Correlative Light and Electron Microscopy
Figure 3: (A) Fluorescence image of vacuole shown in top right box of Figure 2. (B) TEM image of same region. (C) The fluorescence/TEM overlay image shows the alignment of the gold particles and the fluorescent signal. (D) is at higher magnification to show the gold more clearly. Scale bar in panel (C) indicates 500 nm and applies to panels (A–C). Scale bar in panel (D) indicates 250 nm.
Figure 4: (A) Fluorescence image of vacuole shown in the bottom box of Figure 2. (B) TEM image of same region. (C) The fluorescence/TEM overlay image shows the alignment of the gold particles and the fluorescent signal. (D) shows an increased magnification to show the gold more clearly. Scale bar in panel (C) indicates 500 nm and applies to panels (A–C). Scale bar in panel (D) indicates 250 nm.
As a negative control cells treated
without primary antibody showed no fluo- rescence nor any gold label (Figure 5) even on the electron dense material within the vacuolar space within the parasitophorous vacuole.
Discussion Tis protocol was developed to facili-
Figure 5: TEM images of no primary sample showing electron dense matrix material (arrow) without gold label at low magnification (A) and high magnification (B). Scale bar in (A) indicates 1 µm. Scale bar in (B) indicates 200 nm.
monoclonal antibody 20C3. Due to the disparity in the sec- tion thicknesses, 70 nm physical in TEM and 200 nm optical in fluorescence, there are areas where there is fluorescence but no gold. We observed gold in adjacent serial sections of these areas (data not shown).
22
tate CLEM immunolabeling experiments for a large population of researchers that have access to standard light microscopes and TEMs but no highly specialized CLEM hardware that has been developed by a variety of companies over the last few years. Te key to this method is the use of a dual-labeled, commercially available secondary antibody, which is conjugated to both an Alexa 488 fluorophore and 10 nm gold. Use of this dual-labeled anti- body ensures that any fluorescent signal and gold label observed is due to labeling by the same agent. Te other key compo-
nent of this method is the use of the photo-etched cover glass, which allows the researcher to find the same field of view/ROI in both fluorescence/bright-field images and TEM images, enabling a fast correlation of the two modalities. Although requiring technical expertise in en face thin sectioning and
www.microscopy-today.com • 2020 July
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