COVID-19 Pandemic: The Circus is Over, for the Moment.

This critical essay responds to the COVID-19 pandemic and subsequent lockdown in Victoria from the perspective of a retired Aboriginal academic and reflects on personal responsibility, Indigenous history, and resilience.

Measurement of cytoplasmic to nuclear translocation.

A method is described for the quantitative assessment of the translocation of signaling molecules from the cytoplasm to the nucleus in cells. This method utilizes fluorochrome-conjugated antibodies to the signaling molecule and a nuclear dye, and it is based on imagery acquired rapidly in flow with the use of a multispectral imaging cytometer. The analysis correlates the spatial distribution of the stained translocating signaling molecule with nuclear staining, and it generates a quantitative score for each cell using Pearson's correlation coefficient.

Quantitative image based apoptotic index measurement using multispectral imaging flow cytometry: a comparison with standard photometric methods.

Morphological characterization by microscopy remains the gold standard for accurately identifying apoptotic cells using characteristics such as nuclear condensation, nuclear fragmentation, and membrane blebbing. However, quantitative measurement of apoptotic morphology using microscopy can be time consuming and can lack objectivity and reproducibility, making it difficult to identify subtle changes in large populations. Thus the apoptotic index of a sample is commonly measured by flow cytometry using a variety of fluorescence intensity based (photometric) assays which target hallmarks of apoptosis with secondary markers such as the TUNEL (Terminal Deoxynucleotide Transferase dUTP Nick End Labeling) assay for detection of DNA fragmentation, the Annexin V assay for surface phosphatidylserine (PS) exposure, and fluorogenic caspase substrates to detect caspase activation.

Cellular image analysis and imaging by flow cytometry.

Imaging flow cytometry combines the statistical power and fluorescence sensitivity of standard flow cytometry with the spatial resolution and quantitative morphology of digital microscopy. The technique is a good fit for clinical applications by providing a convenient means for imaging and analyzing cells directly in bodily fluids. Examples are provided of the discrimination of cancerous from normal mammary epithelial cells and the high-throughput quantitation of fluorescence in situ hybridization (FISH) probes in human peripheral blood mononuclear cells.

Quantitative analysis of protein co-localization on B cells opsonized with rituximab and complement using the ImageStream multispectral imaging flow cytometer.

Binding of the chimeric, humanized anti-CD20 mAb Rituximab (RTX) to B lymphocytes activates complement and promotes covalent deposition of C3 fragments (C3b/iC3b) on cells. Previous fluorescence microscopy studies, based on examination of B cell lines and of blood samples from RTX-treated CLL patients, suggest that C3b/iC3b is closely associated with cell-bound RTX. We examined Raji cells opsonized with serum and RTX with the ImageStream imaging flow cytometer.

Sensitivity measurement and compensation in spectral imaging.

Background: The ImageStream system combines advances in CCD technologies with a novel optical architecture for high sensitivity and multispectral imaging of cells in flow. The sensitivity and dynamic range as well as a methodology for spectral compensation of imagery is presented.

Methods: Multicolored fluorescent beads were run on the ImageStream and a flow cytometer.

Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow.

Nuclear translocation of NF-kappaB initiates transcription of numerous genes, many of which are critical to host defense. Fluorescent image-based methods that quantify this event have historically utilized adherent cells with large cytoplasm-to-nuclear area ratios. However, many immunologically relevant cells are naturally non-adherent and have small cytoplasm-to-nuclear area ratios.

Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer.

Background: Here we demonstrate the ability of the ImageStream 100 Multispectral Imaging Cytometer to discriminate between live, necrotic, and early and late apoptotic cells, using unique combinations of photometric and morphometric features.

Methods: Live, necrotic, and early and late apoptotic cells were prepared and analyzed by immunofluorescence microscopy, conventional flow cytometry, and imaging flow cytometry, both as single populations and as a heterogeneous mixture of cells.

Results: Live (annexin V(-), 7-AAD(-)) and early apoptotic (annexin V(+), 7-AAD(-)) cells were readily identifiable using either conventional or ImageStream based flow cytometric methods.