You've set up hundreds of drops, waited days, imaged the plate under visible light, and what stares back at you is a sea of murky, featureless wells. No crystals. Or so it seems.
The problem isn't your protein. It's your imaging. And it's costing you hits you don't even know you're missing.
Why Visible Light Isn't Enough Anymore
Automation has transformed every stage of the protein crystallization pipeline. Purification systems reliably produce large quantities of high-quality protein. Liquid dispensers and drop setters like Formulator and NT8 set up hundreds of conditions with nanoliter precision. Rock Imagers schedule and capture images without any manual intervention.
But detecting what's actually growing in those drops is where visible light hits its ceiling. It struggles with microcrystals smaller than a few microns and crystals buried in lipidic cubic phase (LCP), and can't reliably distinguish protein crystals from salt or amorphous precipitate.
The result? Promising conditions get scored as negative and abandoned. The optimization that could have led to a structure never happens.
SONICC: Seeing What Other Modalities Miss
SONICC® (Second-Order Nonlinear Imaging of Chiral Crystals) is not a refinement of conventional imaging. It is a fundamentally different approach, combining two nonlinear optical techniques into a single system:
SHG (Second Harmonic Generation) fires infrared laser pulses at 1,064 nm onto the sample. When those pulses hit a chiral crystal (which includes virtually all protein crystals), two infrared photons are converted into a single green photon. This frequency-doubling effect is instantaneous and highly specific. The result is a high-contrast signal that lights up crystalline material against a near-black background. Because only chiral, non-centrosymmetric structures generate an SHG signal, amorphous precipitates and most salt crystals produce nothing. However, some non-protein crystals can still produce an SHG signal. That is where UV-TPEF comes to the rescue, adding another layer of specificity to help distinguish protein crystals from other SHG-active materials.
UV-TPEF (UV Two-Photon Excited Fluorescence) works on a different principle: detecting the natural fluorescence of aromatic amino acids, primarily tryptophan, within protein molecules. Unlike conventional UV imaging, UV-TPEF uses longer excitation wavelengths, making it gentler on samples, compatible with a wider range of plate types, and capable of confocal-like imaging contrast.
Used together, SHG and UV-TPEF do something neither can do alone: they tell a protein crystal apart from a chiral salt crystal. Some salts form chiral structures and generate an SHG signal, a false positive that, without a second confirmation, can send you down the wrong path. UV-TPEF resolves that ambiguity. By confirming whether the signal is genuinely protein-derived, it adds the layer of certainty that crystal identification actually demands (Fig. 1).
The detection limit makes the case clear. SONICC routinely identifies crystals down to 1 micrometer, crystals that are completely invisible under brightfield, that would never be found manually, and that would otherwise be written off as empty conditions. Under SONICC, they become unmistakable. That sensitivity makes it particularly powerful in LCP experiments, where the matrix itself obscures conventional imaging, and in serial femtosecond crystallography, where the entire experiment is built around crystals too small to see any other way.
Figure 1. Lipidic cubic phase (LCP) experiment imaged by visible light, SHG, and UV-TPEF modes
Where SONICC Makes the Difference
The capabilities of SONICC aren't theoretical. They're being put to work right now across leading research institutions and pharmaceutical companies.
At the National Crystallization Center at HWI, Dr. Sarah Bowman's team images high-density 1536-well plates using SONICC to identify nanocrystals (Fig. 2), which conventional imaging would miss entirely1.
Figure 2. Crystal detection in 1536-well plates using SONICC
At Arizona State University's Biodesign Institute, Professor Petra Fromme's group relies on SONICC to identify nanocrystals for serial femtosecond crystallography (Fig. 3). The same team has taken it further, using SONICC to detect protein crystals forming inside living insect cells that would have been invisible under standard imaging2.
Figure 3. Nanocrystal identification with SONICC for serial femtosecond crystallography
At Merck, Associate Principal Scientist Dr. Paul Reichert’s group uses SONICC to identify and confirm the crystallinity of monoclonal antibody pembrolizumab after studying different variables in ground and space experimentation that play an important role in crystallization3 (Fig. 4). As different variables can affect crystallizability of macromolecules, therefore it is important to track effect of different variables in an efficient and reliable manner and SONICC makes that analysis possible with speed and selectivity that no conventional method can match.
Figure 4. Analysis of pembrolizumab crystallization experiments using SONICC
Stop Abandoning Hits You Haven't Found Yet
Every missed microcrystal is a condition that could have been optimized. Every false negative is time lost chasing new screens instead of refining a real hit.
SONICC doesn't just improve your imaging; it expands what your crystallization campaigns are capable of finding. Whether you're working in LCP or pushing toward serial femtosecond crystallography, SONICC gives you the sensitivity to see the full picture.
References
- Budziszewski, G. R., Snell, M. E., et al. High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography. JoVE (Journal of Visualized Experiments), (193), e65211 (2023).
https://dx.doi.org/10.3791/65211 - Nagaratnam, N., Tang, Y., et al. Enhanced X-ray diffraction of in vivo-grown µNS crystals by viscous jets at XFELs. Acta Crystallographica Section F: Structural Biology Communications 76(6), 278–289 (2020).https://doi.org/10.1107/S2053230X20006172
- Reichert, P., Prosise, W., et al. Pembrolizumab microgravity crystallization experimentation. npj Microgravity 5(1), 28 (2019).https://doi.org/10.1038/s41526-019-0090-3
If you are interested in knowing more about what SONICC can do, please visit our publication page.