ZANG group research

department of chemistry and biochemistry SIU

Scanning Confocal Microscopy (SCM)

Scanning confocal microscope (SCM) utilizes a high NA (≥ 1.25) objective for focusing both excitation and emission beams, which are separated by a dichroic beam splitter (see the scheme).

Confocal is defined as sharing the same focus. What this means in the fluorescence microscope is that the final emission has the same focus point as the excitation light in the object. The microscope is able to filter out the out-of-focus light from above and below the point of focus in the object by means of a confocal "pinhole" situated in front of the detector. As for an ordinary optical microscope, the diffraction limit (λ/2) represents the ultimate of the optical resolution of SCM, i.e. the focused beam spot in the specimen is in the size scale of the excitation wavelength. Thus, for guaranteeing only one molecule in the focused laser spot (a few hundreds nanometer size), the sample under investigation should be extremely diluted. Normally, the concentration of the solution used for spin-coating molecules on the transparent substrate is in the range of 10^-10 M depending on the spin speed, the molecule affinity to the surface, and the solvent versatility.

In our lab, the SCM is modified from NSOM, from which one can take the advantage of the ScanMaster linearization technology developed for Aurora NSOM. This technology allows for easier and more accurate tip positioning. In the case of investigating the electric field effect of the optical and electronic properties of the sample, a bias voltage can be applied between the NSOM tip and the sample substrate. By running the instrument in NSOM mode, the tip can be held at a constant proximity to the substrate, and thus the applied bias can be linearly controlled by a DC supplier. 

Compared to NSOM scanning, SCM provides high throughput sample screening and much stronger excitation. For those materials or molecules with weak fluorescence, SCM normally afford better sensitivity than NSOM. In our lab, most of the single-molecule spectroscopy studies, which do not require topographic information, are carried out with SCM scanning.

(updated on june 22, 2004)