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

1) The manuscript claims "...silver emissions offer greater spatial resolution and are more photostable than fluorophores...' but such a claim is not verified. While the images presented are beautiful and their stability without question, their resolution is not compared to similar high-quality fluorescence images. This comparison should be made and quantified.

We initially made the claim that silver emissions offer greater spatial resolution, because silver nanoparticle plasmon resonance is known to have a high signal-to-background ratio and extinction coefficient, and thus is more detectable (bright). This detectability enables small anatomical features to be more easily seen and imaged. In retrospect, we should have used the term “detectability” and not spatial resolution; thus we have modified our original assertion throughout the revised manuscript. Depending on objective lenses, light sources and mounting medium used, the diffraction limits of resolution for conventional microscopy, including the LSCM, are between 150-200 nm. The spectral LSCM we used cannot determine whether small neuronal fibers, for example, from a silver-stained preparation versus a fluorophore-labeled one are any better resolved below ca. 200 nm. Both silver- and fluorophore-labeling methods can offer high quality images using the LSCM as now stated in the text (paragraph four, Discussion). However, because the labeling we present involves metal particles, instead of less bright photo-bleachable fluorophores, such specimens can be probed with electron beams and newer super-resolution techniques that may well indicate that sub-cellular anatomical features can be better resolved using silver nanoparticles as compared to traditional fluorophores. Because such studies are beyond the scope of our report here, we have opted not to state that our image resolution is better than fluorescent labeling. In addition, we have added a section in the Discussion that presents the potential of super-resolution microscopy techniques for imaging silver or gold nanoparticles (paragraph four, Discussion).

2) Please amplify the supplemental methods section to more thoroughly describe post-capture image processing. For example, in Figure 2 the signal from the ganglion's perinerurial sheath was removed (in FIJI) and this process should be explained more fully and perhaps illustrated as a supplemental figure.

In response to the request for amplification on supplemental methods to more thoroughly describe post-capture imaging and the optical removal of the perineurial sheath covering the ganglion (for Figure 2), we have provided a new detailed section (in the Materials and Methods) with the heading, “Image processing and spectral unmixing for 3D rendering”. In addition to step-by-step procedures for post-capture imaging, we provide a new supplement figure (Figure 2—figure supplement 1), which illustrates the image processing from the raw state to the processed image.

3) Please indicate whether the laser lines and detectors required are standard on most LSCMs.

We further clarified that the laser lines and PMT detectors are standard on all commercially available spectral LSCMs. For non-spectral LSCMs, we mention that the excitation laser lines are standard, but that the optical filter sets to capture light shorter than the excitation wavelength are not standard. If the LSCM were not tunable, a spectral detector or optical setup would need to be procured from the manufacturer of a given confocal microscope (subheading “Laser scanning confocal imaging”).