SONICC (Second Order Nonlinear Imaging of Chiral Crystals) definitively identifies chiral crystals with SHG (Second Harmonic Generation) technology and discerns between protein and salt crystals with UV-TPEF (Ultraviolet Two-Photon Excited Fluorescence) technology.
Crystals appear white against a stark black background, helping you to identify crystals even in murky environments. SONICC can detect extremely thin crystals, microcrystals <1 μm, and crystals obscured in birefringent LCP.
Major pharmaceutical and academic research labs worldwide find SONICC to be extremely successful, cost effective, and efficient.
The unique imaging properties of SONICC allow crystal detection in almost any optical environment, including opaque and turbid environments. Only chiral crystals, such as proteins, produce a signal using second harmonic generation (SHG). This imaging technology reveals very thin protein crystals, as seen to the left, or those buried under precipitant.
SONICC can detect nano-crystals with the use of high numerical aperture objectives. Small crystals that may be indistinguishable from precipitant can be clearly differentiated as shown in the image to the left. This positive hit indicates a condition for further optimization, which may have been missed without SONICC technology.
SONICC technology produces high contrast black and white images, making it easier to find hidden microcrystals.
The grids to the left show a 96-well plate imaged by SONICC in SHG and in visible light. Toggle between the SHG and Visible buttons to see how SONICC reveals crystal hits. As you can see, each well containing crystals is instantly apparent when imaged with SHG.
The UV-TPEF (Ultraviolet Two-Photon Excited Fluorescence) mode is analogous to traditional UV fluorescence and creates images based on the fluorescence of UV excited amino acids such as tryptophan. UV-TPEF is a multiphoton imaging technique that uses longer wavelengths of excitation versus traditional linear imaging, which provides greater plate compatibility, less damage to your samples, and confocal imaging.
SONICC can be built into a Rock Imager 1000 which allows you to store and automatically image up to 1,000 plates. Two plates can be imaged at the same time - one in visible and one in SONICC. This allows for over 200 96-well plates to be imaged in visible and more than 40 plates in SONICC in just one day.
The extremely low detection limit of SONICC makes it the best imaging technique for LCP plates. Small crystals, buried in turbid lipidic cubic phase can easily be visualized with SONICC. Drop location and auto focus are both used to quickly and accurately find the LCP drop. The accompanied visible imaging techniques of crossed-polarized imaging offer complimentary information all with extremely fast imaging times.
Image quality is retained even with fans circulating, motors running and air flowing. Without our dedicated design towards minimizing vibration, image quality would be severely affected resulting in poor image quality despite good optics. Innovative mechanical design, advanced vibration damping materials, and special motor tuning ensure extremely low vibration to conserve image quality and minimize disturbance to the protein drops.
The SONICC benchtop system is designed to precisely monitor and regulate temperature throughout the enclosure. A network of sensors measures and records temperatures with 0.1°C resolution. An air recirculation system maintains an even temperature distribution. SONICC benchtop uses a Peltier heat exchanger to regulate temperature in the range from 5° C below ambient to 7° C above ambient to within +/-0.5° C. If SONICC is built into a RI-1000, then a chiller option is available where the temperature can be regulated from 4° C to 19° C with ambient temperature from 16° C to 30° C.
SONICC, Second Order Non-linear Imaging of Chiral Crystals, uses a femtosecond pulsed laser to exploit the frequency-doubling effect found in the majority of protein crystals, and produces high-contrast images with negligible background signal. SONICC has two imaging methods, Second Harmonic Generation (SHG) and Ultraviolet Two-Photon Excited Fluorescence (UV-TPEF). The SHG channel probes crystallinity, and the UV-TPEF channel is specific to proteinaceous samples.
SHG imaging identifies chiral crystals by imaging the sample with infrared light and detecting the frequency-doubled response, only present in chiral crystals. Chiral crystals are those that lack an internal plane of symmetry, so only crystals that lack inversion symmetry will produce signal. Most salt crystals are symmetric and therefore generate no SHG, whereas all protein crystals are chiral and will generate SHG signal.
Nonlinear effects such as SHG require high electric fields, thus requiring the use of a femtosecond (fs) laser. The laser operates with a pulse width of 200 fs and has high peak powers resulting in nonlinear effects, but the pulses are short enough to reduce damage associated with localized heating. Further efforts are taken to reduce sample damage by scanning the laser beam quickly so that it does not remain in one spot long enough to heat the sample.
SHG results from all chiral crystals, including some salts and small molecules that form noncentrosymmetric crystals. These chiral salt and small molecule crystals will result in false positives when imaged in SHG mode. To combat this, SONICC is also equipped with Ultraviolet Two-Photon Excited Fluorescence (UV-TPEF) imaging.
UV-TPEF imaging is analogous to traditional UV fluorescence and creates images based on the fluorescence of UV excited amino acids such as tryptophan. UV-TPEF is a multi-photon technique, and therefore uses longer wavelengths of excitation versus traditional linear imaging. This provides significant advantages including compatibility with more plates, less damage to protein and confocal imaging.
In order to probe a sample’s fluorescence, the laser is doubled with a NLO (nonlinear optical) crystal from 1064 nm to 532 nm. The green light (532 nm) is then used to image the sample. The two photon equivalent of the green light is 266 nm, which excites any tryptophan amino acids that are present in the sample. The two-photon excited fluorescence (350 – 400 nm) is then collected and used to create a fluorescence image.
The combined effect of SHG and UV-TPEF imaging is so precise that SONICC can detect microcrystals (<1 um) and distinguish between crystal showers and amorphous aggregates.
