Spectrum of the pH at given time points (6 h and 10 h p.i.) in apoptotic and non-apoptotic infected monocytes.

<p>The dashed lines (shown in blue and green) are the spectra of pH in wild-type (wt.) and mutant infection in non-apoptotic monocyte at 6 h p.i. when their pH showed approximately similar values (6.3 to 6.5). The constant lines represent the wild-type or mutant infections in apoptotic monocyte (treated with STS) comparing two time points of 6 and 10 h p.i. The most acidic pH (less than 4) was recorded 10 h p.i. in the apoptotic monocyte infected with mutant whereas at the same time point the apoptotic monocyte infected with wild-type showed a higher pH around 5.3.Regardless of the type of infection, the signals related to acidic pH in non-apoptotic cells were generally less intense than in apoptotic cells. 10 h p.i., the apoptotic cells infected by melanin-free <i>pksP</i> mutant conidia were far more acidic compared to cells containing wild-type conidia.</p

Hyperspectral Imaging Using Intracellular Spies: Quantitative Real-Time Measurement of Intracellular Parameters <i>In Vivo</i> during Interaction of the Pathogenic Fungus <i>Aspergillus fumigatus</i> with Human Monocytes

<div><p>Hyperspectral imaging (HSI) is a technique based on the combination of classical spectroscopy and conventional digital image processing. It is also well suited for the biological assays and quantitative real-time analysis since it provides spectral and spatial data of samples. The method grants detailed information about a sample by recording the entire spectrum in each pixel of the whole image. We applied HSI to quantify the constituent pH variation in a single infected apoptotic monocyte as a model system. Previously, we showed that the human-pathogenic fungus <i>Aspergillus fumigatus</i> conidia interfere with the acidification of phagolysosomes. Here, we extended this finding to monocytes and gained a more detailed analysis of this process. Our data indicate that melanised <i>A</i>. <i>fumigatus</i> conidia have the ability to interfere with apoptosis in human monocytes as they enable the apoptotic cell to recover from mitochondrial acidification and to continue with the cell cycle. We also showed that this ability of <i>A</i>. <i>fumigatus</i> is dependent on the presence of melanin, since a non-pigmented mutant did not stop the progression of apoptosis and consequently, the cell did not recover from the acidic pH. By conducting the current research based on the HSI, we could measure the intracellular pH in an apoptotic infected human monocyte and show the pattern of pH variation during 35 h of measurements. As a conclusion, we showed the importance of melanin for determining the fate of intracellular pH in a single apoptotic cell.</p></div

Effects of chloroquine treatment and phagosomal acidification on <i>A</i>.<i>fumigatus</i> infection in an apoptotic cell.

<p>The acidified pH of the apoptotic cell in the presence of chloroquine was maintained after infection. As it can be concluded, <i>A</i>.<i>fumigatus</i> only interferes with pH if the phagolysosme stays intact.</p

Kinetics of phagolysosomal pH upon infection.

<p><b>(A)</b> Phagosomal pH in an apoptotic monocyte containing labelled wild-type conidia in comparison to an apoptotic monocyte carrying <i>pksP</i> mutant conidia. <b>(B)</b> Data represent the mean ₊ SD from three experiments. <b>(C)</b> Phagosomal pH in the survived apoptotic monocyte infected with labelled wild-type conidia. <b>(D)</b> Data represent the mean ₊ SD of cytosolic pH from three experiments.</p

Effect of bafilomycin A1 treatment and inhibition of phagosomal acidification.

<p>Despite the inhibitory effect of bafilomycin on phagosomes-lysosome degradation, in the presence of melanin the engulfment of conidia and inhibition of intra-cellular acidification were not affected and the conidia were localised in the phagolysosome. Wild-type infection prevented acidification of phagosome, while the <i>pksP</i> mutant was not able to recover the acidic pH.</p

Spectrum of the pH at given time points (6 h and 10 h p.i.) in apoptotic and non-apoptotic infected monocytes.

<p>The dashed lines (shown in blue and green) are the spectra of pH in wild-type (wt.) and mutant infection in non-apoptotic monocyte at 6 h p.i. when their pH showed approximately similar values (6.3 to 6.5). The constant lines represent the wild-type or mutant infections in apoptotic monocyte (treated with STS) comparing two time points of 6 and 10 h p.i. The most acidic pH (less than 4) was recorded 10 h p.i. in the apoptotic monocyte infected with mutant whereas at the same time point the apoptotic monocyte infected with wild-type showed a higher pH around 5.3.Regardless of the type of infection, the signals related to acidic pH in non-apoptotic cells were generally less intense than in apoptotic cells. 10 h p.i., the apoptotic cells infected by melanin-free <i>pksP</i> mutant conidia were far more acidic compared to cells containing wild-type conidia.</p

Principle of hyperspectral imaging (HSI) as a combination of conventional image processing and classical spectroscopy.

<p><b>(A)</b> Hyperspectral data cube includes a set of data that are layered on top of one another. Each pixel in the cube consists of an entire spectrum and the resulting image represents the corresponding wavelength band. Typically in hyperspectral imagery, the spatial information is collected in the X-Y plane and spectral information represented in the Z-direction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163505#pone.0163505.ref044" target="_blank">44</a>]. <b>(B)</b> HSI detector attached to side camera port of an inverted microscope. This combination comprises the imaging spectrograph with sensitive monochrome camera. The CCD camera attached to the spectrograph and the main body through a port. It records the spectral images in a time interval of microsecond-scale. The fluorescence light source is located in the back of camera segment and is adjustable to give an ample light intensity. The front DSLR camera obtains real images of the sample that is located on the heating stage. The filter shield contains combination of filters to detect different fluorescent probes with different excitation/emission rate. <b>(C)</b> Different spectral responses at distinct positions within the detection line. The monocyte is placed within the object field. The multiple fluorescence spectra of cell, conidium or their surroundings are simultaneously captured. The schematic shows a line of areas, and each line covers more than one pixel.</p