50 Years of Image Analysis

Fluorescence microscopy is one of the most powerful tools for elucidating the cellular functions of proteins and other molecules. In many cases, the function of a molecule can be inferred from its association with specific intracellular compartments or molecular complexes, which is typically determined by comparing the distribution of a fluorescently labeled version of the molecule with that of a second, complementarily labeled probe. Although arguably the most common application of fluorescence microscopy in biomedical research, studies evaluating the "colocalization" of two probes are seldom quantified, despite a diversity of image analysis tools that have been specifically developed for that purpose. Here we provide a guide to analyzing colocalization in cell biology studies, emphasizing practical application of quantitative tools that are now widely available in commercial and free image analysis software.

A practical guide to evaluating colocalization in biological microscopy.

Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large number of studies have focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, in which the preferential associations between cholesterol and saturated lipids drive the formation of relatively packed (or ordered) membrane domains that selectively recruit certain lipids and proteins. Recent studies have yielded new insights into this mechanism and its relevance in vivo, owing primarily to the development of improved biochemical and biophysical technologies.

The mystery of membrane organization: composition, regulation and roles of lipid rafts

HIV-1 exploits multiple host proteins during infection. We performed a large-scale small interfering RNA screen to identify host factors required by HIV-1 and identified more than 250 HIV-dependency factors (HDFs). These proteins participate in a broad array of cellular functions and implicate new pathways in the viral life cycle. Further analysis revealed previously unknown roles for retrograde Golgi transport proteins (Rab6 and Vps53) in viral entry, a karyopherin (TNPO3) in viral integration, and the Mediator complex (Med28) in viral transcription. Transcriptional analysis revealed that HDF genes were enriched for high expression in immune cells, suggesting that viruses evolve in host cells that optimally perform the functions required for their life cycle. This effort illustrates the power with which RNA interference and forward genetics can be used to expose the dependencies of human pathogens such as HIV, and in so doing identify potential targets for therapy.

https://science.sciencemag.org/content/sci/319/5865/921.full.pdf

Identification of Host Proteins Required for HIV Infection Through a Functional Genomic Screen

Identification of host proteins required for HIV infection through a functional genomic screen

The Pearson correlation coefficient (PCC) and the Mander's overlap coefficient (MOC) are used to quantify the degree of colocalization between fluorophores. The MOC was introduced to overcome perceived problems with the PCC. The two coefficients are mathematically similar, differing in the use of either the absolute intensities (MOC) or of the deviation from the mean (PCC). A range of correlated datasets, which extend to the limits of the PCC, only evoked a limited response from the MOC. The PCC is unaffected by changes to the offset while the MOC increases when the offset is positive. Both coefficients are independent of gain. The MOC is a confusing hybrid measurement, that combines correlation with a heavily weighted form of co-occurrence, favors high intensity combinations, downplays combinations in which either or both intensities are low and ignores blank pixels. The PCC only measures correlation. A surprising finding was that the addition of a second uncorrelated population can substantially increase the measured correlation, demonstrating the importance of excluding background pixels. Overall, since the MOC is unresponsive to substantial changes in the data and is hard to interpret, it is neither an alternative to nor a useful substitute for the PCC. The MOC is not suitable for making measurements of colocalization either by correlation or co-occurrence.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/cyto.a.20896

Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander's overlap coefficient.

https://www.cell.com/developmental-cell/pdf/S1534-5807(07)00257-2.pdf

Sequential Pulses of Apical Epithelial Secretion and Endocytosis Drive Airway Maturation in Drosophila

