Fixation artifacts and how to minimize them

Sample preparation is the first step for having high-quality images that will impress everyone, but it is often overlooked. Many times I have tried to help others improve their microscope images, only to find out that improvement was not possible due to the quality of the sample. No matter how expensive your microscope is, you can’t achieve an ideal image without a well-prepared sample. The first step in sample preparation, if not performing live-cell imaging, is to fix your sample. There are many different methods, and the “best” depends on your samples and what your final goal may be. Permeabilization choice is also critical for sample preparation, but will not be covered.

The goal of performing fixation on your cells or tissue is to preserve the sample in as close as possible to a “life-like state.” Fixation of tissues has been commonplace for over a 100 years, with formalin being introduced in 1893 [1].  Since then, a large variety of chemicals and procedures have been developed to improve the quality of the prepared sample.  Currently, the types of fixatives can be divided into four categories: cross-linking, dehydration, heat, and acids  [2].  We are going to focus on the most common categories, cross-linking and dehydration.  

Formaldehyde is the most commonly used fixative due to its ability to preserve a wide variety of tissue components. Most microscopists will have used formaldehyde at some point in their careers. Often, formaldehyde and the protocol to use it may be passed down from person to person in the lab, without optimization for a particular sample.  Protocols may not take into consideration duration, temperature, concentration, and pH that can all affect the quality of sample fixation.  In fact, Schnell et al. demonstrated a striking difference in localization and intensity of immunofluorescence between different fixation methods and even between cell lines utilizing the same method [2].  For this reason, it is critical to test multiple fixation methods whenever you begin a new experiment.

Aldehydes

Cross-linkers are from the aldehyde family and commonly refer to two different chemicals.  These are formaldehyde, which can be in the form of formalin or paraformaldehyde (PFA), and glutaraldehyde (GA), a bifunctional aldehyde. Both chemicals fix samples by cross-linking proteins through amino groups, effectively stabilizing the structure of the cell.

Difficulties that arise with aldehydes are epitope preservation, cellular structure alteration, and poor fixation of membranes. Cross-linking of proteins by GA will cause changes in protein structure, potentially altering the epitope you are targeting with antibodies.  Briefly, an epitope is the specific part of a molecule where an antibody attaches. 

Organic solvents  

Organic solvents fix samples through a dehydration method, which precipitates proteins and extracts lipids.  The most common organic solves used for fixation of samples are methanol, ethanol, and acetone.  Methanol fixation can be performed with ice-cold methanol in just a few minutes with cultured cells and has the added benefit of permeabilization of the cell.  The other advantage is that alcohols are much safer to use in the laboratory than aldehydes.

The drawbacks of using organic solvents are that they can extract cytosolic or nuclear proteins, and the sample undergoes a round of dehydration and rehydration, which can disrupt organelles and overall cellular structure. 

Specific cellular components

Both cross-linkers and organic solvents have pros and cons, so deciding which is best for you is an experiment-specific question and is primarily driven by which cellular components are essential to answer the biological question.  

Mitochondria

Mitochondria are known to be very sensitive to fixation artifacts [3]. The mitochondrial structure is best preserved with PFA when observed with super-resolution imaging methods. Organelle shrinkage and membrane disruption occur with GA and methanol, respectively.  Full protocols have been outlined by Whelan et al. [4].

Cytoskeleton

For many years general wisdom was that methanol was a good method to fix the cytoskeleton because the structure of the cytoskeleton was less affected by precipitation [5]. Super-resolution microscopy has demonstrated that we need a more nuanced approach. Methanol works great for imaging of microtubules and intermediate filaments, such as cytokeratin and vimentin, but careful preparation with PFA and GA will also provide excellent results.  Actin labeled with phalloidin, on the other hand, is imaged best with GA fixation [6,4]. PFA works for fixation of actin if you don’t need to visualize individual actin filaments.

