) The first project is a portable AI fluorescence microscope based on a webcam and the NVIDIA Jetson Nano (NJN) with real-time analysis functionality. The system was 3D printed, weighing ~250 grams with a size of 145mm × 172 mm × 144 mm (L×W×H) and costing ~$400. It achieves a physical magnification of ×5 and can resolve 228.1 lp/mm USAF features. The system can recognise and count fluorescent beads and human red blood cells (RBCs). 2) I developed a smartphone-based optical sectioning microscope using the HiLo technique. To our knowledge, it is the first smartphone-based HiLo microscope that offers low-cost optical-sectioned widefield imaging. It has a 571.5 μm telecentric scanning range and an 11.7 μm axial resolution. I successfully used it to realize optical sectioning imaging of fluorescent beads. For this system, I developed a new low-cost HiLo microscopy technique using microlens arrays (MLAs) with incoherent light-emitting diode (LED) light sources. I conducted a numerical simulation study assessing the integration of uncoherent LEDs and MLAs for a low-cost HiLo system. The MLA can generate structured illumination in HiLo. How the MLA’s geometry structure and physical parameters affect the image performance were discussed in detail. This PhD thesis explores the advancement of low-cost portable microscopes (LPMs) through the integration of 3D printing, optimized optical design, and artificial intelligence (AI) techniques to enhance their real-time analysis capabilities. The research involved a comprehensive literature review on LPMs and their applications, leading to the development of two innovative prototype LPMs, alongside a theoretical study. These works contribute significantly to the field by not only addressing the technical and financial barriers associated with advanced microscopy but also by laying the groundwork for future innovations in portable and accessible biomedical imaging. Through its focus on simplification, affordability, and practicality, the research holds promise for substantially expanding the reach and impact of diagnostic imaging technologies, especially in those resource-limited areas

Nonmechanical parfocal and autofocus features based on wave propagation distribution in lensfree holographic microscopy

Performing long-term cell observations is a non-trivial task for conventional optical microscopy, since it is usually not compatible with environments of an incubator and its temperature and humidity requirements. Lensless holographic microscopy, being entirely based on semiconductor chips without lenses and without any moving parts, has proven to be a very interesting alternative to conventional microscopy. Here, we report on the integration of a computational parfocal feature, which operates based on wave propagation distribution analysis, to perform a fast autofocusing process. This unique non-mechanical focusing approach was implemented to keep the imaged object staying in-focus during continuous long-term and real-time recordings. A light-emitting diode (LED) combined with pinhole setup was used to realize a point light source, leading to a resolution down to 2.76 μm. Our approach delivers not only in-focus sharp images of dynamic cells, but also three-dimensional (3D) information on their (x, y, z)-positions. System reliability tests were conducted inside a sealed incubator to monitor cultures of three different biological living cells (i.e., MIN6, neuroblastoma (SH-SY5Y), and Prorocentrum minimum). Altogether, this autofocusing framework enables new opportunities for highly integrated microscopic imaging and dynamic tracking of moving objects in harsh environments with large sample areas

Roadmap on digital holography [Invited]

This Roadmap article on digital holography provides an overview of a vast array of research activities in the field of digital holography. The paper consists of a series of 25 sections from the prominent experts in digital holography presenting various aspects of the field on sensing, 3D imaging and displays, virtual and augmented reality, microscopy, cell identification, tomography, label-free live cell imaging, and other applications. Each section represents the vision of its author to describe the significant progress, potential impact, important developments, and challenging issues in the field of digital holography

New implementations of phase-contrast imaging

Phase-contrast imaging is a method of imaging widely used in biomedical research and applications. It is a label-free method that exploits intrinsic differences in the refractive index of different tissues to differentiate between biological structures under analysis. The basic principle of phase-contrast imaging has inspired a lot of implementations that are suited for different applications. This thesis explores multiple novel implementations of phase-contrast imaging in the following order. 1, We combined scanning Oblique Back-illumination Microscope (sOBM) and confocal microscope to produce phase and fluorescence contrast images in an endomicroscopy configuration. This dual-modality design provides co-registered, complementary labeled and unlabeled contrast of the sample. We further miniaturized the probe by dispensing the two optical fibers in our old design. And we presented proof of principle demonstrations with ex-vivo mouse colon tissue. 2, Then we explored sOBM-based phase and amplitude contrast imaging under different wavelengths. Hyperspectral imaging is achieved by multiplexing a wide-range supercontinuum laser with a Michaelson interferometer (similar to Fourier transform spectroscopy). It features simultaneous acquisition of hyperspectral phase and amplitude images with arbitrarily thick scattering biological samples. Proof-of-principle demonstrations are presented with chorioallantoic membrane of a chick embryo, illustrating the possibility of high-resolution hemodynamics imaging in thick tissue. 3, We focused on increasing the throughput of flow cytometry with principle of phase-contrast imaging and compressive sensing. By utilizing the linearity of scattered patterns under partially coherent illumination, our cytometer can detect multiple objects in the same field of view. By utilizing an optimized matched filter on pupil plane, it also provides increased information capacity of each measurement without sacrificing speed. We demonstrated a throughput of over 10,000 particles/s with accuracy over 91% in our results. 4, A fourth part, which describes the principle and preliminary results of a computational fluorescence endomicroscope is also included. It uses a numerical method to achieve sectioning effect and renders a pseudo-3D image stack with a single shot. The results are compared with true-3D image stack acquired with a confocal microscope

PRINCIPLES FOR NEW OPTICAL TECHNIQUES IN MEDICAL DIAGNOSTICS FOR mHEALTH APPLICATIONS

Medical diagnostics is a critical element of effective medical treatment. However, many modern and emerging diagnostic technologies are not affordable or compatible with the needs and conditions found in low-income and middle-income countries and regions. Resource-poor areas require low-cost, robust, easy-to-use, and portable diagnostics devices compatible with telemedicine (i.e. mHealth) that can be adapted to meet diverse medical needs. Many suitable devices will need to be based on optical technologies, which are used for many types of biological analyses. This dissertation describes the fabrication and detection principles for several low-cost optical technologies for mHealth applications including: (1) a webcam based multi-wavelength fluorescence plate reader, (2) a lens-free optical detector used for the detection of Botulinum A neurotoxin activity, (3) a low cost micro-array reader that allows the performance of typical fluorescence based assays demonstrated for the detection of the toxin staphylococcal enterotoxin (SEB), and (4) a wide-field flow cytometer for high throughput detection of fluorescently labeled rare cells. This dissertation discusses how these technologies can be harnessed using readily available consumer electronics components such as webcams, cell phones, CCD cameras, LEDs, and laser diodes. There are challenges in developing devices with sufficient sensitivity and specificity, and approaches are presented to overcoming these challenges to create optical detectors that can serve as low cost medical diagnostics in resource-poor settings for mHealth

Developments in Transduction, Connectivity and AI/Machine Learning for Point-of-Care Testing

We review some emerging trends in transduction, connectivity and data analytics for Point-of-Care Testing (POCT) of infectious and non-communicable diseases. The patient need for POCT is described along with developments in portable diagnostics, specifically in respect of Lab-on-chip and microfluidic systems. We describe some novel electrochemical and photonic systems and the use of mobile phones in terms of hardware components and device connectivity for POCT. Developments in data analytics that are applicable for POCT are described with an overview of data structures and recent AI/Machine learning trends. The most important methodologies of machine learning, including deep learning methods, are summarised. The potential value of trends within POCT systems for clinical diagnostics within Lower Middle Income Countries (LMICs) and the Least Developed Countries (LDCs) are highlighted