Correlative Light and Electron Microscopy
in 0.1 M sodium cacodylate buffer for 30 minutes, rinsed with 0.1 M sodium cacodylate buffer, post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate for 30 minutes, rinsed with buffer, and then rinsed with water. Cells were then en bloc stained with 2% uranyl acetate (aqueous), dehydrated through a graded series of ethanol (30, 50, 70, 95, 100, 100%), followed by propylene oxide and infiltrated with LX112 epoxy resin (Ladd Research Industries, Burlington, VT). Te regions of interest (ROIs) imaged with fluorescence were located using a light microscope, and BEEM capsules were inverted over these areas. Samples were polymerized at 60°C for 60 hours, and blocks were popped off the cover slip. Te general region of interest (ROI) was cut out and remounted on a flat BEEM capsule for sectioning. Trimming to the specific ROI was done using a Trimtool 45° (Diatome), and blocks were then serial thin sectioned (70 nm) en face on a Leica UC7 ultramicrotome using a Diatome Ultra 35° knife. Sections were collected on formvar-coated slot grids and stained with uranyl acetate and lead citrate. Imaging was performed on a JEOL 1200EX TEM, and micrographs were recorded using Kodak 4489 film.
Results Cells were grown on photo-etched, gridded cover slips
with alpha-numeric coordinates to facilitate relocating the same cells of interest within a grid. T. gondii cells selected for fluorescent imaging contained toxoplasma vacuoles that were well labeled for MAG2 protein located in the matrix of the toxoplasma cysts [5]. Fluorescence and bright-field (phase con- trast) images were acquired on a standard epi-fluorescence microscope. First, images were acquired at low magnification (20× objective) to show an overview of the area of interest, including a number and a letter (see Figure 1A, overlay of bright- field and fluorescent image acquired with a 20× objective with the number 8 and letter M of the etched grid clearly visible). Ten, a smaller area of that field of view was imaged again as a z-stack in fluorescence and brightfield (phase contrast) using a 60× objective (this subsection is indicated as the black square in Figure 1A). Subsequently, the cells were fixed with
glutaraldehyde, post-fixed with osmium tetroxide, processed for electron micros- copy using a standard protocol, and seri- ally thin-sectioned. Due to the presence of the alpha-numeric grid, we were able to relocate the same cells by using the cor- responding 20× magnification fluores- cent/bright-field images and the gaps leſt in the sections from the etching on the cover slips. Tis is demonstrated in Figure 1B, which shows an early serial section of the fluorescent/bright-field sample shown in 1A. Arrow heads in A and B indicate the location of the corner of the etched
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grid and the bottom of the etched number 8. Once the general area of interest had been identified, we were able to find the cells in subsequent sections in the TEM that corresponded to the cells present in the 20× magnification fluorescent/bright- field image (Figure 1C). Aſter high-resolution images of cells were acquired on the
TEM, we generated overlays of the 60× magnification fluores- cence/bright-field images and the TEM images of the same cells (Figure 2). Te morphology of the parasites is clearly vis- ible in the bright-field image (Figure 2A) as well as the TEM image (Figure 2C). To align the images, we stacked the images in Adobe Photoshop and used the borders of the parasites in brightfield and TEM as a guide for alignment. Te fluorescence image (Figure 2B) was passively aligned along with the bright- field image. In addition, we also had to move through the z-dimension of the fluorescence/bright-field z-stack using NIH ImageJ to select a single slice fluorescence/bright-field image that showed the best alignment of the morphologies with the TEM image. In order to show the gold labeling more clearly, we selected
two areas in Figure 2 (see box for areas of selection) and show these at higher magnification in Figures 3 and 4. Specific gold label could be seen on structures between the parasites. Tere was no gold outside of the cells and very little within the parasites. Te gold label and the fluorescent label correlate well, allowing us to conclude that the electron-dense material between the parasites is the MAG2 protein that is labeled by
Figure 1: Images required for correlation of light and electron microscopy. Phase/fluorescence overlay (A) showing the markings on the etched cover slips and the location of the cell of interest (arrow). Boxed area is shown in (B) as a TEM image; arrowheads indicate alignment of cover glass etching in first serial section. Low-magnification TEM image (C) of the cells, including cell of interest (arrow) in a subsequent serial thin section. Scale bar indicates 20 µm.
Figure 2: Phase (A), fluorescence (B), and TEM (C) images of the same cell containing multiple parasi- tophorous vacuoles of Toxoplasma gondii. Boxes show areas of interest further detailed in Figures 3 and 4. Scale bar shown in (C) indicates 2 µm and applies to all panels.
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