“Fixing cells” in biology refers to a fundamental laboratory technique used to preserve biological samples, such as cells and tissues. This process stabilizes cellular components, preventing degradation and preparing them for detailed study under various microscopic techniques. Fixation halts biological activity and maintains the structural integrity of the sample, allowing researchers to examine its intricate details without immediate decay.
Why Cells Are Fixed
The primary purpose of cell fixation is to prevent decomposition and maintain cellular and tissue architecture. Once removed from a living organism, cells and tissues degrade rapidly through processes like autolysis (self-digestion by enzymes) and putrefaction (caused by bacterial action). Fixation immediately terminates these destructive biochemical reactions and enzymatic activity, preserving the sample’s integrity. It also stabilizes cellular structures, maintaining the spatial relationships of components for accurate observation. Furthermore, fixation prepares cells for subsequent laboratory procedures like sectioning, staining, and mounting, which would otherwise damage unfixed material.
Common Fixation Methods
Cell fixation can be broadly categorized into chemical and physical methods. Chemical fixation involves immersing the sample in a solution. Aldehyde fixatives, such as formaldehyde and glutaraldehyde, form covalent cross-links between proteins, creating a stable, insoluble network that locks cellular components in place. Formaldehyde is common for general histology, while glutaraldehyde is favored for electron microscopy due to its ability to preserve fine ultrastructure.
Coagulating fixatives, including alcohols like ethanol and methanol, and acetone, denature and precipitate proteins. This effectively dehydrates cells and causes proteins to aggregate into a more solid structure. While effective for applications like blood smears or nucleic acid studies, they can cause more shrinkage and hardening of tissues compared to aldehyde fixatives.
Physical fixation, particularly cryofixation, involves rapidly freezing the biological material. This method immobilizes cellular components by bringing them to cryogenic temperatures very quickly, ideally without damaging ice crystals. Cryofixation is useful for preserving delicate structures and for high-resolution techniques like electron microscopy, as it stops all cellular processes almost instantaneously and minimizes chemical alterations.
What Happens to Cells During Fixation
During chemical fixation, several molecular and structural changes occur. One primary effect is the denaturation and cross-linking of proteins. Aldehyde fixatives react with amino groups on proteins, forming stable chemical bonds that link adjacent protein molecules. This stabilizes protein structures and prevents their degradation or movement.
Enzyme inactivation is another outcome of fixation. By altering protein structures, fixatives stop the activity of enzymes that would otherwise break down cellular components, preventing autolysis. Some fixatives, particularly organic solvents, also cause membrane permeabilization. They dissolve membrane lipids, making cell membranes porous and allowing larger molecules, such as antibodies or dyes, to access intracellular structures during subsequent staining.
While fixation aims to preserve cells, it can introduce morphological changes. Cells may experience slight shrinkage or swelling, and their overall shape can be subtly altered depending on the specific fixative and duration. Although fixatives are chosen to minimize these artifacts, understanding these potential changes is important for accurate interpretation of microscopy results.
Where Fixed Cells Are Used
Fixed cells are used across numerous scientific and medical disciplines. Microscopy, including light and electron microscopy, heavily relies on fixed cells to visualize cellular and subcellular structures. Fixation ensures the delicate internal organization of cells remains stable for high-resolution imaging.
In histology and pathology, fixed tissues are routinely processed to create thin sections that are then stained and examined to diagnose diseases and study tissue architecture. Fixed cells are also essential for immunohistochemistry and immunofluorescence, techniques that use antibodies to detect specific proteins or antigens. Fixation preserves these antigens, allowing them to be recognized by antibodies.
Additionally, fixed cells are utilized in flow cytometry, a technique that analyzes the physical and chemical characteristics of cell populations. Fixing cells for flow cytometry stabilizes them, making it possible to store samples and analyze them later without significant degradation. The choice of fixation method often depends on the specific cellular component or process being studied and the subsequent analytical technique.