The correlation coefficient r is a measure of similarity used to compare regions of interest in image pairs. In fluorescence microscopy there is a basic tradeoff between the degree of image noise and the frequency with which images can be acquired and therefore the ability to follow dynamic events. The correlation coefficient r is commonly used in fluorescence microscopy for colocalization measurements, when the relative distributions of two fluorophores are of interest. Unfortunately, r is known to be biased understating the true correlation when noise is present. A better measure of correlation is needed. This article analyses the expected value of r and comes up with a procedure for evaluating the bias of r, expected value formulas. A Taylor series of so-called invariant factors is analyzed in detail. These formulas indicate ways to correct r and thereby obtain a corrected value free from the influence of noise that is on average accurate (unbiased). One possible correction is the attenuated corrected correlation coefficient R, introduced heuristically by Spearman (in Am. J. Psychol. 15:72-101, 1904). An ideal correction formula in terms of expected values is derived. For large samples R tends towards the ideal correction formula and the true noise-free correlation. Correlation measurements using simulation based on the types of noise found in fluorescence microscopy images illustrate both the power of the method and the variance of R. We conclude that the correction formula is valid and is particularly useful for making correct analyses from very noisy datasets.

Acute cholesterol depletion is generally associated with decreased or abolished T cell signalling but it can also cause T cell activation. This anomaly has been addressed in Jurkat T cells using progressive cholesterol depletion with methyl-beta-cyclodextrin (MBCD). At depletion levels higher than 50% there is substantial cell death, which explains reports of signalling inhibition. At 10–20% depletion levels, tyrosine phosphorylation is increased, ERK is activated and there is a small increase in cytoplasmic Ca²⁺. Peripheral actin polymerisation is also triggered by limited cholesterol depletion. Strikingly, the lipid raft marker GM1 aggregates upon cholesterol depletion and these aggregated domains concentrate the signalling proteins Lck and LAT, whereas the opposite is true for the non lipid raft marker the transferrin receptor. Using PP2, an inhibitor of Src family kinase activation, it is demonstrated that the lipid raft aggregation occurs independently of and thus upstream of the signalling response. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains. That cholesterol depletion and not unspecific effects of MBCD was behind the reported results was confirmed by performing all experiments with MBCD–cholesterol, when no net cholesterol extraction took place. We conclude that non-lethal cholesterol depletion causes the aggregation of lipid rafts which then induces T cell signalling.

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.

The plasma membrane of eukaryotic cells contains nanodomains known as lipid rafts. Cholesterol depletion is a widely used technique for studying lipid rafts and their involvement in cellular processes. Cholesterol depletion has been reported to cause both increased and abolished T cell signaling. The abolished cell signaling upon cholesterol depletion is likely to be caused by substantial cell death as demonstrated by cell viability measurements. We have investigated how cholesterol depletion alters T cell activation by analyzing Jurkat T cells upon extraction of 10, 20, 30, 40 and 50% of total cholesterol using methyl β cyclodextrin (MBCD), a protocol in which cholesterol depletion does not have any adverse effect on cell viability.Upon cholesterol depletion peripheral actin polymerization and aggregation of the lipid raft marker GM1 in the plasma membrane is observed. The aggregation of GM1 upon cholesterol depletion is dependent on signaling protein Lck. The aggregated GM1 domains colocalize with signaling proteins such as Lck and LAT. To confirm that the effects seen by cholesterol depletion using cyclodextrin are actually due to cholesterol depletion and not cyclodextrin treatment itself, control experiments having Jurkat T cells treated with MBCD-cholesterol complexes to keep the cellular cholesterol content at equilibrium. A larger fraction of ordered (lo) plasma membrane is observed upon cholesterol depletion, a study performed by using laurdan. A relative membrane order is given by normalized ratio of the two emission regions termed as general polarization (GP). GP is defined analogously to fluorescence polarization by measuring the intensities (I) between 385 and 470 nm and 480 and 508 nm. Change in the membrane order and increased peripheral actin polymerization indicates that actin polymerization is in correlation to the formation of liquid ordered (lo) domains in the plasma membrane upon cholesterol depletion. Our results conclude that limited cholesterol depletion leads to T cell activation and an increase in the amount of liquid ordered domains in the plasma membrane. This activation is followed by aggregation of GM1 enriched domains.

Plasma membrane nanodomains, referred to as lipid rafts, more ordered than the bulk membrane play an important role in T cell signalling by forming signalling platforms in activated T cells. However, the existence of lipid rafts in resting T cells is contentious. Using laurdan, a membrane probe whose peak emission wavelength depends on the lipid environment, evidence is presented for the existence of ordered nanodomains in resting T cells.

T cell signalling can be initiated by stimulating the T cell receptor (TCR), crosslinking the lipid raft markers GM1 (sphingolipid) or glycosylphosphatidylinositol (GPI) anchored proteins. The aggregation of lipid raft components induces the same response in Jurkat T cells as the ligation of an antigen to the TCR. Changes in membrane order linked with reorganization of the plasma membrane upon Jurkat T cell activation were followed at 37°C. Fluorescent images were analyzed for generalised polarisation values - a measure of the relative abundance of liquid ordered and liquid disordered domains. TCR patching does not increase the overall membrane order suggesting that membrane domains of high order are brought together in the patches. This supports the existence of small ordered membrane domains in resting T cells that aggregate upon activation. Patching of GM1, the GPI-anchored protein CD59 and the non lipid raft marker CD45 significantly increases the overall membrane order. So does general crosslinking of membrane components with Concanavalin A. Remodelling of the actin cytoskeleton is an integral part of TCR signaling and T cell activation. Disrupting actin polymerization using latrunculin B decreases membrane order and stabilizing actin filaments with jasplakinolide increases membrane order. An increase in membrane order appears to be a general effect of plasma membrane component patching and is likely due to a global induction of actin polymerization at the plasma membrane.

Mahammad, S. and Parmryd, I. Methyl-b-cyclodextrin does not preferentially target lipid raft cholesterol. The 48th Conference on the Bioscience of Lipids, Turku, Finland, September 2007