title: Toward-1mm depth precision with a solid state full-field range imaging system

authors: Dorrington, Adrian A.

subjects: solid-state

abstract: Previously, we demonstrated a novel heterodyne based solid-state full-field range-finding imaging system. This system is comprised of modulated LED illumination, a modulated image intensifier, and a digital video camera. A 10 MHz drive is provided with 1 Hz difference between the LEDs and image intensifier. A sequence of images of the resulting beating intensifier output are captured and processed to determine phase and hence distance to the object for each pixel. In a previous publication, we detailed results showing a one-sigma precision of 15 mm to 30 mm (depending on signal strength). Furthermore, we identified the limitations of the system and potential improvements that were expected to result in a range precision in the order of 1 mm. These primarily include increasing the operating frequency and improving optical coupling and sensitivity. In this paper, we report on the implementation of these improvements and the new system characteristics. We also comment on the factors that are important for high precision image ranging and present configuration strategies for best performance. Ranging with sub-millimeter precision is demonstrated by imaging a planar surface and calculating the deviations from a planar fit. The results are also illustrated graphically by imaging a garden gnome.

text: Toward-1mm depth precision with a solid state full-field range imaging system Previously, we demonstrated a novel heterodyne based solid-state full-field range-finding imaging system. This system is comprised of modulated LED illumination, a modulated image intensifier, and a digital video camera. A 10 MHz drive is provided with 1 Hz difference between the LEDs and image intensifier. A sequence of images of the resulting beating intensifier output are captured and processed to determine phase and hence distance to the object for each pixel. In a previous publication, we detailed results showing a one-sigma precision of 15 mm to 30 mm (depending on signal strength). Furthermore, we identified the limitations of the system and potential improvements that were expected to result in a range precision in the order of 1 mm. These primarily include increasing the operating frequency and improving optical coupling and sensitivity. In this paper, we report on the implementation of these improvements and the new system characteristics. We also comment on the factors that are important for high precision image ranging and present configuration strategies for best performance. Ranging with sub-millimeter precision is demonstrated by imaging a planar surface and calculating the deviations from a planar fit. The results are also illustrated graphically by imaging a garden gnome.

year: 2006

title: Quiescence induces epigenetic changes in bovine fibroblasts and improves their reprogramming into cloned embryos

authors: Kallingappa, Prasanna Kumar

subjects: Quiescence, Epigenetics, Reprogramming, Somatic cell nuclear transfer (SCNT), Histone Methylation, Histone Acetylation, Histone varients, DNA methylation, Polycomb group (PcG) proteins

abstract: Cloning by somatic cell nuclear transfer (SCNT) forces cells to lose their lineage-specific epigenetic marks and become totipotent again. This reprogramming process often results in epigenetic and transcriptional aberrations that compromise development. Development rates after SCNT can thus serve as a functional assay for genome-wide epigenetic reprogramming. Dolly the sheep, the first mammalian SCNT clone, was derived from a donor cell that was induced into quiescence by serum starvation. We hypothesized that quiescence alters the epigenetic status of donor cells and elevates their reprogrammability. In order to test this idea, we compared chromatin composition and cloning efficiency of serum-starved, quiescent (G₀), bovine fibroblasts vs non-starved, diploid G₁ controls. Mechanically synchronized G₁ cells were generated by mitotic shake-off and harvested within 3 h post-mitosis. Based on morphological assessment and EdU incorporation during continuous labeling, >93% of cells were captured in G₁.
Using quantitative confocal immunofluorescence microscopy and fluorometric ELISA, we show that G₀ fibroblasts were significantly hypomethylated at lysines (K) of histone 3 (H3), specifically H3K4me3, H3K9me2, H3K9me3 and H3K27me3, but not H3K9me1. Histone acetylation was reduced at H3K9 and H4K5, increased at H3K12 and remained unchanged at H3K16. G₀ cells also significantly reduced DNAme. In addition, they significantly down-regulated the nuclear abundance of RNA polymerase II, histone variant H2A.Z, as well as Polycomb group (PcG) proteins EED, SUZ12, PHC1 and RING2. Histone variant H3.3, PcG proteins EZH2 and histone deacetylase HDAC1 did not change compared to the G₁ controls. Following NT into metaphase-arrested oocytes, G₀ DNA condensed slower than that of G₁ cells, indicating a more relaxed chromatin configuration. After seven days of in vitro culture, H3K9me3, but not H3K4me3, H3K27me3, SUZ12 and RING2, remained hypomethylated in G₀- vs G₁-derived NT blastocysts, both in the inner cell mass and trophectoderm. Furthermore, G₀ donors significantly improved development into cloned blastocysts. In conclusion, quiescence induced long-term epigenetic changes, specifically H3K9me3 hypomethylation, that correlated with increased donor cell reprogrammability.

