Those conditions that are determined as optimal for crystallization per FRAP are subsequently used as the basis for a crystallization screen. After an extended period of time, the crystallization plates are imaged in the hopes of finding well-formed crystals. Imaging LCP plates is not trivial however, making it often difficult to find protein crystals that have formed within the drops. LCP itself can be turbid and opaque, obscuring small crystals buried within it. The optical quality of the plates used is therefore extremely important when imaging LCP drops. The use of glass sandwiches for holding LCP crystallization drops has been proven to be a reliable method for imaging LCP drops.5,6 The smooth surfaces of the glass flatten the drops so as to minimize the bumps and ridges formed during dispensing. Also, the glass itself has negligible birefringence and thus does not interfere with birefrigent imaging.
Conventional brightfield microscopy cannot always be used as a reliable method for screening LCP plates for crystals. Oftentimes the LCP drop is very turbid and optically obscures the crystals, making it virtually impossible to distinguish a protein crystal from the medium. The use of crossed polarizers significantly improves the ability to detect crystals. The birefringent properties of protein crystals allow them to be distinguished from the LCP (Figure 6a). However, the alignment of the crystal does impact the degree of birefringence, so often they can be missed because of their orientation. Also, the LCP commonly becomes birefrigent itself, making it impossible to identify crystals (Figure 6b).
Ultra-Violet Fluorescence (UV) can be used to detect protein crystals even if the drops are turbid and birefringent. This imaging technique takes advantage of the UV fluorescence from aromatic amino acids like tryptophan present in most proteins. UV works particularly well for distinguishing between salt and protein crystals. As can be seen in Figure 7, no UV fluorescence is detected from the large salt crystal in the top of the drop, whereas small protein crystals fluoresce strongly and can be visualized within the drop.
Visible, birefrigent and UV imaging are good tools for a scientist in trying to find crystals formed within their LCP drops. However, they are all limited when it comes to detecting small crystals. In order to detect significant birefringence, the crystals need to be at least 10 um in size, and the fact that lipids often become birefrigent detracts from using this technique in isolation. UV is advantageous in that it isn’t impacted from the turbidly or birefringence of the LCP. However it is limited in that all protein, whether solubilized or crystallized will be detected. The background from the solubilized protein decreases the contrast significantly and false positives can result from aggregated proteins.
A new imaging technique called SONICC (Second Order Nonlinear Optical Imaging of Chiral Crystals), recently developed by Garth Simpson at Purdue University and distributed exclusively by Formulatrix, provides a solution for high sensitivity imaging of protein crystals.7 SONICC uses multiphoton microscopy to specifically detect ordered crystals, while producing negligible background signal. SONICC relies on the underlying principle of Second Harmonic Generation, where two low-energy photons under intense electric fields combine to form a higher energy photon. This process occurs only in non-centrosymmetric ordered crystals and is therefore specific to the vast majority of chiral protein crystals. However, unlike with UV fluorescence, solubilized or aggregated proteins do not result in signal. LCP drops imaged with brightfield, crossed-polarizers, and UV fluorescence are compared to SONICC imaging in Figure 8.8 SONICC can clearly identify both large crystals as well as small nanocrystals formed in the LCP, regardless of drop turbidity.
The SONICC imager from Formulatrix employs a drop location algorithm to accurately locate LCP drops. It subsequently adjusts the magnification of both the visible and SONICC optical paths so that the maximum magnification can be used to image the drop (Figure 9). 96 well drop plates can be imaged in less than 5 minutes in visible and 15 minutes in SONICC, with a SONICC imaging rate of two frames per second.
The SONICC imager also has an imaging mode to detect UV fluorescence called UV-TPEF (ultra violet two-photon excited fluorescence). Some salt crystals including lithium sulfate and ammonium phosphate form noncentrosymmetric crystals and generate SHG. The use of UV-TPEF aids in the discrimination of protein from salt crystals. Similar to standard UV fluorescence as described above, this imaging mode also takes advantage of the intrinsic aromatic amino-acid residues (ex. Tryptophan) found in proteins. When excited with UV radiation (~266 nm) these aromatic residues within the protein molecules fluoresce, making it very easy to determine whether or not a crystal is proteinaceous. In order to excite this fluorescence, the laser in SONICC 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 is 266 nm which excites any tryptophan amino acids that are present. The two-photon excited fluorescence (325 – 500 nm) is then collected and used to create a fluorescence image. It should be noted that it is not necessary for the protein to be crystalline in order to fluoresce. In this way the SHG and UV-TPEF imaging modes act as complements to each other. The SHG channel probes crystallinity and the UV-TPEF channel is specific to proteinaceous samples. Examples of images acquired with the Formulatrix imager are shown in Figure 10.
- Cherezov, V., and M. Caffrey. Nano-volume plates with excellent optical properties for fast, inexpensive crystallization screening of membrane proteins. J. Appl. Cryst. 2003.36: 1372-1377.
- Cherezov, V., Peddi, A., Muthusubramaniam, L., Zheng, Y.F., and M. Caffrey. A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases. Acta Crystallogr. D Biol. Crystallogr. 200460: 1795-1807.
- Kissick, D. J.; Gualtieri, E. J.; Simpson, G. J.; Cherezov, V., Nonlinear optical imaging of integral membrane proteins in lipidic mesophases. Anal. Chem. 2010, 82(2), 491-497.
- Wampler, R. D.; Kissick, D. J.; Dehen, C. J., Gualtieri, E. J.; Grey, J. L.; Wang, H.; Thompson, D. H.; Cheng, J.; Simpson, G.J., Selective detection of protein crystals by second harmonic microscopy. Journal of American Chemical.