Microplastics are particles that are < 5 mm in size and come from a wide range of sources. The global distribution in terrestrial and aquatic environments indicates they are likely to cause harm to living organisms. They are used in a variety of personal care products and kitchen scourers. To advance further studies, different approaches have been developed in recent years. In this research, a comparison of methods and analytical techniques were applied to characterise microplastics in two toothpastes and two facial scrubs. The analysis of microplastics was determined using light microscopy, laser diffraction, Fourier-transform infrared spectroscopy. This research reports for the first time, the application of Imaging flow cytometry to characterise microplastics, and was explored to characterise smaller sized particles in each product. The methods developed where validated by characterising particles abraded from kitchen scourers. Two market leading and three chain store brands of kitchen scourers were utilised for the characterisation of microplastics. The application of the different techniques indicated differences in the size, number and morphological characteristics of the particles analysed. The different approaches developed for particle extraction, and the analytical techniques had an apparent influence on the results produced. Currently, there are no universally accepted laboratory protocol and analytical techniques to characterise microplastics. However, this research can serve as a reference point to promote more studies on laboratory methods and analytical techniques to characterise microplastics, with the hope of understanding better these complex particles.

Cell painting uses fluorescent agents to label the compositions or organelles of cells to evoke morphological profiling. Imaging flow cytometry (IFC) is a multi-channel imaging technique to acquire individual cell images, including the brightfield and multiple single fluorescence channels. Thus, it is necessary to assess whether cell painting combined with IFC can provide sufficient phenotypic information to distinguish morphological changes in cells. This thesis investigated changes in morphological characteristics and classification of images under different drug perturbations by employing this novel combination. The analysis procedure of IFC images of U-2 OS cells and leukemia blood cells was the focus of this thesis, which could be broadly divided into two stages. The first stage was the preprocessing of images, exploring a preprocessing framework that uses montage processing of cell images to reshape the specification of individual cell images and involve cell segmentation. The following stage was image analysis, which could be further branched into two approaches. The first approach consisted of quantifying brightfield features using CellProfiler (CP) and performing feature classification using CellProfiler Analyst (CPA). Three machine learning classifiers in CPA were utilized: Random Forest, AdaBoost, and Gradient Boosting. The investigation found that the brightfield intensity, size of the cells, and texture complexity were the most distinguishing features. The second approach employed a convolutional neural network model to conduct image classification from two image resources: the brightfield images and the merged brightfield and fluorescence channel images. This study found that brightfield images alone for phenotypic classification were not sufficient, but the accuracy of classification can be further improved by superimposing fluorescence information into the brightfield images. Nevertheless, the availability of IFC for differentiation of cell phenotypic changes under different drug effects is still proven to be viable. Furthermore, this thesis also discussed some measures to improve the image analysis procedure both regarding the image preprocessing and image analysis stages.

Protecting drinking water supplies from pathogens such as Cryptosporidium parvum is a major concern for water treatment plants worldwide. The sensitivity and specificity of current detection methods are largely determined by the effectiveness of the concentration and separation methods used. In this study, disposable microfluidic micromixers were fabricated to effectively isolate Cryptosporidium parvum Oocysts from water samples, while allowing direct observation of Oocysts captured in the device using high quality immunofluorescence microscopy. In parallel, quantitative analysis of the capture yield was carried out by analyzing the waste from the microfluidics outlet with an Imaging Flow Cytometer. At the optimal flow rate, capture efficiencies higher than 95% were achieved in spiked samples, suggesting that scaled microfluidic isolation and detection of Cryptosporidium parvum will provide a faster and more efficient detection method for Cryptosporidium compared to other available laboratory-scale technologies.

