Researchers have discovered a new method of visualizing neurons that promises to benefit neuroscientists and cell biologists alike by using spectral confocal microscopy to image tissues impregnated with silver or gold.
Rather than relying on the amount of light reflecting off metal particles, this novel process involves delivering light energy to silver or gold nanoparticles deposited on neurons and imaging the higher energy levels resulting from their vibrations, known as surface plasmons.
This technique is particularly effective as the light emitted from metal particles is resistant to fading, meaning that decades-old tissue samples achieved through other processes, such as the Golgi stain method from the late 1880s, can be imaged repeatedly.
Laser Scanning Confocal Microscope
The new process was achieved by using spectral detection on a Laser Scanning Confocal Microscope (LSCM), first made available in the late 1980s and, until now, used most extensively for fluorescent imaging.
Paired with such methods, silver- and gold-based cell labeling is poised to unlock new information in a myriad of archived specimens. Furthermore, silver-impregnated preparations should retain their high image quality for a century or more, allowing for archivability that could aid in clinical research and disease-related diagnostic techniques for cancer and neurological disorders.
“For the purposes of medical diagnostics, older and newer specimens could be compared with the knowledge that signal intensity would remain fairly uniform regardless of sample age or repeated light exposure,"
says contributing author Karen Mesce from the University of Minnesota.
“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. 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 brightfield 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 the spectral 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 collecting the light energy emitted from vibrating surface plasmons in the spectral LSCM, the team obtained spectacular three-dimensional computer images of silver and gold-impregnated neurons. This holds enormous potential for stimulating a re-examination of archived preparations, including Golgi-stained and cobalt/silver-labelled nervous systems.
Additionally, by using a number of different metal-based cell-labeling techniques in combination with the new LSCM protocols, tissue and cell specimens can be generated and imaged with ease and in great three-dimensional detail.
Changes in even small structural details of neurons can be identified, which are often important 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,"
says co-first author Karen Thompson from Agnes Scott College.
“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,” adds Mesce.
Karen J Thompson et al. Plasmon resonance and the imaging of metal-impregnated neurons with the laser scanning confocal microscope eLife (2015). DOI: 10.7554/eLife.09388