Plasmon resonance and the imaging of metal-impregnated neurons with the laser scanning confocal microscope

The staining of neurons with silver began in the 1800s, but until now the great resolving power of the laser scanning confocal microscope has not been utilized to capture the in-focus and three-dimensional cytoarchitecture of metal-impregnated cells. Here, we demonstrate how spectral confocal microscopy, typically reserved for fluorescent imaging, can be used to visualize metal-labeled tissues. This imaging does not involve the reflectance of metal particles, but rather the excitation of silver (or gold) nanoparticles and their putative surface plasmon resonance. To induce such resonance, silver or gold particles were excited with visible-wavelength laser lines (561 or 640 nm), and the maximal emission signal was collected at a shorter wavelength (i.e., higher energy state). Because the surface plasmon resonances of noble metal nanoparticles offer a superior optical signal and do not photobleach, our novel protocol holds enormous promise of a rebirth and further development of silver- and gold-based cell labeling protocols. DOI: http://dx.doi.org/10.7554/eLife.09388.001
eLife digest A fresh slice of brain tissue has a fairly uniform appearance, even when viewed under a microscope. To study the neurons and other cells in the brain, scientists must therefore first prepare tissue samples using methods that make it easier to see certain kinds of cells, or particular features of them. One method that has been available for over a century is to use metal particles to stain some of the cells. For example, when the Spanish anatomist Santiago Ramón y Cajal investigated how brain cells – or neurons – are organized in the brain in the late 1880s, he made the cells visible by staining them with silver. This silver staining technique, called the Golgi method, bears the name of Camillo Golgi who first discovered it. Both Golgi and Ramón y Cajal are considered to be the pioneers of neuroscience, and shared the Nobel Prize in Physiology or Medicine in 1906. Over the years, silver staining was superseded by the use of fluorescent probes. Light travels in the form of waves, and different colors of light have different wavelengths (the distance between the peaks of the wave). Shining light of one specific color onto a fluorescent probe causes it to emit light of a longer wavelength. By detecting this emitted light, it is possible to visualize structures that contain the probes. In the late 1980s, the invention of the laser-scanning confocal microscope allowed highly detailed three-dimensional reconstructions of individual neurons to be obtained using these fluorescent labels. Unfortunately, the lifespan of fluorescent probes is limited by the fact that their fluorescence decreases with repeated use, in a process called photobleaching. Traditional silver stains avoid this problem, but standard confocal microscopy cannot obtain good images from metal-stained cells. Now, Thompson, Harley et al. have overcome this problem by using the confocal microscope in a new way to detect emitted light with shorter wavelengths than the light that was initially absorbed (rather than the longer wavelength light normally detected). This protocol produced highly detailed three-dimensional images of individual metal-stained neurons that had been impregnated with silver or gold particles. The short wavelength light is thought to result from the activity of free electrons called plasmons that are present on the surface of small metal particles (nanoparticles) that are about one millionth of a centimeter in size. When plasmons absorb radiation of a specific wavelength, they vibrate rapidly and emit their excess energy in the form of light. Medieval craftsmen unknowingly exploited this same phenomenon when they added silver and gold particles to molten stained glass, producing windows with vivid red and yellow colors that are still vibrant today. A return to metal-based staining of brain tissue could produce similar longevity for today’s tissue samples. Equally, the procedure developed by Thompson, Harley et al. opens up the possibility of revisiting archived material with the tools of modern confocal microscopy. DOI: http://dx.doi.org/10.7554/eLife.09388.002