Microscopy101
Strategies to Effi ciently Locate Cultured Cells of Interest on Transmission Electron Microscopy Grids
Linda White , * Jeffrey Prince , and Kathryn Tosney Biology Department , University of Miami , Cox Science Center , 1301 Memorial Dr. , Coral Gables , FL 33146
*
lwhite@miami.edu Introduction
In transmission electron microscopy (TEM), fi nding a cell of interest can be time-consuming and, given the high cost of beam time, expensive. T e diffi culty is exacerbated if each grid has only one cell of interest. An example might be when a particular cell has been recorded with time-lapse microscopy before and during an experimental treatment that culminated in fi xation. T e sample may even have been subjected to post-fi xation immuno-TEM procedures, increasing the importance of locating the cell. Here we report a strategy for fi nding our targets expeditiously.
Materials and Methods Grids . To assure the most effi cient use of beam time, we have developed a method of culturing cells on special formvar- coated electron microscopy (TEM) grids and pre-examining them with light microscopy. It is crucial to use gold grids when seeding live cells (copper and nickel grids are toxic to cells) and to have some asymmetry that is visible both in EM and light microscopy. Grids are available in which each square of the grid is numbered, but we eschewed these because we thought that the numbers occluded too much of the fi eld of view. Instead, we use grids in which the center is marked with an “ L ” shape (PELCO 50 mesh gold grids from Ted Pella, Inc.; #1GG-50; Redding, CA). Grid preparation . To prepare the grids, they are fi rst coated with 0.5% formvar (Electron Microscopy Sciences, Hatfi eld, PA) by inverting a cluster of 2–3 grids onto an island of formvar that is fl oating on sterilized water [ 1 ]. Each island of formvar with grids attached is then picked up upside-down with an acid-washed 22 × 22 mm coverslip (Gold Seal #3406) such that the formvar-coated grids are facing upward ( Figure 1A ). T e coverslips, draped with the formvar-coated grids are allowed to air dry overnight in a dust-free environment. T irty-fi ve millimeter poly- styrene Petri dishes (BD Falcon #351008) are prepared by punching a 14 mm hole in the bottom with a punch-and-die press. Each grid/formvar/coverslip assembly is attached, formvar-side up, using a non- toxic medical silicone adhesive (#A-100, Factor II, Inc., Lakeside, AZ; Figure 1B ).
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T e adhesive is allowed to cure for at least 24 hours at room temperature. T e hole in the plastic Petri dish with the glass coverslip glued to the bottom creates a well in the dish, which holds about 0.2 ml. To prepare the dish for cell culture, the insides of the assembled Petri dishes are sterilized for 15 minutes in a biosafety cabinet equipped with an ultraviolet germicidal light, and a suitable substrate is applied for cell survival. Because we examine the cell biology of neuronal growth cone motility, for our purposes we fi rst coat the sterile dishes containing formvar-coated grids with 0.2 ml of sterile 0.01% poly-L-ornithine for 1 hour at room temperature (Sigma, St. Louis, MO), gently rinse with water, coat with 20 μ g of laminin (Invitrogen/ Life Technologies, Grand Island, NY) in 0.2 ml sterile phosphate-buff ered saline for 1 hour at room temperature, and fi nally gently wash with three changes of sterile phosphate-buff ered saline. T e last rinse is leſt on the dishes so that the laminin will not dry out. A sterile transfer pipet or sterile pipet tip is used rather than using a vacuum device to gently remove the rinses because the force of the suction can easily remove the formvar fi lm. T ese assemblies could potentially be used for any cell type and are also suitable for time-lapse microscopy using an inverted light microscope or for correlative light and electron microscopy. For our studies, we remove the last saline rinse and seed a triturated suspension of chicken embryo dorsal root ganglion neurons in cell culture medium into the center of the coverslip and allow the cells to attach overnight. T e cells on the grids are fi xed while attached to the coverslips in the Petri dishes, then they are removed for processing, either for TEM or for
Figure 1 : (A) A sketch of the side view of a Petri dish/coverslip/formvar-coated grid assembly. Medical adhesive is placed around the bottom edge of the hole punched in a 35 mm plastic Petri dish. Then a coverslip containing formvar-coated grids is glued grid side up over the hole, creating a small well in the center of the dish containing the formvar-coated grids. The Petri dishes are stackable and have a small outer rim around the bottom so that the Petri dish does not rest on the coverslip. (B) A photograph of a completed dish assembly.
doi: 10.1017/S1551929515000541
www.microscopy-today.com • 2015 July
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