Cell fixation is a foundational process in biology that preserves cells and tissues for observation by maintaining their structure and biochemical makeup. The procedure acts as a biological snapshot, halting cellular processes to lock a sample in a state that resembles its living condition. This preservation is a preparatory step for analysis, especially for viewing samples under a microscope.
The Purpose of Cell Fixation
Once a cell or tissue is removed from its native environment, it begins to break down through two processes: autolysis, where the cell’s own enzymes digest its components, and putrefaction, the breakdown by microorganisms. Fixation’s primary objective is to stop these destructive processes.
Beyond preventing decay, fixation stabilizes the cell’s internal and external structures. The goal is to preserve morphology, which includes the shape and size of the cell and its organelles. This ensures the spatial relationships between components are maintained as they were in the living state, allowing for the study of a static, representative image of cellular life.
This stabilization also prepares the sample for subsequent treatments. A fixed cell is more robust and can withstand procedures like dehydration and embedding in wax. It also renders the cell permeable to various stains and dyes, which are necessary to make specific features like proteins or DNA visible for microscopic examination.
Chemical Fixation Methods
The most common approach to cell preservation involves chemical fixatives, categorized by their mechanism. One group is cross-linking fixatives, such as formaldehyde and glutaraldehyde. These chemicals form covalent bonds between proteins and other molecules, creating an interconnected web that locks all cellular components into their positions.
This method preserves the fine structural details of a cell, known as its ultrastructure. The bonds create a stable matrix that withstands further processing with minimal distortion. Formaldehyde penetrates tissues quickly, while glutaraldehyde forms stronger cross-links, making it a choice for high-resolution electron microscopy.
Another category is precipitating, or dehydrating, fixatives, which includes alcohols like ethanol and methanol, as well as acetone. These chemicals remove water from cells, causing proteins to denature and precipitate. This coagulation immobilizes the proteins and other cellular constituents.
While precipitating fixatives can cause some alteration of cellular structures, they are better at preserving the chemical reactivity of molecules known as antigens. This makes them useful for techniques like immunohistochemistry, where antibodies detect and label specific proteins.
Physical Fixation Methods
Physical techniques can also fix biological samples. Heat fixation is a simple method used for preparing smears of microorganisms like bacteria on a microscope slide. The process involves passing the slide briefly through a flame, which kills the organisms and adheres them to the glass.
This technique is effective for attachment and killing, but has drawbacks. The heat alters and distorts the internal structures of the cells, providing a poor representation of their living state. Its application is limited to basic bacteriological studies for observing the shape, size, and arrangement of whole cells.
A more sophisticated physical method is cryofixation, which involves freezing the sample at extremely high speeds. This can be done by plunging the tissue into a cryogen like liquid nitrogen or impacting it against a copper block cooled to very low temperatures. The rapid freezing turns cellular water into a glass-like, non-crystalline solid, a process known as vitrification.
The advantage of cryofixation is its speed. By stopping all biological processes nearly instantaneously, it provides the most accurate preservation of cellular structures possible. This makes it a useful technique for high-resolution imaging methods like electron microscopy.
Choosing the Right Fixation Technique
Selecting the appropriate fixation method depends on the specific goals of the scientific inquiry. The choice is guided by the biological component being studied and the analytical technique that will be used.
For instance, visualizing the fine ultrastructure of organelles requires a method that offers excellent structural preservation, such as using a cross-linking fixative. Conversely, if the objective is to detect a specific protein with antibodies, a precipitating fixative might be preferred to avoid altering the protein’s shape in a way that prevents antibody binding.
The planned analysis is also a guiding factor. Basic light microscopy of a bacterial smear may only require simple heat fixation. In contrast, sensitive applications like immunofluorescence or high-resolution electron microscopy demand methods that preserve both molecular reactivity and structural integrity at the nanoscale.
Every fixation method introduces some level of change to the cell, known as artifacts. An artifact is any distortion in the final image that was not present in the living specimen. Understanding and minimizing these artifacts is a consideration for researchers to ensure the data accurately reflects the cell’s true biology.