Development of a low-cost automated sample presentation and analysis system for counting and classifying nematode eggs : a thesis presented in partial fulfilment of the requirements for the degree of Master of Engineering in Mechatronics at Massey University, Manawatu, New Zealand

This thesis discusses the concept development and design of a low-cost, automated, sample presentation system for faecal egg counting, and classification. The system developed uses microfluidics to present nematode eggs for digital imaging to produce images suitable for image analysis and classification. The system costs are kept low by using simple manufacturing methods and commonly available equipment to produce microfluidic counting chambers, which can be interfaced with conventional microscopes. This thesis includes details of the design and implementation of the software developed to allow capture and processing of images from the presentation system. This thesis also includes details on the measures taken to correct for the optical aberrations introduced by the sample presentation system

Optical imaging techniques in microfluidics and their applications

Microfluidic devices have undergone rapid development in recent years and provide a lab-on-a-chip solution for many biomedical and chemical applications. Optical imaging techniques are essential in microfluidics for observing and extracting information from biological or chemical samples. Traditionally, imaging in microfluidics is achieved by bench-top conventional microscopes or other bulky imaging systems. More recently, many novel compact microscopic techniques have been developed to provide a low-cost and portable solution. In this review, we provide an overview of optical imaging techniques used in microfluidics followed with their applications. We first discuss bulky imaging systems including microscopes and interferometer-based techniques, then we focus on compact imaging systems that can be better integrated with microfluidic devices, including digital in-line holography and scanning-based imaging techniques. The applications in biomedicine or chemistry are also discussed along with the specific imaging techniques

Exploiting open source 3D printer architecture for laboratory robotics to automate high-throughput time-lapse imaging for analytical microbiology

Growth in open-source hardware designs combined with the low-cost of high performance optoelectronic and robotics components has supported a resurgence of in-house custom lab equipment development. We describe a low cost (below USD700), open-source, fully customizable high-throughput imaging system for analytical microbiology applications. The system comprises a Raspberry Pi camera mounted on an aluminium extrusion frame with 3D-printed joints controlled by an Arduino microcontroller running open-source Repetier Host Firmware. The camera position is controlled by simple G-code scripts supplied from a Raspberry Pi singleboard computer and allow customized time-lapse imaging of microdevices over a large imaging area. Open-source OctoPrint software allows remote access and control. This simple yet effective design allows high-throughput microbiology testing in multiple formats including formats for bacterial motility, colony growth, microtitre plates and microfluidic devices termed ‘lab-on-a-comb’ to screen the effects of different culture media components and antibiotics on bacterial growth. The open-source robot design allows customization of the size of the imaging area; the current design has an imaging area of ~420 × 300mm, which allows 29 ‘lab-on-a-comb’ devices to be imaged which is equivalent 3480 individual 1μl samples. The system can also be modified for fluorescence detection using LED and emission filters embedded on the PiCam for more sensitive detection of bacterial growth using fluorescent dyes

Development of a 3D printer using scanning projection stereolithography

We have developed a system for the rapid fabrication of low cost 3D devices and systems in the laboratory with micro-scale features yet cm-scale objects. Our system is inspired by maskless lithography, where a digital micromirror device (DMD) is used to project patterns with resolution up to 10 µm onto a layer of photoresist. Large area objects can be fabricated by stitching projected images over a 5cm2 area. The addition of a z-stage allows multiple layers to be stacked to create 3D objects, removing the need for any developing or etching steps but at the same time leading to true 3D devices which are robust, configurable and scalable. We demonstrate the applications of the system by printing a range of micro-scale objects as well as a fully functioning microfluidic droplet device and test its integrity by pumping dye through the channels

Interferometric detection and enumeration of viral particles using Si-based microfluidics

Single-particle interferometric reflectance imaging sensor enables optical visualization and characterization of individual nanoparticles without any labels. Using this technique, we have shown end-point and real-time detection of viral particles using laminate-based active and passive cartridge configurations. Here, we present a new concept for low-cost microfluidic integration of the sensor chips into compact cartridges through utilization of readily available silicon fabrication technologies. This new cartridge configuration will allow simultaneous detection of individual virus binding events on a 9-spot microarray, and provide the needed simplicity and robustness for routine real-time operation for discrete detection of viral particles in a multiplex format.This work was supported in part by a research contract with the ASELSAN Research Center, Ankara, Turkey, and in part by the European Union's Horizon 2020 FET Open program under Grant 766466-INDEX. (ASELSAN Research Center, Ankara, Turkey; 766466-INDEX - European Union's Horizon 2020 FET Open program)First author draf

