Abstracts

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Alexis Perez Gonzalez, PhD

Manager, Melbourne Cytometry Platform at University of Melbourne

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The results presented and discussed in this lecture derive from a benchmark study comparing the sensitivity of Aurora spectral analysers against conventional cytometers at the University of Melbourne Cytometry Platform. Fluorescence sensitivity was assessed via Rainbow 8-peaks beads dim peaks resolution and stain index of human CD4+ cells stained with a broad collection of fluorochrome conjugates (26), differing in excitation, emission wavelengths, brightness and spectral signatures. The comparison of CD4-fluor brightness on fresh vs. fixed human blood revealed the negative effect of autofluorescence on signal dynamic range, resolution and thus the perceived brightness and ranking of fluorochromes. The operational brightness of fluorochromes in cytometry is not only dependent on properties of the dye (extinction coefficient and quantum efficiency) and the instrument (optical configuration and detectors sensitivity), but on the autofluorescence of the cells they label. The autofluorescence extraction feature gives Cytek Aurora a clear advantage in the resolution of fluorescence carried by highly autofluorescent cells, with the instrument showing the best performance across platforms in terms of human CD4 resolution on autofluorescence-rich regions. The benchmark was further extended into a biologically relevant setting, with the comparison of dim tdTomato resolution in Cytek Aurora 5L vs. Beckman Coulter CytoFLEX 6L, as part of a study on the tissue and cellular distribution of a barely known innate immunity marker. Our results reveal the critical role of autofluorescence extraction on the detection of dim fluorescence on highly autofluorescent cell subsets, and the clear advantage this feature gives Aurora instruments over highly sensitive platforms (i.e. CytoFLEX) that lack the autofluorescence extraction feature or the capacity to measure intrinsic cellular autofluorescence in fine details. The lecture will discuss the theoretical basis for the impact of Autofluorescence on signal resolution, describe several strategies for the isolation of unique autofluorescence signatures and illustrate methods for subset-specific autofluorescence extraction in both spectral and conventional analysers.

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Daniel Chiu, PhD

Professor of Chemistry and Bioengineering at The University of Washington

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Fluorescence based techniques, from cellular imaging to molecular assays, have become an indispensible tool kit in both basic cellular studies and in clinical applications and in vitro diagnostics. Organic dyes appear to be the most versatile fluorophores used so far in these applications. But the intrinsic limitations of conventional dyes, such as low absorptivity, limited multiplexing capability, and poor photostability, have posed difficulties for the further development of high-sensitivity techniques and high-throughput assays. As a result, there has been considerable interest in brighter and more stable fluorescent probes. We have developed and reported a new class of fluorescent probes based on semiconducting polymer nanoparticles called Pdots. This talk will highlight some of our recent advancements in developing new generations of Pdots for biomedical applications, with particular emphasis on flow cytometry.

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Christopher Hall, BSc(Hons), MSc

Senior Flow Cytometry Technical Specialist at the Flow Cytometry Facility at the Babraham Institute

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With the proliferation of full spectrum flow cytometers on the market (and in our facility at Babraham) I have been investigating how unmixing works and if there are any novel (or forgotten) analysis methodologies that could be applied to full spectrum flow cytometry data.  In this talk I will discuss these with you. 

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