Environmental monitoring involves the quantification of microscopic cells and particles such as algae, plant cells, pollen, or fungal spores. Traditional methods using conventional microscopy require expert knowledge, are time-intensive and not wellsuited for automated high throughput. Multispectral imaging flow cytometry (MIFC) allows measurement of up to 5000 particles per second from a fluid suspension and can simultaneously capture up to 12 images of every single particle for brightfield and different spectral ranges, with up to 60x magnification. The high throughput of MIFC has high potential for increasing the amount and accuracy of environmental monitoring, such as for plant-pollinator interactions, fossil samples, air, water or food quality that currently rely on manual microscopic methods. Automated recognition of particles and cells is also possible, when MIFC is combined with deep-learning computational techniques. Furthermore, various fluorescence dyes can be used to stain specific parts of the cell to highlight physiological and chemical features including: vitality of pollen or algae, allergen content of individual pollen, surface chemical composition (carbohydrate coating) of cells, DNA- or enzyme-activity staining. Here, we outline the great potential for MIFC in environmental research for a variety of research fields and focal organisms. In addition, we provide best practice recommendations.

Light allows to non-invasively study the complex and dynamic biological phenomenon undergoing within cells and tissues in their native state. The development of super-resolution microscopes in recent years has helped to overcome the fundamental limitation imposed by Abbe’s diffraction limit, thereby revolutionizing the field of molecular and cellular biology. With the advancement of various super-resolution techniques (like STED, PALM, and 4Pi) it is now possible to visualize the nanometeric cellular structures and their dynamics in real time. The limitations of existing fluorescence microscopy techniques are: poor axial resolution when compared to their lateral counterpart, and their inability to produce high resolution images of dynamic samples. This thesis covers two broadly connected areas of fluorescence imaging techniques while addressing these limitations. First, the PSF engineering and spatial filtering technique for axial super-resolution microscopy and second, the integration of light sheet illumination PSF with microfluidic cytometry for imaging cells on-the-go. The first chapter gives an explicit description on the fundamentals of fluorescence imaging. This introductory chapter includes a variety of optical microscopes, PSF engineering, the resolution limit imposed by the wave nature of light, the photochemistry of the fluorescent dyes, and their proper selection for fluorescence experiments. In addition to the state-of-art imaging techniques, namely Laser Scanning Confocal Microscopy and Light Sheet Microscopy, this chapter also gives a brief explanation on the evolution of imaging cytometry techniques. Their high speed analytic capability (i.e sorting and counting) makes this technique an important tool in health care diagnosis and other various biomedical applications. The chapter ends with a discussion on the operating principle of the flow cytometers and their limitations. The second chapter in this thesis describes the spatial filtering technique for engineering the PSF to eliminate the side-lobes in the system PSF of the 4Pi Confocal Microscopes. Employing an amplitude mask with binary light transmission windows (also called binary filters), the incident light is structured to minimize the secondary lobes. These lobes are responsible for exciting the off-focal planes in the specimen, hence provide incorrect map of the fluorophore distribution in the object. The elimination of the side-lobes is essential for the artifact-free axial super-resolution microscopy. This second chapter describes the spatial filtering technique in details (its mathematical formulation, application in fluorescence microscopy for generation of desired PSF including Bessellike beam). Specifically, spatial filtering technique is employed in 4Pi type-C Confocal Microscope. The spatial mask used results in the reduction of the side-lobes in 1PE case while they are nearly eliminated in 2PE variant of the proposed technique. The side-lobes are reduced by 46% and 76% for 1PE and 2PE when compared to the existing 4Pi type-C Confocal Microscope system. Moreover, OTF of the proposed system confirms the presence of higher frequencies in the Fourier domain indicating high resolution imaging capability. Apart from the resolution in lateral and axial dimension, achieving high resolution while imaging dynamic samples is another challenge that is limiting the field of fluorescence microscopy to flourish. The third and fourth chapters are entirely dedicated towards the work that was carried out to develop imaging techniques on a microfluidic platform for imaging dynamic samples. The fusion of microscopy and flow cytometry has given rise to