Cell fixation is a fundamental laboratory process employed to prepare biological specimens, such as cells and tissues, for detailed examination. It involves treating samples to preserve their intricate structures as close to their living state as possible. This preparatory step is crucial for various scientific analyses, allowing researchers to study cellular components without degradation. The process ensures that the spatial relationships within cells remain intact, which is essential for accurate observation.
The Purpose of Fixation
Studying living cells directly presents challenges because biological processes continue rapidly after a sample is removed from its living environment. Cells can quickly undergo autolysis, a self-digestion process where enzymes break down cellular components. Without intervention, tissues and cells also face putrefaction from microbial contamination, leading to a loss of structural integrity.
Fixation halts these destructive processes by rapidly inactivating enzymes and inhibiting microbial growth. It “freezes” the biological material in time, stabilizing cellular and subcellular structures. This stabilization allows researchers to handle, process, and section the samples for microscopic examination or other analytical techniques without alteration or decay. The preserved state ensures observations accurately reflect the original cellular morphology.
Fixation also strengthens the sample, making it more resistant to the physical and chemical stresses of subsequent laboratory procedures. These procedures often include dehydration, embedding, and sectioning, which could otherwise distort or destroy delicate cellular architecture. By rigidifying the cell structure, fixation ensures the sample remains intact throughout the preparation workflow.
The Science Behind Fixation
Fixation works by altering proteins within the cells and tissues. One mechanism involves cross-linking, where fixative molecules form stable covalent bonds between protein molecules. This creates a network that rigidifies the cell’s internal framework, anchoring soluble proteins to the cytoskeleton and lending mechanical strength.
Denaturation is another mechanism, employed by some chemical fixatives. These agents remove water and disrupt protein structure, causing precipitation. This change stabilizes cellular components and prevents enzymatic activity. Fixative selection influences how cellular components are preserved and their accessibility for subsequent analytical techniques.
Both cross-linking and denaturation prevent cellular component breakdown and maintain morphology. The goal is to stabilize proteins and nucleic acids, ensuring they remain in their original locations and forms. This molecular stabilization allows scientists to visualize cellular structures that would otherwise degrade rapidly.
Approaches to Cell Fixation
Cell fixation employs two strategies: chemical and physical. The choice between these methods depends on the research question, the type of sample, and the downstream applications. Each utilizes distinct mechanisms for cellular preservation.
Chemical fixation involves immersing the sample in a solution containing chemical fixatives. Formaldehyde, used as 10% neutral buffered formalin or 4% paraformaldehyde, is a common chemical fixative. It works by forming methylene bridges (cross-links) between protein molecules, by reacting with amino groups. Formaldehyde penetrates tissues effectively and preserves general tissue architecture, making it suitable for many applications.
Glutaraldehyde is another aldehyde fixative, known for stronger cross-linking. Its larger molecular size allows thorough preservation of cellular ultrastructure (e.g., organelles and membrane details), making it useful for electron microscopy. However, its strong cross-linking can mask specific molecular targets, sometimes necessitating additional steps to unmask them for antibody-based detection.
Organic solvents like ethanol and methanol are another class of chemical fixatives. These agents work by dehydrating cells and denaturing proteins, causing precipitation. While they fix cells quickly and are useful for preserving nucleic acids and certain antigens, they may lead to cellular shrinkage and lipid extraction compared to aldehyde fixatives. Methanol is used for blood films and 95% ethanol for cytology smears.
Physical fixation preserves samples by rapidly altering their physical state, typically through extreme temperatures. Heat fixation, applied to bacterial smears, involves quickly passing a sample over a flame to dry and adhere cells to a slide, denaturing proteins. Cryo-fixation (freezing) involves rapidly cooling samples to very low temperatures, using liquid nitrogen. This method minimizes chemical alterations and preserves delicate structures and antigenicity, though it risks ice crystal formation that could damage cellular architecture.
Where Fixation is Used
Cell fixation is a foundational step across scientific and medical disciplines, enabling detailed structural and molecular analysis. Its broad applicability stems from its ability to stabilize cells, making them amenable to downstream techniques.
In histology and pathology, fixation is important for preparing tissue samples for microscopic examination. Pathologists rely on fixed tissues to diagnose diseases by observing cellular and tissue morphology under a microscope. Fixed tissues can be thinly sliced and stained to highlight specific features, allowing identification of abnormalities.
Fixation is also used for microscopy techniques, including light and electron microscopy. For light microscopy, fixed samples allow visualization of cellular components and overall tissue architecture. Electron microscopy, which requires fine structural preservation, utilizes specific fixatives like glutaraldehyde to maintain the integrity of subcellular organelles.
Immunohistochemistry (IHC) and immunofluorescence (IF) depend on proper fixation to preserve antigens within cells and tissues. These techniques use antibodies to detect specific proteins, and fixation ensures target proteins remain in their native locations and maintain their ability to bind antibodies. While some fixatives can mask antigens, appropriate fixation protocols balance structural preservation with antigen accessibility.
In flow cytometry, cells are fixed to stabilize them for analysis of surface and intracellular markers. Fixation in flow cytometry allows safe handling of potentially biohazardous samples and extends the time samples can be stored before analysis, though some fluorescent dyes may be affected. This stabilization is important for consistent and accurate measurement of cellular properties.