Time of flight mass spectrometry imaging of samples fractured in situ with a spring-loaded trap system

Abstract

An in situ freeze fracture device featuring a spring-loaded trap system has been designed and characterized for time of flight secondary ion mass spectrometry (TOF SIMS) analysis of single cells. The device employs the sandwich assembly, which is typically used in freeze fracture TOF SIMS experiments to prepare frozen, hydrated cells for high-resolution SIMS imaging. The addition of the spring-loaded trap system to the sandwich assembly offers two advances to this sample preparation method. First, mechanizing the fracture by adding a spring standardizes each fracture by removing the need to manually remove the top of the sandwich assembly with a cryogenically cooled knife. A second advance is brought about because the top of the sandwich is not discarded after the sandwich assembly has been fractured. This results in two imaging surfaces effectively doubling the sample size and providing the unique ability to image both sections of a cell bifurcated by the fracture. Here, we report TOF SIMS analysis of freeze fractured rat pheochromocytoma (PC12) cells using a Bi cluster ion source. This work exhibits the ability to obtain single cell chemical images with subcellular lateral resolution from cells preserved in an ice matrix. In addition to preserving the cells, the signal from lipid fragment ions rarely identified in single cells are better observed in the freeze-fractured samples for these experiments. Furthermore, using the accepted argument that K(+) signal indicates a cell that has been fractured though the cytoplasm, we have also identified different fracture planes of cells over the surface. Coupling a mechanized freeze fracture device to high-resolution cluster SIMS imaging will provide the sensitivity and resolution as well as the number of trials required to carry out biologically relevant SIMS experiments.

Figure 1

Schematic of the freeze fracture device in an opened and a closed view. Screws holding the stainless steel tabs are colored red, silicon shards yellow and the sample area between the two shards orange. The two mounting holes are colored white.

Figure 2

High lateral resolution image showing intra cellular structures in three PC12 cells. A) The image shows the distribution of the phosphocholine head group at m/z 184 and B) H₃0⁺ at m/z 19. The image was taken in burst alignment mode, 256×256 pixels with a current of 0.04 pA on the TOFSIMS IV instrument. The image was imported into MATLAB where it was normalized to tic, binned (2 × 2) and median filtrated (3 × 3). The lowest 15 % of the signal is not shown. The image area is 116 × 116 μm.

Figure 3

Mirror image of several PC12 cells. A) Image of m/z 184 taken on the big shard with three cell clusters zoomed in. B) Image of m/z 184 taken on the same spot on the opposite shard. The images were imported into MATLAB where they were normalized to total ion current, binned (2 × 2) and median filtrated (2 × 2). The lowest 15 % of the signal in A) is not shown. The maximum intensity of the pallet in A) is twice the pallet in B). The images were recorded in burst alignment mode with a surface of 500 × 500 μm.

Figure 4

Localization of lipid fragments in freeze fractured hydrated PC12 cells. Images A to E were acquired on the hydrated sample (at −110° C) and images F to J after the sample was dried. Images A and F show the distribution of m/z 124, B and G of m/z 142, C and H of m/z 166, D and I of m/z 184, and E and J of m/z 224. All images were taken in bunched mode, 256×256 pixels and with a current of 0.1 pA. The images are taken directly from the ionimage software. The imaged area is 136 × 136 μm².

Figure 5

Signal intensity in marked region of interest (ROI). A) Hydrated PC12 cells imaged at m/z 184, ROI marked by the circle B) Dried PC12 cells, at same position as in A, imaged at m/z 184. ROI marked by the circle C) Signal intensities from selected lipid fragments normalized to total ion signal are compared in the region of interest from A and B. D) Spectrum from ROI in panel A. E) Spectrum from ROI in panel B.

Figure 6

The plane of fracture through the PC12 cells exposes different chemistry. A) An overlay of red (K⁺), blue (Na⁺) and green (PC fragment at m/z 184) is shown. Five ROI’s are marked in colored circles. The scale bar is 20 μm. B) Bar graph showing the intensity normalized to total ion current of four different ions. The color code in the graph follows that of the ROI in image 6A, giving the intensities of the ions in the different ROI’s. m/z 23 (Na⁺) and m/z 39 (K⁺) correspond to the y-axis on the left, m/z 86 (PC lipid fragment) and m/z 184 (PC lipid fragment) correspond to the y-axis on the right.

Figure 7

The distribution of A) m/z 184 and B) m/z 142 over a freeze fractionated hydrated PC12 cell. The image (raw data) was imported into MATLAB where it was normalized to total ion current, binned (3 × 3) and median filtrated (3 × 3). The lowest 15 % of the signal is not shown. Scale bar represents 10 μm. C) Graph of line scans from two PE fragments, at m/z 124 and 142, and two PC fragments, at m/z 184 and 224. The six pixels wide line scan was performed as marked in A) and B). The primary y-axis show PC data and the secondary y-axis show PE data. To fit the scale of the y-axis the data from PE at m/z 124 is multiplied by 3 and the data from PC at m/z 224 is multiplied by 9. The image was taken in burst alignment mode and the area of the image is 85 × 85 μm.