High-resolution fluorescence microscopy of myelin without exogenous probes
Pia Crone Christensen1, Craig Brideau1, Kelvin W C Poon1
1Department of Clinical Neurosciences, Hotchkiss Brain Institute, University of Calgary, Alberta, Canada.
Insights
We developed a fast, inexpensive method to visualize myelin using glutaraldehyde fixation and its natural autofluorescence. This technique provides detailed myelin morphology images and can be combined with immunohistochemistry for co-localization studies.
Area of Science:
- Neuroscience
- Biochemistry
- Microscopy
Background:
- Myelin is crucial for nerve impulse conduction in vertebrates.
- Myelin's lipid-rich structure complicates traditional staining and imaging methods.
- Disturbances in myelin integrity impair axonal function.
Purpose of the Study:
- To develop a reliable and efficient method for visualizing myelin structure.
- To overcome challenges associated with myelin staining and immunoprobing.
- To enable high-resolution imaging of myelin morphology in nervous system tissues.
Main Methods:
- Utilized glutaraldehyde fixation to induce strong, broad-spectrum myelin autofluorescence.
- Employed spectral confocal microscopy to capture high-resolution 3D images (400-750 nm).
- Applied mathematical post-processing to separate autofluorescence from exogenous probes and enable immunohistochemistry.
Main Results:
- Demonstrated a fast, inexpensive, and reliable method for myelin visualization.
- Achieved detailed imaging of myelin morphology leveraging intrinsic autofluorescence.
- Successfully co-localized myelin morphology with specific proteins using immunohistochemistry.
- Showcased the technique's utility in a lysolecithin model of de- and remyelination.
Conclusions:
- Glutaraldehyde-induced myelin autofluorescence is a valuable tool for high-resolution imaging.
- This method offers a practical solution for studying myelin structure and pathology.
- The technique facilitates detailed morphological analysis and protein co-localization in myelinated tissues.
Abstract:
Myelin is a critical element of the central and peripheral nervous systems of all higher vertebrates. Any disturbance in the integrity of the myelin sheath interferes with the axon's ability to conduct action potentials. Thus, the study of myelin structure and biochemistry is critically important. Accurate and even staining of myelin is often difficult because of its lipid-rich nature and multiple tight membrane wraps, hindering penetration of immunoprobes. Here we show a method of visualizing myelin that is fast, inexpensive and reliable using the cross-linking fixative glutaraldehyde that produces strong, broad-spectrum auto-fluorescence in fixed tissue. Traditionally, effort is generally aimed at eliminating this auto-fluorescence. However, we show that this intrinsic signal, which is very photostable and particularly strong in glutaraldehyde-fixed myelin, can be exploited to visualize this structure to produce very detailed images of myelin morphology. We imaged fixed rodent tissues from the central and peripheral nervous systems using spectral confocal microscopy to acquire high-resolution 3-dimensional images spanning the visual range of wavelengths (400-750 nm). Mathematical post-processing allows accurate and unequivocal separation of broadband auto-fluorescence from exogenous fluorescent probes such as DAPI and fluorescently-tagged secondary antibodies. We additionally show the feasibility of immunohistochemistry with antigen retrieval, which allows co-localization of proteins of interest together with detailed myelin morphology. The lysolecithin model of de- and remyelination is shown as an example of a practical application of this technique, which can be routinely applied when high-resolution microscopy of central or peripheral myelinated tracts is required.
