I am trying to look at the topography of various surfaces under confocal microscope and would like to coat silicion wafer and/or glass surfaces with a uniform layer of florescent molecule. Some covalent bonding preferably, don't want the flourescent coating to come off after washing steps.
I don't know much about this project, but I would question whether you need fluorescence to look at surface topography. You can try DIC or polarized light as well as the right type of electron microscopy if you need to look at very small scale. Have you been to the CMIF at NCSU? https://www.ncsu.edu/project/cmif-micro/
There are many ways to covalently bond fluorescent dyes to a silicon oxide surface, be it glass or SiO2 in Si.
1) clean it
2) coat/ couple with siloxane (APTES aminopropyl triethoxy silane, GOPTS glycidyl oxypropyl triethoxy silane, or other)
3) couple appropriate dye (e.g. FITC on APTES, dansyl cadaverine on GOPTS)
However, these simple procedures will result in a quite dense population of the dye molecules which gives you a quenching of the fluorescence. That means you will have to dilute them e.g. by a co-coating of the siloxane with e.g. TEOS (tetraethoxysilane) or an incomplete dye coupling reaction. In any case it will require quite some efforts to get this working.
The answer from Andreas Holländer is very promising. FITC works very well for e.g. particles (Fpc study). The covering of the surface will also result in a too bright signal for reasonable measurements (Two color).
I suggest performing/investigating two different approaches.
1.
Coating (straightforward vapour deposition (i.a. APTES paper) of normal deposition (gelest catalog)) resulting in of Aminoproylsiloxane with have to be “dilute” using a non-reactive monoorganylXXsilane with similar properties (first case vapour pressure and second case reactivity (or both) like propyltriethoxysilane), I do not see how TEOS dilute the density). Then modify the surface using 5-Fluorescein-Isothiocyanate.
2.
Perform a full coating by mono-Aminoproylsiloxane and a modification using a mixture of 5-Fluorescein-Isothiocyanate and ethylisothiocyanat.
Note that the surface properties of the deposition are strongly dependent on the history (“clean it” APTES paper) of your SiO2-surface (wet, dry preparation)
http://www.gelest.com/goods/pdf/couplingagents.pdf
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Use a short organic molecular (mercapto) o silanization process.
Gelest has some fluorescent organosilanes that can be attached to these surfaces. You can read about them in a brochure "Silane Coupling Agents" page 65. This can be downloaded from the Gelest website, www.gelest.com
Are you trying to get topography (i.e. surface roughness) this way? Just FYI, I suggest that tools such as SPM or a 3D laser scanning confocal microscope (not a biological confocal microscope) will give you true topography without the need to coat the surface.
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