Selective detection of protein crystals by second harmonic microscopy.
The unique symmetry properties of second harmonic generation (SHG) microscopy enabled sensitive and selective imaging of protein microcrystals with negligible contributions from solvated proteins or amorphous protein aggregates. In studies of microcrystallites of green fluorescent protein (GFP) prepared in 500 pL droplets, the SHG intensities rivaled those of fluorescence, but with superb selectivity for crystalline regions. GFP in amorphous aggregates and in solution produced substantial background fluorescence, but no detectable SHG. The ratio of the forward-to-backward detected SHG provides a measure of the particle size, suggesting detection limits down to crystallites 100 nm in diameter under low magnification (10x). In addition to being sensitive and highly selective, second-order nonlinear optical imaging of chiral crystals (SONICC) is directly compatibility with virtually all common protein crystallization platforms.
Wampler
Ronald D
RD
Department of Chemistry, Purdue University, West Lafayette, Indiana 47906, USA.
Kissick
David J
DJ
Dehen
Christopher J
CJ
Gualtieri
Ellen J
EJ
Grey
Jessica L
JL
Wang
Hai-Feng
HF
Thompson
David H
DH
Cheng
Ji-Xin
JX
Simpson
Garth J
GJ
D003460:Crystallization; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods; D008859:Microscopy, Polarization / Q000379:methods*; D009113:Muramidase / Q000032:analysis / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013997:Time Factors
D049452:Green Fluorescent Proteins; D009113:Muramidase