Spectral confocal microscopy has been used to visualize neurons with the help of silver- and gold-based cell labeling. The development enables imaging of archived tissue samples, which could aid long-term clinical research efforts and diagnostics for cancer and neurological disorders. The staining of neurons with silver began in the 1800s, and now researchers from the University of Minnesota and Agnes Scott College in Decatur, Ga., have coupled the technique with spectral confocal microscopy, which has typically been used for fluorescence imaging. Silver-impregnated dendrites from an insect motor neuron, captured through 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 561- or 640-nm wavelengths to induce plasmon resonances. The maximal emission signal was collected at a shorter wavelength (that is, at a higher energy state). Surface plasmon resonances of noble metal nanoparticles offer a superior optical signal and do not photobleach, the researchers said. Silver-impregnated preparations should retain their high image quality for a century or more, allowing for archiving. "With the prediction that superior-resolution microscopic techniques will continue to evolve, older archived samples could be reimagined with newer technologies and with the confidence that the signal in question was preserved," said Minnesota professor Karen Mesce. "The progression or stability of a cancer or other disease could therefore be charted with accuracy over long periods of time." To appreciate the enhanced image quality produced by the new technique, the team first examined a conventional bright-field image of a metal-labelled neuron within a grasshopper's abdominal ganglion, a type of mini-brain which, even at that size, presented out-of-focus structures. They then imaged the same ganglion with a spectral laser scanning confocal microscope (LSCM), adjusted to the manufacturer's traditional fluorescence settings, resulting only in strong natural fluorescence and a collective dark blur in place of the silver-labelled neurons. However, after using the spectral LSCM to collect the light energy emitted from the labels' vibrating surface plasmons, the team obtained high-quality 3D computer images of silver and gold-impregnated neurons. The team believes that by using a number of different metal-based cell-labeling techniques in combination with the LSCM protocols, tissue and cell specimens could be generated and imaged in 3D with ease. Changes in small structural details of neurons could be identified, which are often indicators of neurological disease, learning and memory, and brain development. "Both new and archived preparations are essentially permanent, and the information gathered from them increases the data available for characterizing neurons as individuals or as members of classes for comparative studies, adding to emerging neuronal banks," said Agnes Scott professor Karen Thompson. "Just as plasmon resonance can explain the continued intensity of the red (caused by silver nanoparticles) and yellow (gold nanoparticles) colors in centuries-old medieval stained glass and other works of art, metal-impregnated neurons are also likely never to fade, neither in the information they provide nor in their intrinsic beauty," Mesce said. Results were published in eLife (doi: http://dx.doi.org/10.7554/eLife.09388).