Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline

Area of Science:

• Crystallography
• Biophysics
• Microscopy

Background:

• Synchrotron X-ray diffraction (XRD) is crucial for protein structure determination.
• Automated crystal centering and identification are bottlenecks in XRD workflows.
• Nonlinear optical (NLO) techniques offer rapid, non-destructive sample analysis.

Purpose of the Study:

• To integrate NLO microscopy with synchrotron XRD on a single platform.
• To develop and evaluate NLO methods for rapid protein crystal detection and centering.
• To assess the performance of NLO-XRD integration for various crystalline and non-crystalline samples.

Main Methods:

• Developed two NLO optical designs (downstream and upstream) for integration with a diffractometer.
• Employed second-harmonic-generation (SHG) and two-photon-excited ultraviolet fluorescence microscopy.
• Evaluated crystal detection and centering using X-ray raster scanning for comparison.
• Analyzed protein crystals (cPAH, TsUCH37, kOR-T4L, intimin) and α-cellulose samples.

Main Results:

• Achieved protein crystal identification with per-pixel integration times of 80–150 µs.
• Demonstrated a ∼10^3–10^4-fold reduction in exposure time compared to XRD raster scanning.
• Successfully performed quantitative centering and analysis of multiple protein samples and α-cellulose.
• Observed good agreement between NLO and XRD measurements for sample positioning.

Conclusions:

• NLO instrumentation integrated with synchrotron XRD enables rapid, automated crystal analysis.
• The combined platform significantly accelerates sample characterization workflows.
• This integrated approach is effective for diverse crystalline and fiber diffraction samples.