Second harmonic Imaging is based on the non-linear effect known as Second Harmonic Generation (SHG) whereby two incident photons from an intense laser pass through a polarisable material and are changed by the sample under investigation (which must not have a centre of symmetry) into one photon that emerges with double the incident energy and frequency. Biological samples can be highly polarisable and can be good centres for SHG signals. SHG does not require excitation of fluorescent molecules and since there are no excited molecular states involved does not suffer from photobleaching and phototoxicity . Thus SHG is advantageous with respect to other forms of fluorescence imaigng in viewing live tissue and in cell investigation.SHG polarisation anisotropy can also be used to determine the absolute orientation and degree of organization of proteins in tissues. 

A MultiView 1000 head sitting on the sample stage of a dual microscope	Second Harmonic signal enhanced by a silver nanoparticle at the tip of a cantilevered nanopipette.

Nanonics' MultiView systems are easily integrated into dual (combined inverted & upright) optical microscopes, in large part due to their compact, optically-open, SPM heads and are thus ideal for integrating scanning probe microscopy (SPM) with non-linear optics. Techniques such as Second Harmonic Generation (SHG), stimulated emission, CARS (Coherent Anti-stokes Raman spectroscopy) and third harmonic generation can be readily performed.

Also in terms of linear microscopy our dual microscope provides direct access to the detector even in the inverted microscope portion of the system offering much higher throughput. Our combined system can also be used for simultaneous reflection and transmission NSOM.

In addition, SHG signals from membrane bound dyes have been shown to be highly dependent upon membrane potential. The second harmonic response of styryl dyes in electric fields have been investigated using Nanonics' systems .SHG has been shown to be a valuable tool for imaging electrical activity in biological specimens and has great potential for investigations into cell activity with the potential to map neuronal activity.

Nanonics patented AFM  probes have a structre which have an exposed probe tip that allows for nanoparticle growth. Nanonics produces silver and gold nanoparticles at the tip of such cantilevered patented AFM probe. It has been shown that these nanoparticles can enhance second-harmonic generation of a molecular system such as a styryl dye.

The Nanonics probe structure allows for an exposed tip that allows for metal nanoparticle growth.

Here is a diagrammatic representation of SHG of a membrane labelled with a styryl dye:

In this example of SHG the sample is a membrane labeled with a styryl dye. For second order phenomena such as second harmonic generation only the asymmetrically oriented molecules will produce a signal

The most intense non-linear second order signals are in the forward direction. All Nanonics' systems have a completely free optical axis which readily allows excitation and collection of non-linear optical phenomena together with scanned probe microscopy data.