Field-portable pixel super-resolution colour microscope.

Based on partially-coherent digital in-line holography, we report a field-portable microscope that can render lensfree colour images over a wide field-of-view of e.g., >20 mm(2). This computational holographic microscope weighs less than 145 grams with dimensions smaller than 17×6×5 cm, making it especially suitable for field settings and point-of-care use. In this lensfree imaging design, we merged a colorization algorithm with a source shifting based multi-height pixel super-resolution technique to mitigate 'rainbow' like colour artefacts that are typical in holographic imaging. This image processing scheme is based on transforming the colour components of an RGB image into YUV colour space, which separates colour information from brightness component of an image. The resolution of our super-resolution colour microscope was characterized using a USAF test chart to confirm sub-micron spatial resolution, even for reconstructions that employ multi-height phase recovery to handle dense and connected objects. To further demonstrate the performance of this colour microscope Papanicolaou (Pap) smears were also successfully imaged. This field-portable and wide-field computational colour microscope could be useful for tele-medicine applications in resource poor settings

3-D volumetric Lensfree imaging using Digital Holographic Microscopy

Despite of advancement in microscopic techniques most of the improved microscopy modalities are complex, bulky and require costly setup. Many of the modalities are not capable of live imaging.Today imaging devices are needed to be compact, cost-effective, portable, light-weight and significantly accurate and easy to handle. Digital holography has overcome few barriers but still demands costly setup. Here we demonstrate the lens free imaging which is based on “Digital In-line holography” principle and does not require bulky and costly setup. The technique uses Light emitting diode (LED) as a source of light and CCD camera to capture the hologram of the object which allow to reconstruct the image using numerical algorithm on computing machines. This technique has many applications including one in telemedicine and can address the issues related to the rural health

Engineering of plasmonic excitations for hand-held and ultra-sensitive biosensors

Thesis (Ph.D.)--Boston UniversityEarly detection and effective diagnosis are important for disease screening and preventing epidemics. Recently, optical biosensors have attracted significant attention, as they are very powerful detection and analysis tools that have variety of applications in homeland security, public and global healthcare, biomedical research and pharmacology. However, most of these biosensors are time-consuming, require costly chemical procedures and bulky instrumentation, and need advanced medical infrastructures with trained laboratory professionals. In order to address these needs, recently lensfree computational on-chip imaging techniques have been introduced to eliminate the need for bulky and costly optical components. However, this technology is limited by the size of the analytes as it uses a lensfree computational technique insufficient for detecting biomolecules down to nm-scale. In order to provide highly sensitive and massively multiplexed detection of biomolecular binding events, fluorescent imaging and surface plasmon resonance (SPR) based platforms are the most favored. However, SPR sensors are limited due to the alignment sensitive prism coupling scheme and bulky instrumentation while the fluorescence imaging suffers from quantitative and qualitative drawbacks of the labeling steps. This thesis focuses on the unique integration of lensfree telemedicine technology and nanostructured plasmonic chip technology to realize ultra-sensitive and label-free biosensing in a high-throughput and massively multiplexed manner for field-settings. Toward this aim, we introduce a handheld on-chip biosensing technology that employs plasmonic microarrays coupled with a lensfree computational imaging system. Employing a sensitive plasmonic array design that is combined with lensfree computational imaging, we demonstrate label-free and quantitative detection of biomolecules with a protein layer thickness down to 3 nm. Integrating large-scale plasmonic microarrays, our platform enables the simultaneous detection of protein mono- and bilayers on the same platform over a wide range of biomolecule concentrations. In this plasmonic device, we also monitor binding dynamics of protein complexes as a function of time by integrating it with microfluidics. Plasmonic antennas utilized in our lensfree platform, supporting very sharp and sensitive spectral feature as well as easily accessible large local electromagnetic fields, are highly advantageous for biosensing applications as they enable stronger interaction between surface waves and biological molecules on the sensing chip

