Open-Channel Microfluidic Membrane Device for Long-Term FT-IR Spectromicroscopy of Live Adherent Cells

Spatially resolved infrared spectroscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal information on functional groups in biomolecules of a sample by their characteristic vibrational modes. One difficulty in performing long-term FT-IR measurements on live cells is the competition between the strong IR absorption from water and the need to supply nutrients and remove waste. In this proof of principle study, we developed an open-channel membrane device that allows long-term continuous IR measurement of live, adherent mammalian cells. Composed of a gold-coated porous membrane between a feeding channel and a viewing chamber, it allows cells to be maintained on the upper membrane surface in a thin layer of fluid while media is replenished from the feeding channel below. Using this device, we monitored the spatiotemporal chemical changes in living colonies of PC12 cells under nerve growth factor (NGF) stimulation for up to 7 days using both conventional globar and high-resolution synchrotron radiation-based IR sources. We identified the primary chemical change cells undergo is an increase in glycogen that may be associated with secretion of glycoprotein to protect the cells from evaporative stress at the air–liquid interface. Analyzing the spectral maps with multivariate methods of hierarchical cluster analysis (HCA) and principal component analysis (PCA), we found that the cells at the boundary of the colony and in a localized region in the center of the colony tend to produce more glycogen and glycoprotein than cells located elsewhere in the colony and that the degree of spatial heterogeneity decreases with time. This method provides a promising approach for long-term live-cell spectromicroscopy on mammalian cell systems

Ambient Infrared Laser Ablation Mass Spectrometry (AIRLAB-MS) of Live Plant Tissue with Plume Capture by Continuous Flow Solvent Probe

A new experimental setup for spatially resolved ambient infrared laser ablation-mass spectrometry (AIRLAB-MS) that uses an infrared microscope with an infinity-corrected reflective objective and a continuous flow solvent probe coupled to a Fourier transform ion cyclotron resonance mass spectrometer is described. The efficiency of material transfer from the sample to the electrospray ionization emitter was determined using glycerol/methanol droplets containing 1 mM nicotine and is ∼50%. This transfer efficiency is significantly higher than values reported for similar techniques. Laser desorption does not induce fragmentation of biomolecules in droplets containing bradykinin, leucine enkephalin and myoglobin, but loss of the heme group from myoglobin occurs as a result of the denaturing solution used. An application of AIRLAB-MS to biological materials is demonstrated for tobacco leaves. Chemical components are identified from the spatially resolved mass spectra of the ablated plant material, including nicotine and uridine. The reproducibility of measurements made using AIRLAB-MS on plant material was demonstrated by the ablation of six closely spaced areas (within 2 × 2 mm) on a young tobacco leaf, and the results indicate a standard deviation of <10% in the uridine signal obtained for each area. The spatial distribution of nicotine was measured for selected leaf areas and variation in the relative nicotine levels (15–100%) was observed. Comparative analysis of the nicotine distribution was demonstrated for two tobacco plant varieties, a genetically modified plant and its corresponding wild-type, indicating generally higher nicotine levels in the mutant

Synchrotron Infrared Measurements of Protein Phosphorylation in Living Single PC12 Cells during Neuronal Differentiation

Protein phosphorylation is a post-translational modification that is essential for the regulation of many important cellular activities, including proliferation and differentiation. Current techniques for detecting protein phosphorylation in single cells often involve the use of fluorescence markers, such as antibodies or genetically expressed proteins. In contrast, infrared spectroscopy is a label-free and noninvasive analytical technique that can monitor the intrinsic vibrational signatures of chemical bonds. Here, we provide direct evidence that protein phosphorylation in individual living mammalian cells can be measured with synchrotron radiation-based Fourier transform-infrared (SR-FT-IR) spectromicroscopy. We show that PC12 cells stimulated with nerve growth factor (NGF) exhibit statistically significant temporal variations in specific spectral features, correlating with changes in protein phosphorylation levels and the subsequent development of neuron-like phenotypes in the cells. The spectral phosphorylation markers were confirmed by bimodal (FT-IR/fluorescence) imaging of fluorescently marked PC12 cells with sustained protein phosphorylation activity. Our results open up new possibilities for the label-free real-time monitoring of protein phosphorylation inside cells. Furthermore, the multimolecule sensitivity of this technique will be useful for unraveling the associated molecular changes during cellular signaling and response processes

Scanning and transmission electron micrographs of biofilms, cells and hami.