Lensless high-resolution on-chip optofluidic microscopes for Caenorhabditis elegans and cell imaging

Low-cost and high-resolution on-chip microscopes are vital for reducing cost and improving efficiency for modern biomedicine and bioscience. Despite the needs, the conventional microscope design has proven difficult to miniaturize. Here, we report the implementation and application of two high-resolution (≈0.9 μm for the first and ≈0.8 μm for the second), lensless, and fully on-chip microscopes based on the optofluidic microscopy (OFM) method. These systems abandon the conventional microscope design, which requires expensive lenses and large space to magnify images, and instead utilizes microfluidic flow to deliver specimens across array(s) of micrometer-size apertures defined on a metal-coated CMOS sensor to generate direct projection images. The first system utilizes a gravity-driven microfluidic flow for sample scanning and is suited for imaging elongate objects, such as Caenorhabditis elegans; and the second system employs an electrokinetic drive for flow control and is suited for imaging cells and other spherical/ellipsoidal objects. As a demonstration of the OFM for bioscience research, we show that the prototypes can be used to perform automated phenotype characterization of different Caenorhabditis elegans mutant strains, and to image spores and single cellular entities. The optofluidic microscope design, readily fabricable with existing semiconductor and microfluidic technologies, offers low-cost and highly compact imaging solutions. More functionalities, such as on-chip phase and fluorescence imaging, can also be readily adapted into OFM systems. We anticipate that the OFM can significantly address a range of biomedical and bioscience needs, and engender new microscope applications

Rapid, efficient, and economical synthesis of PET tracers in a droplet microreactor: application to O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET).

BackgroundConventional scale production of small batches of PET tracers (e.g. for preclinical imaging) is an inefficient use of resources. Using O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET), we demonstrate that simple microvolume radiosynthesis techniques can improve the efficiency of production by consuming tiny amounts of precursor, and maintaining high molar activity of the tracers even with low starting activity.ProceduresThe synthesis was carried out in microvolume droplets manipulated on a disposable patterned silicon "chip" affixed to a heater. A droplet of [18F]fluoride containing TBAHCO3 was first deposited onto a chip and dried at 100 °C. Subsequently, a droplet containing 60 nmol of precursor was added to the chip and the fluorination reaction was performed at 90 °C for 5 min. Removal of protecting groups was accomplished with a droplet of HCl heated at 90 °C for 3 min. Finally, the crude product was collected in a methanol-water mixture, purified via analytical-scale radio-HPLC and formulated in saline. As a demonstration, using [18F]FET produced on the chip, we prepared aliquots with different molar activities to explore the impact on preclinical PET imaging of tumor-bearing mice.ResultsThe microdroplet synthesis exhibited an overall decay-corrected radiochemical yield of 55 ± 7% (n = 4) after purification and formulation. When automated, the synthesis could be completed in 35 min. Starting with < 370 MBq of activity, ~ 150 MBq of [18F]FET could be produced, sufficient for multiple in vivo experiments, with high molar activities (48-119 GBq/μmol). The demonstration imaging study revealed the uptake of [18F]FET in subcutaneous tumors, but no significant differences in tumor uptake as a result of molar activity differences (ranging 0.37-48 GBq/μmol) were observed.ConclusionsA microdroplet synthesis of [18F]FET was developed demonstrating low reagent consumption, high yield, and high molar activity. The approach can be expanded to tracers other than [18F]FET, and adapted to produce higher quantities of the tracer sufficient for clinical PET imaging

Linear scanning ATR-FTIR for chemical mapping and high-throughput studies of Pseudomonas sp. biofilms in microfluidic channels

A fully automated linear scanning attenuated total reflection (ATR) accessory is presented for Fourier transform infrared (FTIR) spectroscopy. The approach is based on the accurate displacement of a multi-bounce ATR crystal relative to a stationary infrared beam. To ensure accurate positioning and to provide a second sample characterization mode, a custom-built microscope was integrated into the system and the computerized work flow. Custom software includes automated control and measurement routines with a straightforward user interface for selecting parameters and monitoring experimental progress. This cost-effective modular system can be implemented on any research-grade spectrometer with a standard sample compartment for new bioanalytical chemistry studies. The system was validated and optimized for use with microfluidic flow cells containing growing Pseudomonas sp. bacterial biofilms. The complementarity among the scan positioning accuracy, measurement spatial resolution and the microchannel dimensions paves the way for parallel biological assays with real-time control over environmental parameters and minimal manual labor. By rotating the channel orientation relative to the beam path, the system could also be used for acquisition of linear biochemical maps and stitched microscope images along the channel length.Comment: 9 pages, 6 figure