Comments on fluorescence cross-correlation spectroscopy in a nanoaperture
A point that is always missed by readers of peer-reviewed articles is the discussion with referees, which I believe often contains interesting comments and should somehow be available to the scientific community. To (locally) correct for this, I give here the reviewers’ remarks concerning our latest Optics Express publication on FCCS.
“[…] The experiments are carefully done, the analysis seems correct, the FCS and FCCS experiments are internally consistent and the results are very convincing. […] This is a straightforward next step of earlier work of the group of Wenger, using nano apertures, which looks very promising and widens the path for the use of FCCS. “
“[…] It is an important work on the field of fluorescence microscopy and is worth publishing in Optics Express. Unfortunately, the manuscript is not written in a well manner. […] If it wasn't for this important topic and the in principle well-done experimental work, I would not recommend this work for publication.”
I obviously do not share his global opinion ! Reviewer #2 then wrote a list of 20 points, which I cannot detail here. Fortunately, my answers and corrections were convincing. I give here a selection of the main discussion :
Q: “How is the influence of the nanoholes on the performance of biological assays in general? Usually, biological and chemical reactions are influenced by a mechanically confined sample volume.”
A: Actually, we don’t know much about this issue, which is well beyond the scope of this paper. We can only say that we never detected any effect of the aperture on the reactions we tested. A nice point with Al is the aluminum oxide layer that forms naturally on top of the film and passivates the surface. Thus we have no reason to think that the aperture has a large influence on the biochemical reactions. A challenging question is to determine by how much the nanoaperture itself affects the molecular diffusion. We are aware of some work preformed by Harold Craighead’s group (Samiee et al, Biophys. J. 88, 2145), but this reference assumes a 1D diffusion. If this seems valid for a 50nm diameter aperture, we question the fact that it still holds for a 340nm diameter hole, which is obviously more complex. We use the general FCS formula for a 3D Brownian diffusion, while letting the aspect ratio s vary freely. The numerical fits converge easily, and remarkably account for the experimental data.