Nucleic acids

Any fixative is fine for visualizing the DNA in the nucleus due to the plethora of DNA. If your research involves specific sequences or structures within nucleic acids, it’s essential to treat the sample gently so as not to damage the sequence or finer features present in the sample. PFA has the ability to cross-link nucleic acids, and this feature is used for experiments using in situ hybridization  [7]. On the other hand, chromosome preparations are often done with a combination of methanol and acetic acid, utilizing the ability of alcohols to disrupt cellular membranes [8]. Due to the highly variable nature to experiments with nucleic acids, it is best to check several sources for the best method. 

Lipid droplets

Organic solvents extract lipids, so they are generally not suggested for research involving lipid droplets.  PFA and GA are both good at stabilizing proteins associated with lipid droplets. The detergent used for permeabilization is the step critical for success when studying lipid droplets and Triton-X should be avoided [9].

Membranes

Fixation artifacts are tricky to avoid with membranes because aldehydes do not cross-link lipids and organic solvents strip samples of lipids.  Samples that study membranes are, therefore, dependent on the embedded proteins stabilizing the local environment. Pereira et al. demonstrated that PFA fixation performed well for transmembrane protein structural fixation, but PFA disrupted localization of the transmembrane protein within the membrane [10]. Another study demonstrated that a combination of PFA fixation followed by methanol was the best method to preserve GFP fluorescence and membrane localization.

In conclusion, there is no perfect method for preserving a cell identical to the living state. Knowing the properties of fixatives and cellular components is an excellent method to narrow down the conditions for optimizing your experiment.  New probes and microscopy methods are quickly evolving. We may find that current methods are insufficient for new technologies.  Fixation protocols will have to keep up to avoid creating artifacts that were not visible to researchers previously. 

Heather Brown-Harding

Microscopy Facility Assistant Director

Biology Department

Wake Forest University, Winston-Salem, NC, USA

Reference list

1. Hussein, I.H., Raad, M. (2015). Once Upon a Microscopic Slide: The Story of Histology. Journal of Cytology & Histology 06: 10.4172/2157-7099.1000377

2. Schnell, U., Dijk, F., Sjollema, K.A., Giepmans, B.N.G. (2012). Immunolabeling artifacts and the need for live-cell imaging. Nature Methods 9: 152–158.

3. Schwerzmann, K., Hoppeler, H., Kayar, S.R. and Weibel, E.R. (1989). Oxidative capacity of muscle and mitochondria: correlation of physiological, biochemical, and morphometric characteristics. Proceedings of the National Academy of Sciences 86: 1583–1587.

4. Whelan, D.R., Bell, T.D.M. (2015). Image artifacts in Single Molecule Localization Microscopy: why optimization of sample preparation protocols matters. Scientific Reports 5: 7924.

5. Dardick, I., Ostrynski, V.L., Ekem, J.K., Leung, R., Burford-Mason, A.P. (1992). Immunohistochemical and ultrastructural correlates of muscle-actin expression in pleomorphic adenomas and myoepitheliomas based on comparison of formalin and methanol fixation. Virchows Archiv 421: 95–104.

6. Vielkind, U., Swierenga, S.H. (1989). A simple fixation procedure for immunofluorescent detection of different cytoskeletal components within the same cell. Histochemistry 91: 81–88.

7. Bacallao, R., Sohrab, S., Phillips, C. (2006). Guiding Principles of Specimen Preservation for Confocal Fluorescence Microscopy, in: Pawley, J.B., Handbook Of Biological Confocal Microscopy, 368–380.

8. Howe, B., Umrigar, A., Tsien, F. (2014). Chromosome Preparation From Cultured Cells. Journal of Visualized Experiments 83: 50203

9. Ohsaki, Y., Maeda, T., Fujimoto, T. (2005). Fixation and permeabilization protocol is critical for the immunolabeling of lipid droplet proteins. Histochemistry and Cell Biology 124: 445–452.

10. Pereira, P.M., Albrecht, D., Culley, S., Jacobs, C., Marsh, M., Mercer, J., Henriques, R. (2019). Fix Your Membrane Receptor Imaging: Actin Cytoskeleton and CD4 Membrane Organization Disruption by Chemical Fixation. Frontiers in Immunology 10: 675

Epitope | Definition of Epitope by Lexico [WWW Document], n.d. . Lexico Dictionaries Engl. URL https://www.lexico.com/en/definition/epitope (accessed 5.27.20).

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