Handheld high-throughput plasmonic biosensor using computational on-chip imaging

We demonstrate a handheld on-chip biosensing technology that employs plasmonic microarrays coupled with a lens-free computational imaging system towards multiplexed and high-throughput screening of biomolecular interactions for point-of-care applications and resource-limited settings. This lightweight and field-portable biosensing device, weighing 60 g and 7.5 cm tall, utilizes a compact optoelectronic sensor array to record the diffraction patterns of plasmonic nanostructures under uniform illumination by a single-light emitting diode tuned to the plasmonic mode of the nanoapertures. Employing a sensitive plasmonic array design that is combined with lens-free computational imaging, we demonstrate label-free and quantitative detection of biomolecules with a protein layer thickness down to 3 nm. Integrating large-scale plasmonic microarrays, our on-chip imaging platform enables simultaneous detection of protein mono- and bilayers on the same platform over a wide range of biomolecule concentrations. In this handheld device, we also employ an iterative phase retrieval-based image reconstruction method, which offers the ability to digitally image a highly multiplexed array of sensors on the same plasmonic chip, making this approach especially suitable for high-throughput diagnostic applications in field settings

Suivi de culture cellulaire par imagerie sans lentille

Biological studies always start from curious observations. This is exemplified by description of cells for the first time by Robert Hooke in 1665, observed using his microscope. Since then the field of microscopy and cell biology grew hand in hand, with one field pushing the growth of the other and vice-versa. From basic description of cells in 1665, with parallel advancements in microscopy, we have travelled a long way to understand sub-cellular processes and molecular mechanisms. With each day, our understanding of cells increases and several questions are being posed and answered. Several high-resolution microscopic techniques are being introduced (PALM, STED, STORM, etc.) that push the resolution limit to few tens of nm, taking us to a new era where ‘seeing is believing'. Having said this, it is to be noted that the world of cells is vast, with information spread from nanometers to millimetres, and also over extended time-period, implying that not just one microscopic technique could acquire all the available information. The knowledge in the field of cell biology comes from a combination of imaging and quantifying techniques that complement one another.Majority of modern-day microscopic techniques focuses on increasing resolution which, is achieved at the expense of cost, compactness, simplicity, and field of view. The substantial decrease in the field of observation limits the visibility to a few single cells at best. Therefore, despite our ability to peer through the cells using increasingly powerful optical instruments, fundamental biology questions remain unanswered at mesoscopic scales. A global view of cell population with significant statistics both in terms of space and time is necessary to understand the dynamics of cell biology, taking in to account the heterogeneity of the population and the cell-cell variability. Mesoscopic information is as important as microscopic information. Although the latter gains access to sub-cellular functions, it is the former that leads to high-throughput, label-free measurements. By focussing on simplicity, cost, feasibility, field of view, and time-lapse in-incubator imaging, we developed ‘Lensfree Video Microscope' based on digital in-line holography that is capable of providing a new perspective to cell culture monitoring by being able to capture the kinetics of thousands of cells simultaneously. In this thesis, we present our lensfree video microscope and its applications in in-vitro cell culture monitoring and quantification.We validated the system by performing more than 20,000 hours of real-time imaging, in diverse conditions (e.g.: 37°C, 4°C, 0% O2, etc.) observing varied cell types and culture conditions (e.g.: primary cells, human stem cells, fibroblasts, endothelial cells, epithelial cells, 2D/3D cell culture, etc.). This permitted us to develop label-free cell based assays to study the major cellular events – cell adhesion and spreading, cell division, cell division orientation, cell migration, cell differentiation, network formation, and cell death. The results that we obtained respect the heterogeneity of the population, cell to cell variability (a raising concern in the biological community) and the massiveness of the population, whilst adhering to the standard cell culture practices - a rare combination that is seldom attained by existing real-time monitoring methods.We believe that our microscope and associated metrics would complement existing techniques by bridging the gap between mesoscopic and microscopic information

MEMS Technology for Biomedical Imaging Applications

Biomedical imaging is the key technique and process to create informative images of the human body or other organic structures for clinical purposes or medical science. Micro-electro-mechanical systems (MEMS) technology has demonstrated enormous potential in biomedical imaging applications due to its outstanding advantages of, for instance, miniaturization, high speed, higher resolution, and convenience of batch fabrication. There are many advancements and breakthroughs developing in the academic community, and there are a few challenges raised accordingly upon the designs, structures, fabrication, integration, and applications of MEMS for all kinds of biomedical imaging. This Special Issue aims to collate and showcase research papers, short commutations, perspectives, and insightful review articles from esteemed colleagues that demonstrate: (1) original works on the topic of MEMS components or devices based on various kinds of mechanisms for biomedical imaging; and (2) new developments and potentials of applying MEMS technology of any kind in biomedical imaging. The objective of this special session is to provide insightful information regarding the technological advancements for the researchers in the community

