Measuring the contribution of aldehyde vs. pyridinium/quinolinium building blocks to the probes' visual signals
The styryl library is a 168 × 8 combinatorial library, so there are 1344 styryl structures. Excluding images with artifacts, extensive pixel saturation, dye precipitates, a total of 1291 images were used for chemical address tag analysis (including images lacking detectable styryl signal in the cellular region). A total of 23 specific probe-associated image-based features and 9 general control image-based features were extracted from the images (). To address the extent to which global trends in probe behavior -apparent in the fluorescence images acquired in the FITC, TRITC and Cy5 acquisition channels of the imaging instrument- can be traced back to additive contributions from aldehyde and pyridinium/quinolinium building blocks of the styryl molecules, multivariate ridge regressions were performed, using each image feature as a response variable, and additive factorial effects for the two building blocks of the styryl molecule as predictors. The correlation coefficients between the observed and predicted image features were calculated, using cross-validation to provide unbiased estimates of the extent to which the image features can be predicted from additive contributions of the different building blocks ().
List of quantitative, visual (image) features analyzed in this study.
Figure 1 Correlation coefficients between actual and predicted values for the 32 image-based features analyzed in this study, using additive factors for the two styryl components as predictors. The bars represent the correlation value (estimated unbiasedly using (more ...)
This quantitative analysis indicated that for many fluorescence intensity-related features, the correlation coefficient between predicted and measured values was strong and significant (ranging from 0.6 to 0.8) (, features 1–11). In contrast, control features showed little correlation (correlation coefficient < 0.1) between predicted and measured values irrespective of wavelength (, features 24–32). Visual inspection of arrays of images sorted according to the sign and magnitude of the regressed coefficients of the total intensity contribution from each aldehyde and pyridinium/quinolinium building blocks confirmed the expected trend: dark images in the top left of the array, with image brightness increasing towards the right and bottom (). Furthermore, it is apparent that total intensity is linked with increased fluorescence in the TRITC channel, with the brightest images being the ones having most intense staining in the red (TRITC) channel, and images of intermediate brightness having most intense staining in the green (FITC) channel.
Figure 2 Array of images acquired from styryl compounds, sorted based on the regression effects of different aldehyde building blocks (rows) and pyridinium/quinolinium building blocks (columns) to the total intensity feature of the styryl molecules. The effect (more ...)
For the spatial features, the relative accumulation of the fluorescence signal intensity in the cells relative to the background was moderately predictable from the regressed, additive contributions of the aldehyde and pyridinium/quinolinium building blocks (, features 12–15). Based on the total intensity of the probe, the correlation coefficient for the CV (, feature 16) and CNR (, feature 20) of probe signal were 0.3 and 0.6 respectively However, in the individual wavelength acquisition channels, only the correlations of CV in the FITC and TRITC channel were as large (, features 17, 18), and the correlation coefficient for the CNR feature was close to zero for each separate wavelength channel (it was positive only for the TRITC channel). To summarize, the total probe signals, several channel specific probe signals, the cellular accumulation of total probe signal relative to the background (for each separate acquisition channels and for the sum of the signal acquired in FITC, TRITC and Cy5 channels) were strongly predictable from additive effects of the molecules' basic building blocks (). The CV and CNR features were moderately predictable based on the total sum of the signals acquired from FITC, TRITC and Cy5 channels, although they were not predictable based on the signal from the individual acquisition channels.
Elucidating chemical address tags with respect to fluorescence signal intensity, spectral and localization features
Next, based on the regression coefficients, the extent to which the different aldehyde and pyridinium/quinolinium building blocks differentially contributed to variations in the observed phenotype was established (). The contribution of the aldehyde and pyridinium/quinolinium building blocks to signal in the total intensity and FITC channel tended to be equally strong (, features 1, 2, 5, 6, 9, 11, 12, 13, 16, 18 and 20), indicating that the different building blocks both contributed as chemical address tags to determine the probes' visual signals in the FITC wavelength. Nevertheless, in the TRITC and Cy5 channel, the pyridinium/quinolinium group generally behaved as the determining chemical address tag relative to the aldehyde group, by showing substantially greater contribution to the image features (, features 3, 4, 7, 8, 14, 15).
