title: A new optical configuration for flow cytometric sorting of bovine spermatozoa by sex

authors: Sharpe, John C.

abstract: The orthogonal axes of illumination, flow, and detection in conventional sorting flow cytometers can limit accuracy or throughput when making fluorescence measurements on aspherical cells. We report a new radially symmetric optical configuration that has been designed to overcome these problems. This system has specifically been developed for high resolution DNA chromosome content measurement in cells with an ultimate goal to separate X- and Y-chromosome-bearing mammalian spermatozoa. 

The new optics design provides radially symmetric illumination and detection of asymmetric particles while retaining the sort capability of a jet-in-air (or cylindrical cuvette) design. A paraboloidal reflector, symmetrical about the sample stream flow axis, both focuses a laser excitation beam and collects cell scatter and fluorescence from the inspection point.

The performance of the new optical configuration has been tested and compared to that of a conventional (orthogonal optical geometry) flow cytometer for a variety of particles and cells. For fluorescence measurements on calibration microspheres the new system produces histograms with similar coefficients of variation to those obtained with the conventional cytometer. Optical artifacts apparent in measurements on flat cells, such as blood cells and mammalian sperm, using conventional optics are overcome by the new configuration. Analysis of chinchilla sperm yields a dual-peaked histogram population that has a coefficient of variation and X-Y split which matches that for a gated (oriented) fraction of the sample as measured by the orthogonal system. Bovine sperm, which are larger and flatter than chinchilla sperm, also produce a single population which, when low sample to sheath differential pressures are used, has coefficients of variation matching those for an oriented subpopulation as measured by conventional optics.

Further modifications to the radial optics system have been carried out to study the effect of cell orientation (with respect to the excitation source) on fluorescence. A ray tracing model has been developed to explain the optical artifacts associated with the measurement of aspherical cells in orthogonal flow systems. This model has been used to verify the experimental findings from our cell orientation studies.
 
The radially symmetric optics we have described can be used to overcome the optical artifacts which decrease the accuracy of optical measurements on aspherical cells. With an added electrostatic sort capability, this system provides the high speed sort rates of conventional jet in air flow cytometers. The system presents an alternative, orientation-independent technique for measuring aspherical cells independent of their orientation. This optical configuration may provide a means for increasing throughput and accuracy for sperm sexing and other applications which require high resolution measurement and sorting of aspherical particles or cells.

text: A new optical configuration for flow cytometric sorting of bovine spermatozoa by sex The orthogonal axes of illumination, flow, and detection in conventional sorting flow cytometers can limit accuracy or throughput when making fluorescence measurements on aspherical cells. We report a new radially symmetric optical configuration that has been designed to overcome these problems. This system has specifically been developed for high resolution DNA chromosome content measurement in cells with an ultimate goal to separate X- and Y-chromosome-bearing mammalian spermatozoa. 

The new optics design provides radially symmetric illumination and detection of asymmetric particles while retaining the sort capability of a jet-in-air (or cylindrical cuvette) design. A paraboloidal reflector, symmetrical about the sample stream flow axis, both focuses a laser excitation beam and collects cell scatter and fluorescence from the inspection point.

The performance of the new optical configuration has been tested and compared to that of a conventional (orthogonal optical geometry) flow cytometer for a variety of particles and cells. For fluorescence measurements on calibration microspheres the new system produces histograms with similar coefficients of variation to those obtained with the conventional cytometer. Optical artifacts apparent in measurements on flat cells, such as blood cells and mammalian sperm, using conventional optics are overcome by the new configuration. Analysis of chinchilla sperm yields a dual-peaked histogram population that has a coefficient of variation and X-Y split which matches that for a gated (oriented) fraction of the sample as measured by the orthogonal system. Bovine sperm, which are larger and flatter than chinchilla sperm, also produce a single population which, when low sample to sheath differential pressures are used, has coefficients of variation matching those for an oriented subpopulation as measured by conventional optics.

Further modifications to the radial optics system have been carried out to study the effect of cell orientation (with respect to the excitation source) on fluorescence. A ray tracing model has been developed to explain the optical artifacts associated with the measurement of aspherical cells in orthogonal flow systems. This model has been used to verify the experimental findings from our cell orientation studies.
 
The radially symmetric optics we have described can be used to overcome the optical artifacts which decrease the accuracy of optical measurements on aspherical cells. With an added electrostatic sort capability, this system provides the high speed sort rates of conventional jet in air flow cytometers. The system presents an alternative, orientation-independent technique for measuring aspherical cells independent of their orientation. This optical configuration may provide a means for increasing throughput and accuracy for sperm sexing and other applications which require high resolution measurement and sorting of aspherical particles or cells.

year: 1997

title: Biological flora of New Zealand 12: Griselinia lucida, puka, akapuka, akakōpuka, shining broadleaf

authors: Bryan, Catherine Louise

subjects: biological flora

abstract: Information on the biology and ecology of Griselinia lucida (Forst. f.) (Griseliniaceae) available through published and unpublished sources is assembled and reviewed. G. lucida is a large shrub hemiepiphyte that grows primarily in trees of wet, lowland forests and also in open coastal and rocky outcrop habitats. Large, bright green, glossy leaves and grooved terrestrial roots that descend from the canopy make this species a very conspicuous member of New Zealand's endemic flora. G. lucida requires warm climates and high atmospheric moisture and is found throughout the North Island and in a limited area of the South Island. G. lucida is one of two native members of the Griselinia genus which includes five South American species. G. lucida is frequently associated with diverse and abundant epiphyte communities. The conservation and restoration of both epiphytic and terrestrial populations is important to ensure functionally diverse ecosystems and accordingly, it is recommended that this species be included in future forest ecological restoration.