text: Quiescence induces epigenetic changes in bovine fibroblasts and improves their reprogramming into cloned embryos Cloning by somatic cell nuclear transfer (SCNT) forces cells to lose their lineage-specific epigenetic marks and become totipotent again. This reprogramming process often results in epigenetic and transcriptional aberrations that compromise development. Development rates after SCNT can thus serve as a functional assay for genome-wide epigenetic reprogramming. Dolly the sheep, the first mammalian SCNT clone, was derived from a donor cell that was induced into quiescence by serum starvation. We hypothesized that quiescence alters the epigenetic status of donor cells and elevates their reprogrammability. In order to test this idea, we compared chromatin composition and cloning efficiency of serum-starved, quiescent (G₀), bovine fibroblasts vs non-starved, diploid G₁ controls. Mechanically synchronized G₁ cells were generated by mitotic shake-off and harvested within 3 h post-mitosis. Based on morphological assessment and EdU incorporation during continuous labeling, >93% of cells were captured in G₁.
Using quantitative confocal immunofluorescence microscopy and fluorometric ELISA, we show that G₀ fibroblasts were significantly hypomethylated at lysines (K) of histone 3 (H3), specifically H3K4me3, H3K9me2, H3K9me3 and H3K27me3, but not H3K9me1. Histone acetylation was reduced at H3K9 and H4K5, increased at H3K12 and remained unchanged at H3K16. G₀ cells also significantly reduced DNAme. In addition, they significantly down-regulated the nuclear abundance of RNA polymerase II, histone variant H2A.Z, as well as Polycomb group (PcG) proteins EED, SUZ12, PHC1 and RING2. Histone variant H3.3, PcG proteins EZH2 and histone deacetylase HDAC1 did not change compared to the G₁ controls. Following NT into metaphase-arrested oocytes, G₀ DNA condensed slower than that of G₁ cells, indicating a more relaxed chromatin configuration. After seven days of in vitro culture, H3K9me3, but not H3K4me3, H3K27me3, SUZ12 and RING2, remained hypomethylated in G₀- vs G₁-derived NT blastocysts, both in the inner cell mass and trophectoderm. Furthermore, G₀ donors significantly improved development into cloned blastocysts. In conclusion, quiescence induced long-term epigenetic changes, specifically H3K9me3 hypomethylation, that correlated with increased donor cell reprogrammability.

year: 2013

title: Studies on the health and bacterial symbionts of  toheroa (Paphies ventricosa)

