Spectral confocal microscopy was used to visualize neurons using silver and gold cell labeling. The development enables imaging of archived tissue samples, which could aid long-term clinical research efforts and the diagnosis of cancer and neurological disorders.
Staining neurons with silver began in the 1800s, and now researchers at the University of Minnesota and Agnes Scott College in Decatur, Georgia, have coupled the technique with spectral confocal microscopy, which has typically was used for fluorescence imaging.
Silver-impregnated dendrites of an insect motor neuron, captured by spectral confocal microscopy. Courtesy of Grant M. Barthel, Karen A. Mesce and Karen J. Thompson.
The imaging process involves the excitation of silver or gold nanoparticles with wavelengths of 561 or 640 nm to induce plasmon resonances. The maximum emission signal was collected at a shorter wavelength (i.e., higher energy state).
Surface plasmon resonances of noble metal nanoparticles provide superior optical signal and do not photobleach, the researchers said. Silver-impregnated preparations should maintain their high image quality for a century or more, allowing for archival storage.
“With the prediction that higher resolution microscopic techniques will continue to evolve, older archived samples could be reimagined with newer technologies and with confidence that the signal in question has been preserved,” said Karen Mesce, a professor at the Minnesota. “The progression or stability of a cancer or other disease could therefore be mapped accurately over long periods of time.”
To appreciate the improved image quality produced by the new technique, the team first examined a conventional bright-field image of a metal-labeled neuron in the abdominal ganglion of a grasshopper, a type of mini- brain which, even at this size, exhibited out-of-focus structures.
They then imaged the same ganglion with a laser spectral scanning confocal microscope (LSCM), adjusted to the manufacturer’s traditional fluorescence parameters, which resulted in only strong natural fluorescence and a collective dark blur instead of neurons. marked with silver.
However, after using the spectral LSCM to collect the light energy emitted by the vibrating surface plasmons of the markers, the team obtained high-quality 3D computer images of neurons imbued with silver and gold.
The team believe that by using a number of different metal-based cell labeling techniques in combination with LSCM protocols, tissue and cell samples could be generated and 3D imaged with ease. Changes in small structural details of neurons could be identified, which are often indicators of neurological diseases, learning and memory, and brain development.
“New and archived preparations are essentially permanent, and the information gathered from them increases the data available to characterize neurons as individuals or as members of classes for comparative studies, adding to neuronal banks emerging,” said Karen Thompson, professor at Agnes Scott.
“Just as plasmon resonance can explain the continued intensity of red (caused by silver nanoparticles) and yellow (gold nanoparticles) colors in centuries-old medieval stained glass windows and other artworks, neurons imbued with metal are also likely to never fade, neither in the information they provide nor in their intrinsic beauty,” Mesce said.
The results were published in eLife (doi: http://dx.doi.org/10.7554/eLife.09388).