Chapter 2. Hybrid nanoparticles for mass spectrometric imaging of single cells. 13
2.3. Results and discussion
2.3.3. Application in the imaging of single cells
Equipped with the controlled release hybrid SiO2/9-AA nanoparticles, we further aimed to apply this material as a matrix in the mass spectrometric imaging of microscopic specimens such as single cells. As a model sample, we selected unicellular algae – Closterium acerosum.
Figure 2.7 Controlled release of 9-AA in the liquid phase. (A and B) Fluorescence spectra (ex = 400 nm) of supernatants collected from the SiO2/9-AA nanoparticle suspensions. (A) Leaching 9-AA from the SiO2/9-AA nanoparticles by addition of 10 µL of 33% NH3(aq) to 200 µL of the 0.5 mg mL-1 nanoparticle suspension in 50% ethanol. The pH of the resulting suspension was 14. (B) Leaching 9-AA from the SiO2/9-AA nanoparticles with 10 µL of pure water. The black lines in (A) and (B) correspond to the supernatant from the 1st washing step, and the red and blue lines correspond to the supernatants obtained after the 2nd and the 3rd washing step, respectively. (C) Photographs of 0.6-mL microcentrifuge tubes containing SiO2/9-AA nanoparticles – after washing with NH3(aq)/water, and centrifugation (10000 rpm, 10 min). Note that the pellets became pale after the treatment with NH3(aq) (C), which indicates the 9-AA had been leached from the SiO2/9-AA nanoparticles.
Figure 2.8 Scanning electron micrographs of SiO2/9-AA nanoparticles following incubation in NH3(aq) solution for 30 min.
This species has rod-shaped cells (~ 350 × 40 µm). Since cell wall in algal cells is rich in anion-forming biomolecules (e.g. galacturonic acid – a component of pectin),58 the SiO2/9-AA nanoparticles can readily attach onto the outer surface of the cell due to electrostatic interactions (Figure 2.9). Figure 2.10 presents optical and fluorescence micrographs of individual C. acerosum cells with/without SiO2/9-AA nanoparticles. Red fluorescence originates from the natural dyes present in the cell, while yellow fluorescence originates from SiO2/9-AA nanoparticles attached onto the cell surface. We also tested the new matrix with another species of algae – Anabaena sp.: also in this case, the SiO2/9-AA nanoparticles readily attached to the outer surface of the cells (Figure 2.11). We noted that, following the incubation with the SiO2/9-AA nanoparticles, heterocysts of Anabaena sp. – which normally do not fluoresce at the
ex = 330-380 nm – started to fluoresce with blue light. One can speculate the cell wall in heterocysts contains more anionic components which can bind a greater amount of the SiO2/9-AA nanoparticles, as compared with the photosynthesizing cells.Following the treatment of C. acerosum cells with SiO2/9-AA nanoparticles (Figures 2.10 and 2.12A), and subsequent exposure of the sample to ammonia vapors, we used
Figure 2.9 Putative mechanism of the adsorption of SiO2/9-AA nanoparticles on algal cells followed by the controlled release of 9-AA.
Figure 2.10 Optical and fluorescence micrographs of individual Closterium acerosum cells with/without SiO2/9-AAnanoparticles. Scale bars: 200 µm.
Figure 2.11 Optical and fluorescence micrographs of Anabaena sp. with/without SiO2/9-AA nanoparticles (0.5 µL, 5 mg mL-1). The fluorescence micrographs were obtained using two different excitation wavelengths (λex = 510-560 and 330-380 nm – middle and right, respectively). Blue arrows indicate heterocysts. Scale bars: 50 µm.
MALDI-MS to obtain spectra of single cells. Figure 2.12B shows a mass spectrum of C.
acerosum with three high signals at the m/z 709.4, 779.5, and 815.5. We further conducted
MS/MS analysis of these three ions by analyzing their post-source decay products using the laser-induced fragmentation technology (LIFT) cell. The presence of three fragments – PO3
-(m/z 97), H2PO4- (m/z 97), and C3H6O5P- (m/z 153) – suggests that the three signals are related to analytes from the group of phospholipids (Figure 2.14). We confirmed the identities of these peaks by performing MALDI-MS analysis with internal calibrants (Table 2.1).
Matching the measured and the predicted m/z values led to a tentative identification of the three metabolites as phosphatidylglycerols.
Figure 2.12 Single-cell MS imaging with SiO2/9-AAnanoparticles used as matrix. (A) Optical and fluorescence micrographs of a single Closterium acerosum cell with the attached SiO2/9-AA nanoparticles. (B) MALDI mass spectrum of a single cell of Closterium acerosum – following the occlusion with SiO2/9-AA nanoparticles, and the release of 9-AA induced by gaseous ammonia. The blank spectrum of SiO2/9-AA nanoparticles shows no peaks overlapping with the three sample-related peaks (Figure 2.13). (C) Mass spectrometric images of a single cell of Closterium acerosum (same as in (A)). The MS images were
obtained in the negative-ion mode by MALDI-time-of-flight (TOF)-MS. Laser beam wavelength: 355 nm;
frequency: 50 Hz; diameter: 10 μm; raster spacing: 15 μm. The red-color dashed line in (A) approximately delimits the MS imaging area in (C). Scale bars: 200 µm.
