Microscopy 101
Figure 5: Use of a small wedge of filter paper to wick away excess fluid and guide a TEM grid into a filter paper-lined petri dish for storage.
seconds. Te lid to the stain station was placed over the top of the station for the duration of this final staining period.
7. Te grid was removed from the drop of stain and the excess stain blotted away using a small wedge of filter paper touched to the edge of the grid. Te entire procedure, from step 1 to step 7, usually takes less than three minutes.
8. Te small wedge of filter paper was then inserted into the opening between the tines of the forceps, just above the grid, to wick up excess fluid and to gently guide the grid from the forceps into the filter paper-lined plastic petri dish (Figure 5). Without this step, capillary action will draw the grid up onto the forceps when the forceps are opened.
Te grid was allowed to dry and was stored away from light
until ready to be viewed with a Termo Fisher Scientific (Phil- ips/FEI) T-12 or TF20 (for LCAT-incubated particles), TEM operated at 100 kV and 200 kV, respectively. Class averages and three-dimensional reconstructions were accomplished using the RELION-3.0 soſtware package [8]. RELION is an open-source soſtware program for performing single-particle analysis.
Figure 4: Depictions of filter paper orientations with respect to blotting TEM grids. A) Orientation of the small wedge of filter paper (Whatman 90 mm) and TEM grid as the filter paper is briefly touched to the edge of a TEM grid to blot away excess fluid. B) Holding the large wedge of filter paper (VWR 18.5 cm) perpendicu- lar to the pipette tip box rack for quickly touching the edge of a TEM grid to the fil- ter paper between the two water washes and after applying the first drop of stain.
drops of water and stain so that the edge of the grid could be rapidly but gently touched to the filter paper for each water wash and first drop of stain (Figure 4B). A thin film of water should remain on the surface of the grid to prevent the sam- ple from drying out.
4. Te grid was then quickly touched to the surface of the second drop of ddH2
the grid quickly touched to the large wedge of filter paper to wick away excess ddH2
O (carbon-side-down) and the edge of O.
5. Following washing, the grid was quickly touched to the sur- face of the first drop of 0.7% UF stain (carbon-side-down), and then the edge of the grid was quickly touched to the large wedge of filter paper.
6. Te grid was then floated on top of the second drop of 0.7% UF stain (carbon-side-down) and allowed to set for 45–60
56
Results Compared to our previous staining method (Figure 6A)
using phosphotungstic acid (PTA), we saw greater surface detail and sharper particle edges on HDL preparations with our new uranyl formate procedure (Figure 6B). Size quantita- tion analyses of mean particle diameters were similar between the two staining methods, suggesting that our modified stain- ing procedure does not dramatically alter particle morphology (Figures 6C and 6D). Te increased surface detail and more defined particle
edges from our modified staining procedure allowed us to perform single-particle analysis on HDL particles using the RELION-3.0 soſtware package [8]. Single-particle analysis is the process by which hundreds to thousands of individual par- ticles are chosen from TEM micrographs, image averaged into two-dimensional class averages that represent unique views of the particle, and then arranged in unique orientations in space to produce a three-dimensional reconstruction of the particle. For a plasma HDL particle preparation, images of the indi- vidual chosen particles were extracted from the micrographs. Two-dimensional class averaging was then performed on the extracted particle images. Te class averages generated from
www.microscopy-today.com • 2020 September
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