Selective detection of protein crystals by second harmonic microscopy

Area of Science:

• Biophysics
• Crystallography
• Microscopy

Background:

• Protein crystallization is crucial for structural biology.
• Distinguishing crystalline proteins from amorphous aggregates is challenging.
• Existing imaging techniques may lack specificity.

Purpose of the Study:

• To evaluate second harmonic generation (SHG) microscopy for imaging protein microcrystals.
• To assess the sensitivity and selectivity of SHG for crystalline versus amorphous or solvated proteins.
• To explore SHG as a tool compatible with protein crystallization platforms.

Main Methods:

• Utilized second harmonic generation (SHG) microscopy.
• Imaged microcrystals of green fluorescent protein (GFP) in pL droplets.
• Compared SHG signals with fluorescence.
• Analyzed forward-to-backward SHG ratios for particle sizing.

Main Results:

• SHG microscopy demonstrated high sensitivity and selectivity for protein microcrystals.
• Negligible SHG signals were detected from amorphous aggregates and solvated proteins.
• SHG intensities were comparable to fluorescence but highly specific to crystalline regions.
• Particle size estimation was possible via SHG ratios, with detection limits down to 100 nm crystallites.
• Second-order nonlinear optical imaging of chiral crystals (SONICC) showed compatibility with standard crystallization platforms.

Conclusions:

• SHG microscopy is a powerful, selective tool for imaging protein microcrystals.
• This technique overcomes limitations of fluorescence imaging by avoiding background noise from non-crystalline forms.
• SONICC offers a sensitive, label-free method for assessing protein crystal formation and size.