Screening of protein crystallization trials by second order nonlinear optical imaging of chiral crystals (SONICC)

Abstract

Second order nonlinear optical imaging of chiral crystals (SONICC) is a promising new method for the sensitive and selective detection of protein crystals. Relevant general principles of second harmonic generation, which underpins SONICC, are reviewed. Instrumentation and methods for SONICC measurements are described and critically assessed in terms of performance trade-offs. Potential origins of false-positives and false-negatives are also discussed.

Figure 1

Comparison of SONICC and conventional optical methods for protein crystal detection of GPCRs, from six representative outcomes of crystallization trials (a–f). Bright field and birefringence (cross-polarized) images were obtained using white-light illumination, with UV and Cy3 fluorescence excited with ~280 nm and ~543 nm light, respectively. All SONICC images were acquired with 800 nm incident light with detection at 400 nm. Adapted from reference .

Figure 2

Diagram of an anharmonic oscillator potential energy well of a 1 dimensional molecule (left, solid) with the harmonic oscillator overlaid for comparison (left, dashed) and the time dependent trace of an oscillation within the well (right, solid). The oscillation can be largely recovered from a weighted sum of several harmonics (right, dashed and dash-dotted).

Figure 3

General instrument design for SONICC. PMT = photomultiplier tube.

Figure 4

Demonstration of rapid plate-reading by SONICC. Top: SONICC data for a 96-well plate. Bottom: comparison of visible, conventional uv-excited fluorescence, two-photon excited UV fluorescence, and SHG for two representative wells. All images were generously provided by Formulatrix, Inc. The identity of the sample is confidential information. Acquisition times were 0.5 s per well for visible, UV, and TPE-UVF and 2.0 s per well for SHG. The field of view of each well is approximately 2 mm on each side. Crystals favorably form on the right edges of the drops. This anomaly is likely due to the dispensing robot not placing the protein and precipitate solution directly on top of one another.