Biology subjects

Gottlieb, D.

Publications and source records attributed to Gottlieb, D..

3 recordsLinked to original sources

bioRxiv2023

FLUTE: a Python GUI for interactive phasor analysis of FLIM data

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique used to probe the local environment of fluorophores. The phasor approach to FLIM data is a fit-free analysis and is increasingly used due to its ease of interpretation. To date, no open-source graphical user interface (GUI) for phasor analysis of FLIM data is available thus limiting the widespread use of phasor analysis in biomedical research. Here we present (F)luorescence (L)ifetime (U)l(t)imate (E)xplorer (FLUTE), a Python GUI that is designed to fill this gap. FLUTE simplifies and automates many aspects of FLIM analysis, such as calibrating the FLIM data, performing interactive exploration of the phasor plot with cursors, displaying the phasor plot and the FLIM images with different lifetime contrasts and calculating the relative concentration of molecular species. The final edited datasets after applying the desired filters and thresholds can be exported for further user specific analysis. FLUTE was tested using several FLIM datasets including autofluorescence of Zebrafish embryos, cells in vitro and intact live tissues. In summary, our user-friendly GUI extends the advantages of phasor plotting by making the data visualization and analysis easy and interactive, allows for analysis of large FLIM datasets and accelerates FLIM analysis for non-specialized labs. Impact statementThis work introduces the first open-source graphical user interface (GUI) for phasor analysis of Fluorescence Lifetime Microscopy (FLIM) data. Phasor analysis is increasingly used for FLIM data analysis in biomedical research as it reduces the complexity of the analysis and provides a powerful visualization of the data content and optimization of data handling with respect to multiexponential fitting. However, the development of quantitative FLIM applications in the life sciences has been until now hampered by the lack of an open source and user-friendly graphical user interface. Here we introduce FLUTE that expands some possibilities of phasor FLIM image processing, accelerates the whole FLIM analysis and simplifies the visualization and the analysis of FLIM data, thus making phasor analysis possible for a broader base of researchers. FLUTE will be of interest to researchers with interests ranging from physics to biology and will facilitate research in several biomedical fields.

bioinformatics↗

bioRxiv2021

Immune Cell Profiling Reveals MAIT and Effector Memory CD4+ T Cell Recovery Link to Control of Cytomegalovirus Reactivation after Stem Cell Transplant

Human cytomegalovirus (HCMV) reactivation is a major opportunistic infection after allogeneic haematopoietic stem cell transplantation and has a complex relationship with post-transplant immune reconstitution. Here, we used mass cytometry to comprehensively define global patterns of innate and adaptive immune cell reconstitution at key phases of HCMV reactivation (before detection, initial detection, peak and near resolution) in the first 100 days post-transplant. In addition to identifying patterns of immune reconstitution in those with or without HCMV reactivation, we found mucosal-associated invariant T (MAIT) cell levels at the initial detection of HCMV DNAemia distinguished patients who subsequently developed low-level versus high-level HCMV reactivation. In addition, early recovery of effector-memory CD4+ T cells distinguished low-level and high-level reactivation. Our data describe distinct immune signatures that emerged with HCMV reactivation post-HSCT, and highlight MAIT cell levels at the initial detection of reactivation as a potential prognostic marker to guide clinical decisions regarding pre-emptive therapy.

immunology↗

bioRxiv2018

Intracellular delivery of mRNA to human primary T cells with microfluidic vortex shedding

Intracellular delivery of functional macromolecules, such as DNA and RNA, across the cell membrane and into the cytosol, is a critical process in both biology and medicine. Herein, we develop and use microfluidic chips containing post arrays to induce microfluidic vortex shedding, or VS, for cell membrane poration that permits delivery of mRNA into primary human T lymphocytes. We demonstrate transfection with VS by delivery of a 996-nucleotide mRNA construct encoding enhanced green fluorescent protein (EGFP) and assessed transfection efficiencies by quantifying levels of EGFP protein expression. We achieved high transfection efficiency (63.6 {+/-} 3.44% EGFP+ viable cells) with high cell viability (77.3 {+/-} 0.58%) and recovery (88.7 {+/-} 3.21%) in CD3+ T cells 19 hrs after VS processing. Importantly, we show that processing cells via VS does not negatively affect cell growth rates or alter cell states. We also demonstrate processing speeds of greater than 2.0 x 106 cells s-1 at volumes ranging from 0.1 to 1.5 milliliters. Altogether, these results highlight the use of VS as a rapid and gentle delivery method with promising potential to engineer primary human cells for research and clinical applications.

bioengineering↗