the celebrated field of imaging flow cytometry. In recent years, the focus has shifted towards miniaturized cytometry devices. Apart from the reduced cost of the sample reagents and the assays, portability and easy handling make the microfluidic devices more relevant to developing countries. The commercially available cytometers are bulky and quite costly. In addition to these practical concerns, they are complex in operation and limited in performance. Most of the existing cytometers use different inlets for sheath and sample flow to achieve the hydrodynamic focusing of the sample assays in a narrow and confined region. The laser beam in the illumination arm interrogates with the flowing samples at this region and the response is captured by the detection optics. The same principle is extensively used in most of the microfluidic based flow cytometers reported till date. Apart from the hydrodynamic force other effects like electro-osmotic, acoustic, and dielectrophoresis have also been exploited to achieve flow focusing in the microfluidic channel. Despite omitting the necessity of external syringe pump as required in pressure driven based cytometers, they all rely upon point-source based excitation scheme and thereby can not interrogate the cells flowing through the entire microfluidic channel. The third chapter describes the integration of light sheet illumination PSF with microfluidic flow cytometry for simultaneous counting and imaging cells on-the-go. The chapter starts with the description on photolithography procedure for preparing SU8 master and PDMS casting procedure adopted to prepare dedicated microfluidic chips for the developed imaging system. The research work reported here demonstrates the proof-ofprinciple of light sheet based imaging flow cytometer. A light sheet fills the entire microfluidic channel and thus omits the necessity of flow focusing and point-scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts-off the incident light, thereby substantially reducing the background in the acquired images. Compared to the existing state-of-the-art techniques, the proposed technique shows marked improvement. Using fluorescently coated Saccharomyces cerevisiae cells, cell counting with throughput as high as 2090 cells/min was recorded. Overall the proposed system is cost-effective and simple in channel geometry. Apart from achieving efficient counting in operational regime of low flow rate, high contrast images of the dynamic samples are also acquired using the proposed cytometry technique. Further, visualization of intra-cellular organelles is achieved during flow in light sheet based high-throughput cytometry system. The fourth chapter demonstrates the proof of concept of light-sheet-based microfluidic cytometer in conjugation with 2π/3 detection system for high-throughput interrogation and high resolution imaging. This system interrogates the flow channel using a sheet of light rather than the existing point-scanning based techniques. This ensures single-shot scanning of specimens flowing through the microfluidic flow channel at variable flow rates. In addition to high throughput counting at low flow rate, visualization of the intra-cellular organelle (mitochondrial network in human cancerous cells) during flow is achieved with sub-cellular resolution. Using mitochondrial network tagged HeLa cells, a maximum count of 2400 cells/min at the optimized flow rate of 700 nl/min was recorded. The 2π/3 detection system ensures efficient photon collection and minimal background caused by scattered illumination light. The other advantage of this kind of detection system which includes 8f detection optics, is the capability to produce variable magnification using the same high NA objective. This thesis opens up in vivo imaging of sub-cellular structures and simultaneous cell counting in a miniaturized flow cytometry system. The developed imaging cytometry technique may find immediate applications in the diverse field of healthcare diagnostics, lab-on-chip technology, and fluorescence microscopy. The concluding chapter summarizes the results with a brief discussion on the future aspects of this field (e.g., live-cell imaging of infectious RBC in microfluidic device and 3D optical sectioning of flowing cells). The field of imaging flow cytometry has immense applications in the overlapping areas of physics and biology. The hydrodynamic forces which are used to achieve flow focusing of the sample assays can have an adverse effect in the cell morphology, thereby altering the cellular functions. Light sheet based cytometry system lifts off the requirement of flow focusing and ensures a single shot scanning of entire samples flowing through the microfluidic channel. The similar concept can be used to study the developmental biology of an entire organism, such as C. elegans. This enables the direct observation of developmental and physiological changes in the entire body. Such an organism can be kept alive for a longer duration in microfluidic chambers, and the neural development and mating behaviors can be extensively studied.

