Abstract
Determination of the intracellular location of proteins is one of the fundamental tasks of microbiology. Conventionally, label-based microscopy and super-resolution techniques are employed. In this work, we demonstrate a new technique that can determine intracellular protein distribution at nanometer spatial resolution. This method combines nanoscale spatial resolution chemical imaging using the photothermal-induced resonance (PTIR) technique with multivariate modeling to reveal the intracellular distribution of cell components. Here, we demonstrate its viability by imaging the distribution of major cellulases and xylanases in Trichoderma reesei using the colocation of a fluorescent label (enhanced yellow fluorescence protein, EYFP) with the target enzymes to calibrate the chemometric model. The obtained partial least squares model successfully shows the distribution of these proteins inside the cell and opens the door for further studies on protein secretion mechanisms using PTIR.
Highlights
Determination of the intracellular location of proteins is one of the fundamental tasks of microbiology
We demonstrate the versatility of our method by analyzing the distribution of cellulases and enhanced yellow fluorescent protein (EYFP) in Trichoderma reesei
QM6a SecEYFP was grown on Mandels−Andreotti medium containing lactose because this carbon source induces cellulase production in T. reesei.[66]
Summary
QM6a SecEYFP was grown on Mandels−Andreotti medium containing lactose because this carbon source induces cellulase production in T. reesei.[66]. To establish a regression from PTIR spectra to fluorescence intensities in the training set, a PLS model was constructed. 2850 local differences in the protein concentration across the hypha and that these are used to model the fluorescence intensity rather than a spurious correlation with an unrelated latent variable or even noise, the model was applied to a sample outside the training set. Using the testing set allows to demonstrate one important property of using PTIR to determine the local fluorescence brightness: by collecting PTIR spectra in a grid, a fluorescence image can be calculated using the PLS model that closely matches the original fluorescence image (see Figure 4).
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