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.
Our latest publication has now been released :
J. Wenger, D. Gérard, P. -F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, " Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture : towards rapid multicomponent screening at high concentrations," Opt. Express 14, 12206-12216 (2006)
It describes the first experimental implementation of nanometric apertures to conduct dual-color fluorescence cross-correlation spectroscopy (FCCS) at high molecular concentrations.
Nanoapertures have already been demonstrated to be useful for single-color FCS analysis [see Levene et al, Science 299, 682-686 (2003)]. Surprisingly, the extension to the dual-color case has never been reported and yet new interesting specificities are described and detailed in this article (simple microscope setup, no confocal pinhole, micromolar concentrations).
As we paid a substantial fee of $1200, this paper is freely available to ALL readers at :
Maybe one of the most crucial part, which will never be published in a paper ! The main picture shows the microscope at the left, the lasers at the center back, and the confocal detection at the forefront right. There are many optical parts, each has a specific role and is manually assembled and aligned.
The second picture focuses on our Zeiss axiovert 35M (inverted) microscope. I should at some time describe it into more details, maybe on a microscope enthusiasts website (micscape).
And finally the microscope objective, which is by far the central component of the setup. We’re using a Zeiss C-Apochromat with 40x magnification, 1.2 numerical aperture, water immersion, coverslip thickness compensation collar and infinite correction. I’m pretty pleased with it for single-color confocal microscopy close to the diffraction limit, but care must be taken to compensate for chromatic aberrations.
To brighten the scope of this blog, here is a new category about various fancy things and thoughts.
My best way to end up a scientific meeting (and the rush of the few days before) is to take advantage of it to discover new places and cultures. Following the 9th Zeiss FCS conference in
My present research is focused on the implementation of nanometric structures to enhance the contrasts (fluorescence, Raman) in optical microscopy. Two goals are aimed:
1- Fundamental Nanophotonics : understand the influence of nanometric structures on the optical emission.
2- Applied Biophotonics : use the nanodevices to improve existing biophotonic techniques (confocal microscopy, fluorescence correlation spectroscopy FCS, fluorescence resonance energy transfer FRET)
I’m presently studying the implementation of single apertures milled in an opaque metallic film. These apertures have dimensions below the optical wavelength (range 100-500 nanometers, which is 100 times smaller than a hair diameter).
We see two funny physical effects :
1- The apertures confine the light to volumes in the attoliter range (10^-18L, or 0.001 micron cube)
2- The fluorescence brightness can be enhanced up to 15 fold : we collect 15 times more photons per single molecule inside the aperture (as compared to a conventional solution).
>>> More information on the MOSAIC website.