Biological research and Clinical Diagnostics heavily rely on Optical Microscopy for analyzing properties of cells. The experimental protocol for con-ducting a microscopy based diagnostic test consists of several manual steps, like sample extraction, slide preparation and inspection. Recent advances in optical microscopy have predominantly focused on resolution enhancement. Whereas, the aspect of automating the manual steps and enhancing imaging throughput were relatively less explored. Cost-e ective automation of clinical microscopy would potentially enable the creation of diagnostic devices with a wide range of medical and biological applications. Further, automation plays an important role in enabling diagnostic testing in resource-limited settings. This thesis presents a novel optofluidics based approach for automation of clinical diagnostic microscopy. A system-level integrated optofluidic architecture, which enables the automation of overall diagnostic work- ow has been proposed. Based on the proposed architecture, three different prototypes, which can enable point-of-care (POC) imaging cytometry have been developed. The characterization of these prototypes has been performed. Following which, the applicability of the platform for usage in diagnostic testing has been validated. The prototypes were used to demonstrate applications like Cell Viability Assay, Red Blood Cell Counting, Diagnosis of Malaria and Spherocytosis. An important performance metric of the device is the throughput (number of cells imaged per second). A novel microfluidic channel design, capable of enabling imaging throughputs of about 2000 cells per second has been incorporated into the instrument. Further, material properties of the sample handling component (microfluidic device) determine several functional aspects of the instrument. Ultrafast-laser inscription (ULI) based glass microfluidic devices have been identi ed and tested as viable alternatives to Polydimethylsiloxane (PDMS) based microfluidic chips. Cellular imaging with POC platforms has thus far been limited to acquisition of 2D morphology. To potentially enable 3D cellular imaging with POC platforms, a novel slanted channel microfluidic chip design has been proposed. The proposed design has been experimentally validated by performing 3D imaging of fluorescent microspheres and cells. It is envisaged that the proposed innovation would aid to the current e orts towards implementing good quality health-care in rural scenarios. The thesis is organized in the following manner : The overall thesis can be divided into two parts. The first part (chapters 2, 3) of the thesis deals with the optical aspects of the proposed Optofluidic instrument (development, characterization and validations demonstrating its use in poc diagnostic applications). The second part (chapters 4,5,6) of the thesis details the microfluidic sample handling aspects implemented with the help of custom fabricated microfludic devices, the integration of the prototype, func-tional framework of the device. Chapter 2 introduces the proposed optofluidic architecture for implementing the POC tool. Further, it details the first implementation of the proposed platform, based on the philosophy of adapting ubiquitously available electronic imaging devices to perform cellular diagnostic testing. The characterization of the developed prototypes is also detailed. Chapter 3 details the development of a stand-alone prototype based on the proposed architecture using inexpensive o -the-shelf, low frame-rate image sensors. The characterization of the developed prototype and its performance evaluation for application in malaria diagnostic testing are also presented. The chapter concludes with a comparative evaluation of the developed prototypes, so far. Chapter 4 presents a novel microfludic channel design, which enables the enhancement of imaging throughput, even while employing an inexpensive low frame-rate imaging modules. The design takes advantage of radial arrangement of microfludic channels for enhancing the achievable imaging throughput. The fabrication of the device and characterization of achievable throughputs is presented. The stand-alone optofluidic imaging system was then integrated into a single functional unit, with the proposed microfluidic channel design, a viscoelastic effect based micro uidic mixer and a suction-based microfluidic pumping mechanism. Chapter 5 brings into picture the aspect of the material used to fabricate the sample handling unit, the robustness of which determines certain functional aspects of the device. An investigative study on the applicability of glass microfluidic devices, fabricated using ultra-fast laser inscription in the context of the microfluidics based imaging flow cytometry is presented. As detailed in the introduction, imaging in poc platforms, has thus far been limited to acquisition of 2D images. The design and implementation of a novel slanted channel microfluidic chip, which can potentially enable 3D imaging with simplistic optical imaging systems (such as the one reported in the earlier chapters of this thesis) is detailed. A example application of the proposed microfludic chip architecture for imaging 3D fluorescence imaging of cells in flow is presented. Chapter 6 introduces a diagnostic assessment framework for the use of the developed of m in an actual clinical diagnostic scenario. The chapter presents the use of computational signatures (extracted from cell images) to be employed for cell recognition, as part of the proposed framework. The experimental results obtained while employing the framework to identify cells from three different leukemia cell lines have been presented in this chapter. Chapter 7 summarizes the contributions reported in this thesis. Potential future scope of the work is also detailed.