<p>Left panels: MSI, right panel: SM. A: Scanning electron micrograph of MSI biofilm, showing SM1 euryarchaeal cells with defined distances and cell-cell connections. Bar: 1 µm. B: Scanning electron micrograph of SM biofilm, showing SM1 euryarchaeal cells with defined distances and fine-structured cell-cell connections. In-between: Bacterial filamentous and rod-shaped cells. Bar: 1 µm. C: Scanning electron micrograph of dividing SM1 euryarchaeal cell (MSI) with cell surface appendages. Bar: 200 nm. D: Scanning electron micrograph of dividing SM1 euryarchaeal cell (SM) with cell surface appendages. Bar: 200 nm. E: Transmission electron micrograph of cell surface appendages (hami) of SM1 euryarchaeal cells from the MSI biofilm. The hami carry the nano-grappling hooks, but besides that appear bare (square), without prickles (Moissl et al 2005). Bar: 100 nm. F: Transmission electron micrograph of cell surface appendages and matrix of SM1 euryarchaeal cells from the SM biofilm. The hami reveal the typical ultrastructure, with nano-grappling hooks and barbwire-like prickle region (square, Moissl et al 2005). Bar: 100 nm.</p

Detailed community profiling using PhyloChip G3 and SR-FTIR.

<p>A: Ordination analysis of PhyloChip G3 data based on weighted UniFrac measure of eOTU abundances followed by non-metric multidimensional scaling (NMDS). Stress for NMDS of archaeal eOTUs (#37): 0.0088. Stress for NMDS of bacterial eOTUs (#1300): 0.0223. B: Heatmap displaying significantly different families found between the two biofilm types, MSI-BF and SM-BF by PhyloChip G3 assay. Significance is based on aggregated HybScores of eOTUs on family level followed by a Welch-test. For false discovery detection please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099801#pone.0099801.s006" target="_blank">Fig. S6</a>. C: Ordination analysis of SR-FTIR data based on a linear discriminant analysis and principal component analysis (PCA-LDA) in the spectral region of 2800–3100 cm⁻¹ on the archaea spectra extracted from the maps from the three different locations. On the right there is the plot of PCA-LDA loadings. PCA-LDA1 explains for the 93.4% of the variance, PCA-LDA2 for 5.3% and PCA-LDA3 for 0.9%. Arrows point to the infrared signals used to explain the difference between the samples: 2975 cm⁻¹, 2965 cm⁻¹, 2924 cm⁻¹ and 2850 cm⁻¹. D: PCA-LDA in the spectral regions of 900–1280 cm⁻¹ and 2800–3100 cm⁻¹ on SR-FTIR spectra of the bacteria “pixels” from the chemical maps of the samples at the three different locations. On the right there is a plot of PCA-LDA loadings in the two spectral region of interest. PCA-LDA1 explains for the 54.5% of the variance, PCA-LDA2 for 28.6% and PCA-LDA3 for 7.3%. Arrows point to the main infrared signals used to explain the difference between the samples: 2958 cm⁻¹, 2925 cm⁻¹, 2870 cm⁻¹ and 2850 cm⁻¹, in the second panel 1250 cm⁻¹, 1110 cm⁻¹, 1080 cm⁻¹ and 1045 cm⁻¹.</p

Quantification of archaeal and bacterial signatures via qPCR, FISH and SR-FTIR (values in brackes give standard deviation).

<p>*data from Probst et al., 2013.</p><p>ND: Not Determined.</p

The conversion of biofilm to string-of-pearls community in the spring water originating from the subsurface.

<p>A: Biofilm. B: Intermediate transition state. C: String-of-pearls community. Row 1: Schematic drawings. Orange: SM1 euryarchaeal cocci, Green: Filamentous, sulfide-oxidizing bacteria. Row 2: Photographs and scanning electron micrograph (2B) of different stages. Row 3: FISH images of different stages (for MSI samples please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099801#pone.0099801-Probst1" target="_blank">[15]</a>; Archaea orange (CY3), Bacteria green (RG)). A: SM-BF, showing high dominance of Archaea. B: Attachment of archaea to filamentous bacteria. C: String-of-pearls communities with large archaeal colony and bacterial mantle. Arrows point to archaeal microcolonies, manteled by filamentous bacteria. It is proposed that attachment of SM1 Euryarchaeota to filamentous bacteria (B) mediates the transition from biofilm (A) to the string-of-pearls community (C). Scale bars: A3: 10 µm, B2: 1 µm B3: 10 mm, C3: 25 µm.</p

Scanning electron micrograph of filamentous bacterium and surrounded and cocooned by the SM1 euryarchaeal cells (SM-BF).

<p>Bar: 1 µm.</p