SONICC FAQs

SHG stands for Second Harmonic Generation and is a nonlinear optical process. In intense electric fields (such as in the presence of a femtosecond laser) the distance between the electrons and the nucleus are distorted (anharmonicity) resulting in nonlinear optical effects such as SHG where the frequency of the outgoing light is doubled that of the incident (i.e. 1064 nm incident results in 532 nm exiting).

A chiral molecule, or in this case a chiral crystal, is a crystal that lacks an internal plane of symmetry, and thus its mirror image is nonsuperimposable on itself. A chiral crystals are symmetric and therefore produce SHG in equal and opposite directions that sum to a net of zero signal.

Almost all molecules that have a chiral center form a chiral crystal, therefore most proteins will form chiral crystals that are detectable via SONICC. Over 99% of the proteins in the PDB have a space group that is detected by SONICC. Those crystals with extremely high symmetry classes will generate less SHG.

They can if they are chiral, but the majority of salts are achrial and therefore do not generate SHG.

Fluorescent imaging takes advantage of either the endogenous fluorescence of the protein or the use of fluorescent tags. Although the fluorescence is bright and easily detectable, it is generated from solubilized and aggregated proteins as well as crystallized proteins. The background from the solubilized protein decreases the S/N significantly and false positives can result from aggregated proteins. SONICC on the other hand is only sensitive to crystallized proteins.

UV fluorescence probes the amino acids present in proteins that are excited in the UV (~280 nm). It does not differentiate between solubilized, aggregated, or crystalline protein. Also, the use of the high energy wavelengths can cause damage to the proteins especially through breakage of disulfide bonds.

For clear birefringent images, crystals usually need to be greater than 30 µm in size, however SONICC can detect down to less than 1 µm. Birefringence can also be seen from salt crystals.

All optically assessable platforms are compatible.

Preliminary experiments show no detectable damage to protein crystals. In one experiment, a protein crystal was imaged on one half with excessive laser input. X-ray diffraction was obtained from both the exposed and un-exposed halves of the crystals. Both sides diffracted to within expected resolution (~2 A) and within statistical variation (i.e. there was no statistical difference between the diffraction of both sides). SONICC has also been utilized to image live cells with no observed impact (they remained adhered to a Poly-Lysine coated slide).

Yes, as long as the fluorescence is stokes shifted by 10 nm, then it will not be detected or interfere with the SHG.

Unfortunately it currently can not, but we are looking into means of assessing quality based on polarization changes of the emitted light.

Theoretically the lower limit of detection can be estimated by the forward to backward ratio of the SHG. Based on the coherence length of the generated SHG and the refractive index of the material this lower limit ranges from 90 nm – 300 nm in thickness. In practice, crystals of the size of 1 µm³ can routinely be detected. 2-D crystals have also been routinely imaged with S/N > 30.

Dependent on the field of view being imaged, pixel sizes range from 3 µm to 6 µm.

The laser focuses to a width of ~100 µm and can image drops greater than 3 mm tall with multiple z-steps.

The current electronic package allows 512 x 512 image acquisition for one z-slice in 500 ms. This corresponds to 8 traces of the fast scanning mirror per line. A one drop 96 well plate can be imaged with SHG in 15 minutes with 8 z-slices and 5 minutes for visible imaging.

Bioavailability is greatly decreased if the APIs crystallize and is therefore extremely important to monitor. Current methods of crystallization detection (thermal, optical, x-ray diffraction and spectrochemical techniques) have a 1% crystallinity limit of detection and are challenging in complex matrixes. Early crystallization detection expedites the process of monitoring APIs and can also give insights crystallization kinetics.

The theoretical detection limit, estimated by the coherence length of SHG and refractive index of griseofulvin is 90 nm in thickness corresponding to ~ 3.4 x 10^-9 % crystallinity. An upper limit of detection would be >5 µm corresponding to % crystallinity of > 6 x 10^-4% which is 5 orders of magnitude improvement from existing techniques.

Raman, differential scanning calorimetry, and X-ray diffraction routinely have detection limits ranging from 0.1 – 10 % crystallinity.