Imaging Pressure, Cells and Light Fields

Imaging systems often make use of macroscopic lenses to manipulate light. Modern microfabrication techniques, however, have opened up a pathway to the development of novel arrayed imaging systems. In such systems, centimeter-scale areas can contain thousands to millions of micro-scale optical elements, presenting exciting opportunities for new imaging applications. We show two such applications in this thesis: pressure sensing in microfluidics and high throughput fluorescence microscopy for high content screening. Conversely, we show that arrayed elements are not always needed for three dimensional light field imaging.Engineering and Applied Science

Fabrication on the microscale : a two-photon polymerized device for oocyte microinjection

Open Access funding enabled and organized by CAUL and its Member Institutions. KRD is supported by a Mid-Career Fellowship from the Hospital Research Foundation (C-MCF-58–2019). KD acknowledges funding from the UK Engineering and Physical Sciences Research Council (grant EP/P030017/1). This study was funded by the Australian Research Council (ARC) Centre of Excellence for Nanoscale BioPhotonics (CE140100003).Purpose Intracytoplasmic sperm injection (ICSI) addresses male sub-fertility by injecting a spermatozoon into the oocyte. This challenging procedure requires the use of dual micromanipulators, with success influenced by inter-operator expertise. We hypothesized that minimizing oocyte handling during ICSI will simplify the procedure. To address this, we designed and fabricated a micrometer scale device that houses the oocyte and requires only one micromanipulator for microinjection. Methods The device consisted of 2 components, each of sub-cubic millimeter volume: a Pod and a Garage. These were fabricated using 2-photon polymerization. Toxicity was evaluated by culturing single-mouse presumptive zygotes (PZs) to the blastocyst stage within a Pod, with several Pods (and embryos) docked in a Garage. The development was compared to standard culture. The level of DNA damage/repair in resultant blastocysts was quantified (γH2A.X immunohistochemistry). To demonstrate the capability to carry out ICSI within the device, PZs were microinjected with 4-μm fluorescent microspheres and cultured to the blastocyst stage. Finally, the device was assessed for oocyte traceability and high-throughput microinjection capabilities and compared to standard microinjection practice using key parameters (pipette setup, holding then injecting oocytes). Results Compared to standard culture, embryo culture within Pods and a Garage showed no differences in development to the blastocyst stage or levels of DNA damage in resultant blastocysts. Furthermore, microinjection within our device removes the need for a holding pipette, improves traceability, and facilitates high-throughput microinjection. Conclusion This novel device could improve embryo production following ICSI by simplifying the procedure and thus decreasing inter-operator variability.Publisher PDFPeer reviewe

The effect of discrete wavelengths of visible light on the developing murine embryo

Open Access funding enabled and organized by CAUL and its Member Institutions KRD is supported by a Mid-Career Fellowship from the Hospital Research Foundation (C-MCF-58–2019). KD is supported by funding from the UK Engineering and Physical Sciences Research Council (EP/P030017/1) and the Australian Research Council (FL210100099). CC acknowledges the support of a PhD scholarship jointly from the University of Adelaide and University of Nottingham. This study was funded by the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). PR acknowledges funding through the RMIT Vice-Chancellor’s Research Fellowship and ARC DECRA Fellowship scheme (DE200100279).Purpose A current focus of the IVF field is non-invasive imaging of the embryo to quantify developmental potential. Such approaches use varying wavelengths to gain maximum biological information. The impact of irradiating the developing embryo with discrete wavelengths of light is not fully understood. Here, we assess the impact of a range of wavelengths on the developing embryo. Methods Murine preimplantation embryos were exposed daily to wavelengths within the blue, green, yellow, and red spectral bands and compared to an unexposed control group. Development to blastocyst, DNA damage, and cell number/allocation to blastocyst cell lineages were assessed. For the longer wavelengths (yellow and red), pregnancy/fetal outcomes and the abundance of intracellular lipid were investigated. Results Significantly fewer embryos developed to the blastocyst stage when exposed to the yellow wavelength. Elevated DNA damage was observed within embryos exposed to blue, green, or red wavelengths. There was no effect on blastocyst cell number/lineage allocation for all wavelengths except red, where there was a significant decrease in total cell number. Pregnancy rate was significantly reduced when embryos were irradiated with the red wavelength. Weight at weaning was significantly higher when embryos were exposed to yellow or red wavelengths. Lipid abundance was significantly elevated following exposure to the yellow wavelength. Conclusion Our results demonstrate that the impact of light is wavelength-specific, with longer wavelengths also impacting the embryo. We also show that effects are energy-dependent. This data shows that damage is multifaceted and developmental rate alone may not fully reflect the impact of light exposure.Publisher PDFPeer reviewe

Low-frequency variation in TP53 has large effects on head circumference and intracranial volume.

Cranial growth and development is a complex process which affects the closely related traits of head circumference (HC) and intracranial volume (ICV). The underlying genetic influences shaping these traits during the transition from childhood to adulthood are little understood, but might include both age-specific genetic factors and low-frequency genetic variation. Here, we model the developmental genetic architecture of HC, showing this is genetically stable and correlated with genetic determinants of ICV. Investigating up to 46,000 children and adults of European descent, we identify association with final HC and/or final ICV + HC at 9 novel common and low-frequency loci, illustrating that genetic variation from a wide allele frequency spectrum contributes to cranial growth. The largest effects are reported for low-frequency variants within TP53, with 0.5 cm wider heads in increaser-allele carriers versus non-carriers during mid-childhood, suggesting a previously unrecognized role of TP53 transcripts in human cranial development