Figure 3 The calculated, relative contribution of the pyridinium/quinolinium building blocks (A) and the aldehyde building blocks (B) towards the 32 image-based features analyzed in this study. The vertical bars represent the partial R2 value, capturing the additional (more ...)
By relating the chemical features of the pyridinium/quinolinium building block to the variations spectral and localization properties, we established the extent to which chemical variations in the building blocks influenced spectral and localization features (). For the pyridinium building block, variation in the chemical structure of the pyridinium/quinolinium group showed good correlation with variations in the image-based features (). In contrast in the case of the aldehyde building block, the relationship between the variation in chemical structure of the building block and variation in the image-based features was minimal ().
Figure 4 Correlation coefficients between the degree of chemical structure variation in the building blocks of the styryl molecules and their contributions towards each image-based feature analyzed in this study. The bars represent the calculated correlation coefficient (more ...)
Relating chemical structure variations to visual signal variations in the context of chemical address tags
Probing how chemical variations in the pyridinium/quinolinium group affected the visual signal of the styryl molecules relative to similar variations in the aldehyde group, the results revealed that changing from a pyridinium to quinolinium exerted a major effect in relation to a phenyl-to-napthalene change in the aldehyde building block (, features 1, 3, 4, 5, 7, 8, 12, 14, 15, 18). In comparison, isomers of pyridinium/quinolinium and aldehyde building blocks exerted comparable effects on the probe's visual signal (). For the aldehyde building blocks, the magnitude of the effect of isomer variants () was similar to the magnitude of the effect of phenyl vs. naphthalene substitutions (). For the pyridinium/quinolinium building blocks, the isomer effect () was generally less than the effect of substituting a quinolinium for pyridinium ().
Figure 5 The relative effect of a pyridinium vs. quinolinium building block (left box plot in each feature) and a phenyl vs. naphthalene aldehyde building block (right box plot in each feature) on each one of the 23 noncontrol image-based features analyzed in (more ...)
Figure 6 The relative effect of a pyridinium/quinolinium building block isomers (left box plot in each feature) and aldehyde building block isomers (right box plot in each feature) on each one of the 23 non-control image-based features analyzed in this study. (more ...)
Using cluster analysis to reveal relationships between chemical address tags
Based on hierarchical clustering (), we analyzed how the quinolinium/pyridinium groups contributed to the image-based features of styryl molecules, in relation to the contribution of the aldehyde groups. A dendrogram () revealed that the pyridinium/quinolinium groups formed distinct clusters with the different aldehyde groups (I, II, III, and IV). Note that the dendrogram divided the building blocks into two major clusters: one formed by group IV and the other one associated with groups I, II and III. Most aldehyde groups clustered with pyridinium/quinolinium groups A, B, C, F, G and H (, groups I, II and III). Nevertheless, a significant number of aldehydes formed a separate cluster with quinolinium groups D or E (, group IV). Visualizing the global pattern of regressed coefficients in a heat map (), group I and II appeared most similar to each other in terms of their contribution towards the staining patterns, with group IV being distinctively different.
Figure 7 Hierarchical cluster analysis of the relationship between the regressed contribution of all the styryl building blocks to the 23 noncontrol image-based features analyzed in this study. A) Dendrogram and heat map visualization of the global relationship (more ...)
Visual inspection of the building blocks in clusters I, II, III and IV () indicated that half of the aldehyde building blocks that appeared closely related to pyridinium/quinolinium groups D or E in terms of their contribution to the styryl molecule's visual signals possessed a nitrogen as part of the conjugated structure (, group IV). As part of the conjugated structure, a nitrogen atom in the aldehyde building block can facilitate the migration of the molecule's positive charge across the central methine bridge of the styryl molecule, through resonance structures that would delocalize the positive charge normally associated with the imminium nitrogen on the pyridinium/quinolinium group.
In terms of the aldehyde groups that were most like pyridinium groups A, B, C, G, or H, many of them contained one or more hydroxyl, methoxy, or ether substituents (, groups I, II). For the aldheyde groups that were most like quinolinium group F (, group III), two out of three were bromobenzene derivatives. Cluster IV also contained two aldehyde building blocks with bromine atoms, while clusters I and II contained none. These chemical functionalities that were prominent in several of the aldehyde groups in each of these clusters while being less represented in other clusters suggest that specific mechanisms can strongly influence image-based features across a large number of probes.