Pushing the boundaries of high content imaging

Abstract

MICROSCOPY performance is routinely assessed based on three criteria (i) Speed, (ii) Resolution, and (iii) Sensitivity. Whilst we, the research community, have access to a spectacular array of advanced equipment, alas the perfect instrument does not exist-we cannot simultaneously have optimal performance in each criterion. For this reason, the ubiquitous triangle of compromise ( Fig. 1) is drawn to demonstrate these limitations. Thankfully, the triangle is continuously shrinking with new technological improvements.There are additional important considerations, like the need keep cells healthy in live-cell imaging experiments; ensuring the phenomenon being observed is not simply a consequence of phototoxicity; or how effective a given technique is in terms of depth-penetration into a 3D sample.Great technological strides have been made in improving instrument performance, but when one criteria is improved it often comes at a cost to another. For example, super resolution microscopy (1) allows us to surpass the diffraction barrier that otherwise limits the resolution (to $250 nm in the lateral, xy, direction), however these techniques are generally slow and less cell-friendly relative to more conventional approaches.In the first part of this special issue (2) we highlight the continuous advancements improving performance for highcontent (HC) and high-throughput (HT) imaging for a variety of applications from basic to applied and clinical research (3), in terms of [i] Reagents and assay tools (4-6), [ii] Microscopy advancements (6,7), [iii] Automation and robotics (7) and [iv] Computational advancements (8,9). Whilst the performance in terms of speed is key for HT imaging, the image quality in terms of both resolution and signal:noise ratio (A.K.A. sensitivity) is critical for HC imaging. The ideal HC instrument requires all three criteria to be enhanced. We want it all and we want it now! As will be clear from Part 2 of the special issue on High Throughput and High Content Imaging and Cellular Informatics, newer demands on HC imaging for live-cells, 3D samples, and whole organisms are becoming increasingly prevalent. This will provide the impetus for continued technological Figure 1. The triangle of compromise of microscopy performance.

Monomeric Targeted Protein Degraders

“…A range of different automated fluorescence imaging systems acquire fluorescence images of the samples stained for the proteins of interest and use image analysis methods to quantify fluorescence intensities (and consequently the level of specific proteins) in different regions of the cells. , In the case of nonadherent cell types, flow cytometry is often a more suitable method than fluorescence imaging. Flow cytometry is a method that can be used to detect protein levels either on the cell surface or within the cell using intracellular staining procedures. − Flow cytometry also uses the quantitation of fluorescence in individual cells as a means to measure protein levels and is potentially more sensitive than fluorescence imaging (although without subcellular localization information). The proteins of interest can be detected using intracellular staining techniques (there are diverse fluorophores that can be used to directly label the primary antibodies), or the proteins of interest can be expressed fused to a variety of different fluorescent proteins .…”

High-Content Screening for the Detection of Drug-Induced Oxidative Stress in Liver Cells

“…After incubation with fluorescent probes, the automated acquisition of fluorescent images in separated channels is performed. Microscopy performance is usually assessed based on three criteria: sensitivity, resolution and speed [ 38 ]. It is impossible to have an optimal performance in each criterion, so a balance between them is pursued based on the compromise triangle of microscopy performance [ 38 ].…”

“…Microscopy performance is usually assessed based on three criteria: sensitivity, resolution and speed [ 38 ]. It is impossible to have an optimal performance in each criterion, so a balance between them is pursued based on the compromise triangle of microscopy performance [ 38 ]. The selection of the objective affects the resolution, field of view and sensitivity, and should also be considered when setting up an HCS assay.…”

“…It should be considered that processing hundreds of images by applying image algorithms requires a powerful infrastructure [ 43 ]. Moreover, the data output of a 3D image-based screen is extremely high, which also poses a challenge in terms of data handling and image analysis [ 38 ].…”

2018: The dog year ahead

“…These were the Issue on Mass Cytometry which was a joint parallel publication with Cytometry Part B: Clinical Cytometry , a premiere! In February, Calvert and colleagues concluded their special issue on high content imaging and cellular informatics of 2016 with Part II . Holden and Popescu highlighted in the issue for the CYTO 2017 World Conference in Boston the field of quantitative phase imaging and holography for label free cellular analysis.…”