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

Karen J Thompson,Cynthia M Harley,Karen A Mesce,Mark A Sanders,Grant M Barthel

doi:10.7554/elife.09388

Abstract

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.

Highlights

In the late 1880s, Santiago Ramon y Cajal began characterizing the cytoarchitecture of different types of brain neurons in a variety of animal species by employing the Golgi stain or ‘black reaction’ method
Fluorescent specimens imaged with the laser scanning confocal microscope (LSCM), made available in the late 1980s (Amos et al, 1987), provided vastly improved results—in-focus 3D reconstructions of individual neurons were obtained
Stacked images from brightfield microscopy offer the promise of improvement (Figure 1b), but they too often lack the detail obtained using the LSCM

Summary

Introduction

In the late 1880s, Santiago Ramon y Cajal began characterizing the cytoarchitecture of different types of brain neurons in a variety of animal species by employing the Golgi stain or ‘black reaction’ method. Targeted neurons were filled with cobalt or nickel ions, and later intensified through the deposition of silver (Pitman et al, 1972; Tyrer and Bell, 1974; Mesce et al, 1993a). The popularity of metal-filling and silver intensification methods gave way to the development and use of fluorescent compounds to label neurons (Stretton and Kravitz, 1968; Stewart, 1981; Mesce et al, 1993b; Vitzthum et al, 1996). A number of fluorophores in wide use showed susceptibility to photobleaching, lessening the ability of fluorescently labeled samples to be archived and repeatedly imaged over time

Methods

Results

Conclusion