For those higher-symmetry crystal classes that are difficult to detect with SHG, use UV-TPEF for imaging. The percentage of those proteins that do not contain tryptophan -- and therefore would not be detectable with UV-TPEF -- are also included in Table 1.
In general, the following can be concluded:
Preliminary experiments show no detectable damage to protein crystals due to the laser. 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 Å) and within statistical variation (i.e. there was no statistical difference between the diffraction of both sides).
SONICC has also imaged live cells with no observed impact. The cells remained adhered to a polylysine coated slide before, during, and after imaging indicating they remained viable.
What is SHG?
SHG stands for Second Harmonic Generation and is a nonlinear optical process. In intense electric fields (i.e. in the presence of a femtosecond laser) the distance between the electrons and the nucleus are distorted (anharmonicity) resulting in non-linear optical effects such as SHG where the frequency of the outgoing light is twice that of the incident (i.e. 1064 nm incident results in 532 nm exiting).
What does “chiral” mean?
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. Achiral crystals are symmetric and therefore produce SHG in equal and opposite directions that sum to a net zero signal.
Are all protein crystals detectable?
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 Protein Data Bank have a space group that is detectable with SONICC. Crystals with extremely high symmetry classes will generate less SHG signal.
Will salts produce signal?
They can if they are chiral, but the majority of salts are achiral and therefore do not generate SHG signal.
How is SONICC different than fluorescent imaging?
Fluorescent imaging takes advantage of either the endogenous fluorescence of the protein or the use of fluorescent tags. Although 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 signal to noise ratio significantly, resulting in false positives. SONICC, on the other hand, is only sensitive to crystallized proteins.
How does SONICC compare to UV imaging?
UV fluorescence probes the amino acids present in proteins that are excited by UV light (~280 nm). The fluorescence does not differentiate between solubilized, aggregated or crystalline protein. Also, the use of high-energy wavelengths can damage proteins, especially through breakage of disulfide bonds.
How does SONICC compare to birefringence imaging?
For clear birefringent images, crystals usually need to be >30 μm. SONICC can detect crystals <1 μm. Birefringence can also be seen from salt crystals.
With which platforms are SONICC compatible?
SONICC is compatible with all optically-accessible platforms.
Can I do TPEF (Two Photon Excited Fluorescence) imaging at the same time as SHG?
Currently, TPEF can be detected, but not simultaneously.
Will the laser damage my crystals?
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 crystal. Both sides diffracted to within expected resolution (~2 Ã) and within statistical variation (i.e. there was no statistical difference between the diffraction of either side). SONICC has also imaged live cells with no observed impact (they remained adhered to a polylysine-coated slide).
Can I use SONICC if my sample is fluorescent?
Yes. As long as fluorescence is Stokes-shifted by 10 nm, the fluorescence will not be detected nor interfere with the SHG signal.
*Please note that each crystal will generate different intensities of SHG signal depending on size, orientation, space group and quality, as well as the acquisition time and incident intensity.
How small of a crystal can SONICC detect?
Theoretically, the lower limit of detection can be estimated by the forward-to-backward ratio of the SHG signal. Based on the coherence length of the generated SHG signal and the refractive index of the material, this lower limit ranges from 90 nm – 300 nm in thickness. In practice, 1 μm3 crystals can be routinely detected. 2D crystals have also been routinely imaged with a signal to noise ratio >30.
What is the spatial resolution?
Pixel sizes range from 3 μm to 6 μm depending on the field of view.
What is the laser´s Z-resolution and how deeply can it penetrate?
The laser focuses to a width of ∼100 μm and can image drops >3 mm tall with multiple Z-steps, called “slices”.
How fast is SONICC?
The current electronic package allows 512 x 512 image acquisition for one Z-slice in 500 ms. This corresponds to eight traces of the fast-scanning mirror per line. A one-drop 96-well plate can be imaged with visible light in 5 minutes and with SHG in 15 minutes with eight Z-slices per drop.
Recent advances in automation technology for drop setting, condition screening and imaging make LCP crystallization considerably more accessible. This application note discusses the three major steps of LCP crystallization, including FRAP, and how automation technology accelerates LCP crystallography.
|Fixed Zoom||Compound Zoom|
|Lens/Objective Options||Asphere 20 mm EFL||Asphere 20 mm EFL||Nikon CFI S Plan Fluor ELWD 20X||40x Nikon CFI S Plan Fluor ELWD 40X|
|Maximum FOL†||2.2 x 2.2 mm||2.2 x 2.2 mm||1.3 x 1.3 mm||0.65 x 0.65 mm|
|Lateral Resolution||4 μm||4 μm||2 μm||1.1 μm|
|Working Distance||13.5 mm||13.5 mm||7.5mm||3.5 mm|
† Continuous zoom is available by controlling the angle of the scanning mirrors.
§ N.A. (numerical aperture) is proportional to the detection limit of SHG.
Plate storage capacity:
|SONICC User's Guide|
Average User Satisfaction Rate
|University of Chicago - Katherine Leon||10/20/2014 5:34 PM|
No additional feedback entered
|Brandeis University - Min-Sung Chris Hong||10/20/2014 5:34 PM|
No additional feedback entered
|Merck - Sangita B Patel||10/20/2014 5:34 PM|
It is amazing to see your crystal light up in UV and SHG, for very small crystals it still is a difficult task but with experience you can resolve lot of confusion about microcrystals
|Major Pharmaceutical Company – anonymous review||04/17/2014 3:50 PM|
We have had a RI-1000 with SONICC for 1.5 years and since then have changed our workflow for viewing images. Very few of us look at visible images at first or even at all when screening. We take everything from SHG & UV-TPEF and put them directly in a random and/or an optimization screen. We also use the instrument to help determine small molecule vs. protein crystal using the UV-TPEF imaging mode.