authors: Bennion, Matthew Joseph

subjects: Shellfish health

abstract: Disease outbreaks in marine species can have devastating consequences for host populations, host-associated species assemblages, ecosystem functioning, and for human users of these marine resources. At present, the risk factors associated with disease outbreaks, such as warming and pollution, are increasing. Given the increasing risk of disease outbreaks and the role that parasites play in marine ecosystems, there is a vital need to gain knowledge of marine diseases for species conservation, and the monitoring, management, and forecasting of disease. The field of marine disease ecology is growing, but many facets of our understanding remain in their infancy. Faced with a lack of baseline health data, efforts to combat disease outbreak in the ocean are impeded, even though the future of food security hinges on an ability to source food from the ocean. With a shift towards aquaculture, in particular bivalve aquaculture, knowledge of ambient health of wild and farmed species will dictate both yield, and our ability to protect wild stocks. 
Disease outbreak poses added risk to species that are already in peril. The toheroa (Paphies ventricosa) is a threatened surf clam endemic to Aotearoa (New Zealand). Once bountiful, overharvesting and population collapse put an end to the large-scale fisheries of the early and mid-1900s. The protections enacted on toheroa populations should have ensured their recovery, but recovery has not occurred. Disease has been suggested as one possible explanation. However, without knowledge of disease dynamics in toheroa populations, restorative efforts risk failure, or worse, inflicting further damage. Intracellular microcolonies of bacteria (IMCs) have been linked to numerous shellfish mass mortality events (MMEs) in Aotearoa in recent years, and were first detected in toheroa in 2017. This discovery raised questions regarding the role IMCs might be playing in preventing the recovery of toheroa populations. 
To address this uncertainty, this thesis set out to investigate what pathogens and parasites are present in toheroa, and determine what effect they might be having on toheroa health. A histopathology survey was conducted to gather fundamental baseline health information on toheroa populations across their entire distribution. IMCs were found to be the only potential pathogens of note. Histology data coupled with Bayesian models indicated that the probability of higher IMC intensity was associated with decreased toheroa condition. Using PCR, DNA sequencing, and in situ hybridisation, IMCs were characterised as bacteria in the genus Endozoicomonas. In many marine organisms, Endozoicomonas spp. are cosmopolitan members of associated-bacterial communities, often fulfilling biogeochemical cycling functions in host tissues. In toheroa populations, Endozoicomonas spp. abundance was found to be seasonal, but site specificity indicated habitat characteristics (specifically, the presence of freshwater outflows) might be a significant driver of their abundance. The bacterial community of toheroa was subsequently investigated using 16S rRNA gene sequencing, and candidates common to other coastal bivalve molluscs were found to be dominant, including Spirochaetaceae, Mycoplasmataceae, and Endozoicomonadaceae (in order of relative dominance). Freshwater outflows appeared to be such a significant driver of microbiome taxonomic dissimilarity, that specimens collected from sites 1240 km apart hosted more similar bacterial communities than specimens from sites with and without streams on the same beach and within the same region. The key indicator taxa driving microbiome dissimilarity were found to be Endozoicomonas spp. and sulfate-reducing bacteria in the phylum Desulfobacterota. 
Gas bubbles were also observed on the shells of toheroa at the same time that IMCs were first detected. At that stage, temperature and total dissolved gaseous pressure was thought to contribute to their manifestation. To gain a better understanding of this phenomenon, a spatio-temporal survey was conducted which revealed an association of high gas bubble intensity with the austral-autumn/winter and freshwater outflows. A new hypothesis is therefore presented that links gas bubbles to sulfate-reducing bacteria and hydrogen sulfide (H₂S), a by-product of anaerobic digestion. Given the apparent influence of freshwater on toheroa intrinsic health, the capacity for streams to deliver pollutants to toheroa beds was assessed. Results gained from inductively coupled plasma-mass spectrometry (ICP-MS) suggest that despite the modified nature of adjacent land, streams do not deliver significant loads of bioavailable trace metals to toheroa beds in the intertidal zone. Instead, nutrition in the form of large phytoplankton blooms in autumn/winter is thought to be the primary source of bioavailable trace metals for toheroa.
No observations of significant pathogenesis, associated with Endozoicomonas species, were made throughout this research. Furthermore, during the timeframe of this research (apart from intermittent mortality events reported), there is no evidence of an ongoing disease epidemic in toheroa populations. While not providing evidence for the factor preventing the recovery of toheroa, this research does have implications for the ongoing conservation and management of toheroa. It seems reasonable to assume that infectious disease is not a major factor contributing to the limited recovery of toheroa throughout Aotearoa. Instead, associations made between freshwater outflows, nutrition, and detritus indicate that habitat requirements provided by freshwater streams are having significant effects on toheroa health, survival, and homeostasis. Furthermore, evidence presented suggests that the Endozoicomonas spp. present in toheroa and other Aotearoa shellfish are likely to be an important endosymbiont. In the future, studies on remaining toheroa populations should prioritise nutritional health. Namely, what role symbionts play in toheroa nutrition, and how niche requirements shape symbiont community composition and function in toheroa tissues. Answers to these questions could profoundly increase the success of toheroa restoration and conservation efforts.