Figure 2.13. MALDI mass spectrum of SiO2/9-AA nanoparticles deposited on an aluminum plate, and incubated with gaseous ammonia (blank). Note that the signal at the m/z: 814.9 – in this blank spectrum – does not completely overlap with the signal at the m/z: 815.5 – recorded when analyzing the sample of Closterium acerosum cells (cf. Figure 2.12).
The three prominent MS signals (m/z 709.5, 779.5, and 815.5) were subsequently monitored in a MALDI imaging sequence (Figure 2.12) using a 10-µm UV laser beam, and a raster with 15-µm spacing. The MS signals follow the contours of single cells as observed in the optical and fluorescence images (Figure 2.12A). Although the coverage of the cell with MALDI matrix is highly homogeneous (Figures 2.12A, 2.10 and 2.14), the distribution of metabolites within the cell – as visualized by MALDI-MS – is seen to represent some heterogeneity (Figure 2.12C): this points out the advantage of performing MALDI-MS imaging with subcellular resolution. For example, the metabolite corresponding to the MS signal at the m/z 779.5 seems to be present in the whole cell, while the metabolites corresponding to the MS signals at the m/z 709.5 and 815.5 appear to be more concentrated in one of the two semicells of the C. acerosum cell under investigation (Figure 2.12C).
Figure 2.14 MALDI-MS/MS spectra of the three ions corresponding to the MALDI images depicted in Figures 2.12 and 2.15: (A) m/z 709.4, (B) m/z 779.5, (C) m/z 815.5. Precursor ions are marked with asterisks (*).
The traces in the left-side column represent full m/z-range spectra whereas the traces in the right-side column display the low-m/z range (within the same spectra). The tandem mass spectra were obtained from the samples of Closterium acerosum (co-crystallized with 9-AA) in the negative-ion mode, using the laser-induced fragmentation technology (LIFT).
Table 2.1 Matching the observed and the predicted m/z values after the analysis of Closterium acerosum cells by negative-ion MALDI-TOF-MS using 9-aminoacridine as matrix, and in the presence of internal calibrants.
The internal calibrant mixture contained adenosine triphosphate, guanosine triphosphate, uridine diphosphate glucose, acetyl coenzyme A and bradykinin acetate (each at the concentration of 8.33 × 10-6 M).
Observed m/z Predicted formula Predicted m/z [M-H]- |∆m| (Da)
709.379 C38H63O10P 709.40861 0.03
779.510 C43H73O10P 779.48686 0.02
815.500 C46H73O10P 815.48484 0.02
High-quality images of single C. acerosum cells were obtained using either 15 and 10-µm laser scan rasters (Figures 2.12C and 2.15, respectively). In the case of Anabaena sp., the method allowed imaging individual chains of cells (thickness < 10 µm; Figure 2.16). Further improvement in lateral resolution is expected after combining the sample preparation using the SiO2/9-AA nanoparticle matrix (proposed here) with state-of-the-art MALDI-MS instruments which use laser beams with diameters smaller than 10 µm.
Coating specimens with chemical matrices is seen to be a big challenge in MALDI imaging. Common coating methods involve pneumatic spray and electrospray deposition [for reviews, see refs 16, 17, 21]. Since matrix compounds are normally dissolved in organic solvents, spray-based methods cause analyte to spread over the sample surface.17,21 Several alternative ways of applying matrix prior to MALDI-MS imaging have been proposed to date:
for example, matrix can be deposited on the sample by using inkjet printing, which produces an array of matrix spots, the method which is mainly applicable for imaging at low lateral resolution.59 The matrix sublimation/recrystallization method ensures small crystal size, high
Figure 2.15 Optical and fluorescence micrographs (top) as well as mass spectrometric images of a single cell of Closterium acerosum (bottom). The fluorescence micrograph (ex = 330-380 nm) shows the presence of SiO2/9-AA nanoparticles (yellow color) on the cell surface (before the exposure to gaseous ammonia).
MS images were obtained in the negative-ion mode by MALDI-TOF-MS. Laser beam wavelength: 355 nm; frequency: 50 Hz; diameter: 10 μm; raster spacing: 10 μm. Scale bars: 200 µm.
homogeneity, and minimum dispersion of analytes; however, this method is not suitable for all kinds of samples and matrices.22,23,25 Here we showed that high lateral resolution can be achieved after replacing conventional matrix application protocols with hybrid nanoparticles used as matrix carriers. Since the nanoparticles are dissolved in water – and the organic matrix release occurs in situ, when triggered by ammonia vapors – this way of preparing samples for MS does not blur the native distributions of the analytes, and the so-called ―sweet spot‖ effect may be reduced.
Figure 2.16 MALDI-MS imaging of small cell ensembles using Anabaena sp. as a model. Optical and fluorescence micrographs (top) obtained using two different excitation wavelengths (λex = 510-560 and 330-380 nm – middle and right, respectively) and MALDI-MS images of Anabaena sp. (bottom) after the treatment with SiO2/9-AA nanoparticles and ammonia vapors. The MS images were obtained in the
negative-ion mode by MALDI-TOF-MS. Laser beam wavelength: 355 nm; frequency: 50 Hz; diameter:
10 μm; raster spacing: 10 μm. The red-color dashed line in approximately delimits the MS imaging area.
Scale bars: 100 μm.