In scientific research, observing cells is fundamental to understanding biological processes. However, living cells are dynamic and prone to degradation once removed from their natural environment. To overcome this challenge, scientists employ cell fixation, a technique that preserves cells and tissues in a stable state for detailed examination. This process allows researchers to study cellular structures and components as they existed at a specific moment in time.
What are Fixed Cells?
Fixed cells are biological samples that have undergone chemical or physical treatment to preserve their structural integrity and molecular components. This preservation halts all biological activity, creating a “snapshot” of their state at the moment of fixation. Without fixation, cellular components like proteins and membranes would rapidly degrade due to enzymatic activity and environmental factors, making meaningful analysis impossible.
Why Fix Cells?
Fixing cells is a routine laboratory procedure serving multiple purposes across scientific disciplines like cell biology, histology, and pathology. A primary reason for fixation is to prevent autolysis, the self-digestion of cells by their own enzymes, and putrefaction, decomposition by microorganisms. By stopping these processes, fixation ensures cellular morphology remains intact and as close to its living state as possible.
This preservation is important for analytical techniques like microscopy, which require clear, stable cellular structures for observation. For instance, in immunofluorescence, fixation immobilizes biological structures and makes them accessible to antibodies used to label specific proteins. In flow cytometry, fixation allows sample storage for later analysis, offers disinfection for infectious samples, and enables the study of intracellular targets like cytokines or nuclear factors by allowing antibody penetration.
How Cells Are Fixed
Cell fixation primarily involves treating biological samples with chemical agents that stabilize cellular components. These chemical fixatives can be broadly categorized into two main types: cross-linking fixatives and precipitating fixatives. Each type works through a distinct mechanism to achieve preservation.
Cross-linking fixatives, such as formaldehyde and glutaraldehyde, operate by forming chemical bonds between proteins and other macromolecules within the cell. Formaldehyde forms methylene bridges by reacting with amino groups of proteins. This process creates a stable network that locks cellular structures in place, effectively “freezing” the cell’s internal arrangement. Glutaraldehyde is another common cross-linking agent, known for its stronger cross-linking capabilities, making it particularly useful for electron microscopy.
Precipitating fixatives, including alcohols like ethanol and methanol, and acetone, work differently. These agents denature proteins by disrupting their three-dimensional structure and removing water from the cells. This denaturation causes proteins to coagulate and precipitate, forming a permeable meshwork that preserves the cellular architecture. While effective, these fixatives can cause more shrinkage and alterations to cell morphology compared to cross-linking agents.
Impact and Considerations of Fixation
While fixation is an important technique for preserving cells, it is a complex physico-chemical process that alters the sample from its living state. Fixation stops all metabolic processes and enzymatic activity, rendering the cells biologically inert. This cessation of life allows for long-term storage and detailed examination without degradation.
However, the chemical reactions involved in fixation can lead to trade-offs and effects on cellular components. For instance, cross-linking fixatives can change protein conformation, reducing antibody binding to specific targets, an issue known as reduced antigenicity. This is relevant for immunohistochemistry and immunofluorescence, where specific protein recognition is essential. Some fixatives, especially aldehydes, can also induce autofluorescence, the natural emission of light by the fixed sample, interfering with fluorescent imaging techniques.
Fixation can also impact cellular dimensions, causing a slight reduction in cell length. This change can be influenced by various steps in the fixation protocol, including wash steps. The choice of fixative also influences the preservation of specific subcellular structures and protein localization. For example, while formaldehyde-based fixatives preserve cellular dimensions well over several days, methanol can lead to a decrease in cell length and cause cell lysis. Researchers must consider these impacts and optimize fixation protocols based on the specific cell type and downstream analytical methods to minimize artifacts and ensure accurate observations.