text: Studies on the health and bacterial symbionts of  toheroa (Paphies ventricosa) Disease outbreaks in marine species can have devastating consequences for host populations, host-associated species assemblages, ecosystem functioning, and for human users of these marine resources. At present, the risk factors associated with disease outbreaks, such as warming and pollution, are increasing. Given the increasing risk of disease outbreaks and the role that parasites play in marine ecosystems, there is a vital need to gain knowledge of marine diseases for species conservation, and the monitoring, management, and forecasting of disease. The field of marine disease ecology is growing, but many facets of our understanding remain in their infancy. Faced with a lack of baseline health data, efforts to combat disease outbreak in the ocean are impeded, even though the future of food security hinges on an ability to source food from the ocean. With a shift towards aquaculture, in particular bivalve aquaculture, knowledge of ambient health of wild and farmed species will dictate both yield, and our ability to protect wild stocks. 
Disease outbreak poses added risk to species that are already in peril. The toheroa (Paphies ventricosa) is a threatened surf clam endemic to Aotearoa (New Zealand). Once bountiful, overharvesting and population collapse put an end to the large-scale fisheries of the early and mid-1900s. The protections enacted on toheroa populations should have ensured their recovery, but recovery has not occurred. Disease has been suggested as one possible explanation. However, without knowledge of disease dynamics in toheroa populations, restorative efforts risk failure, or worse, inflicting further damage. Intracellular microcolonies of bacteria (IMCs) have been linked to numerous shellfish mass mortality events (MMEs) in Aotearoa in recent years, and were first detected in toheroa in 2017. This discovery raised questions regarding the role IMCs might be playing in preventing the recovery of toheroa populations. 
To address this uncertainty, this thesis set out to investigate what pathogens and parasites are present in toheroa, and determine what effect they might be having on toheroa health. A histopathology survey was conducted to gather fundamental baseline health information on toheroa populations across their entire distribution. IMCs were found to be the only potential pathogens of note. Histology data coupled with Bayesian models indicated that the probability of higher IMC intensity was associated with decreased toheroa condition. Using PCR, DNA sequencing, and in situ hybridisation, IMCs were characterised as bacteria in the genus Endozoicomonas. In many marine organisms, Endozoicomonas spp. are cosmopolitan members of associated-bacterial communities, often fulfilling biogeochemical cycling functions in host tissues. In toheroa populations, Endozoicomonas spp. abundance was found to be seasonal, but site specificity indicated habitat characteristics (specifically, the presence of freshwater outflows) might be a significant driver of their abundance. The bacterial community of toheroa was subsequently investigated using 16S rRNA gene sequencing, and candidates common to other coastal bivalve molluscs were found to be dominant, including Spirochaetaceae, Mycoplasmataceae, and Endozoicomonadaceae (in order of relative dominance). Freshwater outflows appeared to be such a significant driver of microbiome taxonomic dissimilarity, that specimens collected from sites 1240 km apart hosted more similar bacterial communities than specimens from sites with and without streams on the same beach and within the same region. The key indicator taxa driving microbiome dissimilarity were found to be Endozoicomonas spp. and sulfate-reducing bacteria in the phylum Desulfobacterota. 
Gas bubbles were also observed on the shells of toheroa at the same time that IMCs were first detected. At that stage, temperature and total dissolved gaseous pressure was thought to contribute to their manifestation. To gain a better understanding of this phenomenon, a spatio-temporal survey was conducted which revealed an association of high gas bubble intensity with the austral-autumn/winter and freshwater outflows. A new hypothesis is therefore presented that links gas bubbles to sulfate-reducing bacteria and hydrogen sulfide (H₂S), a by-product of anaerobic digestion. Given the apparent influence of freshwater on toheroa intrinsic health, the capacity for streams to deliver pollutants to toheroa beds was assessed. Results gained from inductively coupled plasma-mass spectrometry (ICP-MS) suggest that despite the modified nature of adjacent land, streams do not deliver significant loads of bioavailable trace metals to toheroa beds in the intertidal zone. Instead, nutrition in the form of large phytoplankton blooms in autumn/winter is thought to be the primary source of bioavailable trace metals for toheroa.
No observations of significant pathogenesis, associated with Endozoicomonas species, were made throughout this research. Furthermore, during the timeframe of this research (apart from intermittent mortality events reported), there is no evidence of an ongoing disease epidemic in toheroa populations. While not providing evidence for the factor preventing the recovery of toheroa, this research does have implications for the ongoing conservation and management of toheroa. It seems reasonable to assume that infectious disease is not a major factor contributing to the limited recovery of toheroa throughout Aotearoa. Instead, associations made between freshwater outflows, nutrition, and detritus indicate that habitat requirements provided by freshwater streams are having significant effects on toheroa health, survival, and homeostasis. Furthermore, evidence presented suggests that the Endozoicomonas spp. present in toheroa and other Aotearoa shellfish are likely to be an important endosymbiont. In the future, studies on remaining toheroa populations should prioritise nutritional health. Namely, what role symbionts play in toheroa nutrition, and how niche requirements shape symbiont community composition and function in toheroa tissues. Answers to these questions could profoundly increase the success of toheroa restoration and conservation efforts.

year: 2021