McAudi Microscope Sample Preparation: Due to the multiple centrifugation steps that can lead to cell loss, it is recommended to attach cells directly to a slide or plastic sheet for optical microscopy. This ensures that the cells are dehydrated and dried along with the support material. The thickness of the slides used in optical microscopy plays an important role in maintaining sample integrity during the process.
To facilitate cell adhesion, a thin film is typically applied to the slide or plastic surface. Here are three common methods:
(1) The simplest method involves using a polyethylene formaldehyde film (formvar). Cleaned slides are immersed in a 0.5–1% polyethylene drenched formalin solution in chloroform. After evaporation, a yellow film forms on the slide. By gently shaking the slide, the film can be evenly distributed and used for cell attachment.
(2) Another effective technique is to use Poly-L-lysine. A 0.1 mL drop of 1 mg/mL Poly-L-lysine dissolved in filtered water is placed on a cleaned slide and spread at 45°C to dry. This method is preferred because Poly-L-lysine has a positive charge, which helps negatively charged cells adhere more effectively to the surface.
(3) For the McAudi microscope, a glycerin-protein solution is prepared by mixing 30–40% plasma with 3–55% glycerin and distilled water. This mixture is spread on a clean glass slide and allowed to dry. The resulting protein membrane is then immersed in 2% glutaraldehyde to fix the proteins. After washing and drying, the membrane is activated by adding plasma 30 minutes before use, followed by a buffer rinse for direct application.
Once the cells are fixed and suspended in filtered water, they are placed onto the film-coated slide or plastic sheet. The slides are then kept in a humid, low-temperature environment (around 4°C) for 30–120 minutes to allow proper cell adhesion. Afterward, the slides are gently shaken with fine forceps and rinsed with double-distilled water to remove unattached cells.
The next step involves dehydration. Slides are placed in a vessel containing 50% acetone (or ethanol). Over time, the concentration of the dehydrating agent is gradually increased every 3 minutes. It’s more convenient to remove half the liquid and replace it with 100% dehydrating agent. After 5–6 cycles, the concentration should exceed 0.5%, ensuring complete cell dehydration.
After critical point drying, the slide or plastic sheet is attached to the sample stub using conductive adhesive, followed by metal coating. The third method also allows for easy cell attachment to the membrane. Cells fixed with low-concentration glutaraldehyde can be suspended on the "activated" protein film. The slide is incubated in 2% glutaraldehyde for 10 minutes, then washed with filtered double-distilled water and processed as described for dehydration and metal coating.
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