text: Biological flora of New Zealand 12: Griselinia lucida, puka, akapuka, akakōpuka, shining broadleaf Information on the biology and ecology of Griselinia lucida (Forst. f.) (Griseliniaceae) available through published and unpublished sources is assembled and reviewed. G. lucida is a large shrub hemiepiphyte that grows primarily in trees of wet, lowland forests and also in open coastal and rocky outcrop habitats. Large, bright green, glossy leaves and grooved terrestrial roots that descend from the canopy make this species a very conspicuous member of New Zealand's endemic flora. G. lucida requires warm climates and high atmospheric moisture and is found throughout the North Island and in a limited area of the South Island. G. lucida is one of two native members of the Griselinia genus which includes five South American species. G. lucida is frequently associated with diverse and abundant epiphyte communities. The conservation and restoration of both epiphytic and terrestrial populations is important to ensure functionally diverse ecosystems and accordingly, it is recommended that this species be included in future forest ecological restoration.

year: 2011

title: Walking into the past to explore the future: Utulizing larval densities to measure the effects of two climate change factors, temperature and salinity on Poorohe (common smelt, Retropinna retropinna) abundance in Te Puuaha o Waikato

authors: Robcke, Sidney

subjects: Common smelt

abstract: Poorohe (common smelt, Retropinna retropinna) are a highly valued kai species in Te Puuaha o Waikato (the lower 66 km of the Waikato River). Fisher whaanau have observed changes in the amount Poorohe that are harvested during annual fish harvests. Since Poorohe are particularly vulnerable to environmental changes, whaanau are particularly concerned about the how climate change might impact Poorohe populations. Climate change vulnerability assessments (CCVA) currently exist for several native New Zealand fish freshwater species; however, one does not yet exist for Poorohe. Previous larval distribution studies in the lower Waikato River only sampled as far downstream as Tuakau bridge (approximately 45km from the river mouth). These studies indicated that important spawning habitats likely existed downstream of Tuakau bridge. Temperature increases and saltwater intrusion have been identified as two climate change factors that will likely impact Poorohe in Te Puuaha o Waikato, because ambient temperatures have increased since the industrial revolution and the proximity of the study area to the ocean. A literature review was used to explore the experiences of fisher whaanau through the past, present and how the experiences will continue to change in a changing climate. The effects of temperature and saltwater intrusion on Poorohe populations were explored through replicating the methods of past research carried out in the 1980s and 2010s. Current river temperatures are in the optimal temperature range (12°C - 18°C) for Poorohe spawning. The saltwater wedge was suppressed by the high river flow during the eight-week sampling period, and it is unclear whether saltwater intrusion has an impact on Poorohe spawning. Larval densities did not significantly increase down river, which suggests large spawning habitat is not found downstream of Tuakau bridge. Furthermore, there were no decreases in larval densities in the two saltwater sites. This study has contributed to the available literature necessary to develop a CCVA for Poorohe and explored the cultural heritage of traditionally significant practices in Te Puuaha o Waikato relating to Poorohe.

text: Walking into the past to explore the future: Utulizing larval densities to measure the effects of two climate change factors, temperature and salinity on Poorohe (common smelt, Retropinna retropinna) abundance in Te Puuaha o Waikato Poorohe (common smelt, Retropinna retropinna) are a highly valued kai species in Te Puuaha o Waikato (the lower 66 km of the Waikato River). Fisher whaanau have observed changes in the amount Poorohe that are harvested during annual fish harvests. Since Poorohe are particularly vulnerable to environmental changes, whaanau are particularly concerned about the how climate change might impact Poorohe populations. Climate change vulnerability assessments (CCVA) currently exist for several native New Zealand fish freshwater species; however, one does not yet exist for Poorohe. Previous larval distribution studies in the lower Waikato River only sampled as far downstream as Tuakau bridge (approximately 45km from the river mouth). These studies indicated that important spawning habitats likely existed downstream of Tuakau bridge. Temperature increases and saltwater intrusion have been identified as two climate change factors that will likely impact Poorohe in Te Puuaha o Waikato, because ambient temperatures have increased since the industrial revolution and the proximity of the study area to the ocean. A literature review was used to explore the experiences of fisher whaanau through the past, present and how the experiences will continue to change in a changing climate. The effects of temperature and saltwater intrusion on Poorohe populations were explored through replicating the methods of past research carried out in the 1980s and 2010s. Current river temperatures are in the optimal temperature range (12°C - 18°C) for Poorohe spawning. The saltwater wedge was suppressed by the high river flow during the eight-week sampling period, and it is unclear whether saltwater intrusion has an impact on Poorohe spawning. Larval densities did not significantly increase down river, which suggests large spawning habitat is not found downstream of Tuakau bridge. Furthermore, there were no decreases in larval densities in the two saltwater sites. This study has contributed to the available literature necessary to develop a CCVA for Poorohe and explored the cultural heritage of traditionally significant practices in Te Puuaha o Waikato relating to Poorohe.

year: 2024-11-23