Roadmap on Label-Free Super-resolution Imaging

Label-free super-resolution (LFSR) imaging relies on light-scattering processes in nanoscale objects without a need for fluorescent (FL) staining required in super-resolved FL microscopy. The objectives of this Roadmap are to present a comprehensive vision of the developments, the state-of-the-art in this field, and to discuss the resolution boundaries and hurdles that need to be overcome to break the classical diffraction limit of the label-free imaging. The scope of this Roadmap spans from the advanced interference detection techniques, where the diffraction-limited lateral resolution is combined with unsurpassed axial and temporal resolution, to techniques with true lateral super-resolution capability that are based on understanding resolution as an information science problem, on using novel structured illumination, near-field scanning, and nonlinear optics approaches, and on designing superlenses based on nanoplasmonics, metamaterials, transformation optics, and microsphere-assisted approaches. To this end, this Roadmap brings under the same umbrella researchers from the physics and biomedical optics communities in which such studies have often been developing separately. The ultimate intent of this paper is to create a vision for the current and future developments of LFSR imaging based on its physical mechanisms and to create a great opening for the series of articles in this field.Peer reviewe

Digital Holography Microscopy at Lab-on-a-Chip scale: novel algorithms and recording strategies

Il lavoro presentato è mirato allo sviluppo di nuove tecniche di microscopia olografica digitale (Digital Holography Microscopy, DHM), e di opportuni algoritmi numerici per lo studio di biomateriali in ambiente microfluidico. Nello specifico vengono affrontate due problematiche di imaging particolarmente rilevanti nello studio di sistemi Lab-on-a-Chip (LoC). Dapprima è stato studiato il problema della microscopia quantitativa di oggetti biologici osservati attraverso mezzi complessi, come soluzioni torbide e substrati diffondenti, dove la formazione dell’immagine è ostacolata da processi di scattering. Lo studio condotto è stato mirato all’analisi di processi di diffusione da layer statico e da mezzo liquido di tipo colloidale, in regime quasi-statico e dinamico. Sono stati sviluppati a tale scopo dei metodi di registrazione e nuovi algoritmi di ricostruzione dell’immagine olografica (Multi-Look Digital Holography, MLDH) che consentono di fornire un imaging quantitativo dei campioni in esame. Di particolare interesse è il caso di volumi di liquido costituiti da globuli rossi: nel lavoro presentato viene dimostrata la possibilità di studiare, mediante MLDH, processi di adesione cellulare di materiale biologico situato in presenza di flussi di globuli rossi ad alta concentrazione. La possibilità di visualizzare e analizzare quantitativamente materiale biologico all’interno di un capillare o una vena, compensando l’effetto di diffusione del sangue, potrebbe in futuro consentire di studiare la formazione all’interno del vaso di coaguli e placche di colesterolo, sintomatici dell’insorgere di malattie cardiache. La stessa tecnica è in grado di recuperare l’informazione distorta a causa della presenza all’interno del canale di ostacoli statici o quasi-statici (dovuti alla formazione di bio-film o sospensioni batteriche, o causata da processi di fabbricazione del canale microfluidico), aumentando così notevolmente la varietà dei processi biologici analizzabili su piattaforme LoC. Nel lavoro viene anche dimostrato come la presenza di un mezzo torbido possa essere sfruttata vantaggiosamente al fine di migliorare la qualità dell’immagine in sistemi di imaging basati su luce coerente. Parallelamente è stata messa a punto una tecnica interferometrica che, sfruttando il movimento dei campioni nei canali microfluidici, consente di sostituire un sensore convenzionale 2D con un sensore lineare, più compatto e integrabile a bordo del chip, e capace di fornire prestazioni superiori in termini di velocità di acquisizione. Il lavoro presentato descrive il processo di sintesi di un nuovo tipo di ologramma (Space-Time Digital Hologram, STDH), che consente di ottenere un Field-of-View (FoV) illimitato nella direzione del flusso e, quindi, di superare il trade-off esistente tra fattore di ingrandimento e FoV, comune ad ogni tecnica di microscopia convenzionale. Viene inoltre dimostrato che un STDH mantiene le caratteristiche e i vantaggi di un ologramma digitale standard, quali la focalizzazione numerica flessibile, che permette di analizzare contemporaneamente tutti gli oggetti presenti in un volume di liquido, e la possibilità di estrarre la segnatura di fase degli stessi