To the Editor: Many contemporary models of the plasma membrane are based on single-particle tracking (SPT) by light microscopy on live cells. Whereas the analysis of single-particle tracks typically presumes that the cell surface is locally flat, both the ability of cells to rapidly swell and electron microscopy evidence suggest considerable folding1. Even the interface between the plasma membrane and a coverslip is not necessarily flat2. However, there is a lack of hard evidence about the topography of living cells. Accordingly, we examined cell topography using hopping-probe ion conductance microscopy (HPICM)3, a highresolution, noncontact method. HPICM, like all scanning-probe microscopes, has a limited speed (Supplementary Fig. 1), but by scanning many small square blocks, rather than sequential lines, HPICM ensures that the relative heights of adjacent pixels are accurate. Of the 70 cell types we examined using this approach on living cells (Supplementary Table 1), none had flat plasma membrane subregions (Fig. 1a,b and Supplementary Figs. 1–3). This observation has serious implications for SPT. It is well established that molecules on cell surfaces appear to diffuse appreciably more slowly than on planar artificial membranes. Similarly, when we analyzed simulated movement over the non-flat surface of a live cell and a fixed cell in two dimensions (2D), the standard form of SPT analysis, the apparent rate of diffusion dropped; the effect varied with the local topography (Fig. 1b,c). Diffusion over simulated geometric surfaces also had a reduction in apparent movement4 (Supplementary Note and Supplementary Figs. 4,5) and diffusion over a real anisotropically textured surface proved to be similarly anisotropic5. When analyzed in 2D, pillars emulated features of hop diffusion6, where barriers divide a surface into separate domains in which movement is locally unconstrained (Fig. 1d) but from which particles may be excluded or within which they may be confined. The vertical sides of pillars appeared to trap particles (Fig. 1d), a phenomenon we call apparent topographical trapping; for thin pillars, this resembled binding (Supplementary Fig. 6). In these simulations, exclusion, confinement and locally unconstrained movement are artifacts produced by following movement over a non-flat surface in 2D. On membrane blebs, hop diffusion vanishes and diffusion rates are similar to those on reconstituted planar membranes7, which is compatible both with the loss of diffusion barriers and with smoothing of the membrane. The impressive precision with which particles can be localized, down to ±1.5 nm but more typically tens of nanometers, depends generally on localization of a tag rather than the molecule of interest. Cellular topography could cause an underappreciated problem in this respect as well. On a smooth surface the tag and molecule probably maintain a stable alignment, but topography produces an offset of up to ± the tag’s radius (Fig. 1e), which is substantial given the typical diameter of tags (5–40 nm). Membrane folds may also trap or exclude tags. Analysis in 2D of movement on non-flat cellular membranes can cause simple diffusion to show apparently complex patterns that necessitate complex explanations. High-resolution tracking in 3D is possible8. As the topography of live cells is measurable, a topographical map might be used to provide a general 40 × 40 × 2.5 μm

Plasma membrane topography and interpretation of single-particle tracks.

Conventionally, the architecture of arteries is based around the close-packed smooth muscle cells and extracellular matrix. However, the adventitia and endothelium are now viewed as key players in vascular growth and repair. A new dynamic picture has emerged of blood vessels in a constant state of self-maintenance. Recent work raises fundamental questions about the cellular heterogeneity of arteries and the time course and triggering of normal and pathological remodelling. A common denominator emerging in hypertensive remodelling is an early increase in adventitial cell density suggesting that adventitial cells drive remodelling and may initiate subsequent changes such as re-arrangement of smooth muscle cells and extracellular matrix. The organization of vascular smooth muscle cells follows regular arrangements that can be modelled mathematically. In hypertension, new patterns can be quantified in these terms and give insights to how structure affects function. As with smooth muscle, little is known about the organization of the vascular endothelium, or its role in vascular remodelling. Current observations suggest that there may be a close relationship between the helical organization of smooth muscle cells and the underlying pattern of endothelial cells. The function of myoendothelial connections is a topic of great current interest and may relate to the structure of the internal elastic lamina through which the connections must pass. In hypertensive remodelling this must present an organizational challenge. The objective of this paper is to show how the functions of blood vessels depend on their architecture and a continuous interaction of different cell types and extracellular proteins.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/expphysiol.2005.030130

Jeremy Adler

Papers

Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander's overlap coefficient.

Sequential Pulses of Apical Epithelial Secretion and Endocytosis Drive Airway Maturation in Drosophila

Plasma membrane topography and interpretation of single-particle tracks.

New aspects of vascular remodelling: the involvement of all vascular cell types

NRP1 presented in trans to the endothelium arrests VEGFR2 endocytosis, preventing angiogenic signaling and tumor initiation.