Polarization-resolved second-harmonic generation microscopy as a method to visualize protein-crystal domains

Abstract

Polarization-resolved second-harmonic generation (PR-SHG) microscopy is described and applied to identify the presence of multiple crystallographic domains within protein-crystal conglomerates, which was confirmed by synchrotron X-ray diffraction. Principal component analysis (PCA) of PR-SHG images resulted in principal component 2 (PC2) images with areas of contrasting negative and positive values for conglomerated crystals and PC2 images exhibiting uniformly positive or uniformly negative values for single crystals. Qualitative assessment of PC2 images allowed the identification of domains of different internal ordering within protein-crystal samples as well as differentiation between multi-domain conglomerated crystals and single crystals. PR-SHG assessments of crystalline domains were in good agreement with spatially resolved synchrotron X-ray diffraction measurements. These results have implications for improving the productive throughput of protein structure determination through early identification of multi-domain crystals.

Imaging the Nonlinear Susceptibility Tensor of Collagen by Nonlinear Optical Stokes Ellipsometry

“…Incorporation of polarization-dependent analysis into SHG imaging has been used for the discrimination of different biological tissues (15), differentiation of tumor and normal tissues (16), and many other biological applications as well as the study of surfaces and crystals (17)(18)(19)(20)(21)(22)(23). Despite the success of previous polarization-dependent SHG imaging approaches, two major limitations exist.…”

Current advances in synchrotron radiation instrumentation for MX experiments

“…UV light provided by a laser or LED induces fluorescence from aromatic amino acids between 300–450 nm, which is readily detected by a standard microscope giving high contrast between crystals and the surrounding mother liquor. Second-order nonlinear optical imaging of chiral crystals (SONICC) has also recently been explored for imaging protein crystals (Kissick et al , 2010, DeWalt et al , 2013, Closser et al , 2013). This allows identification of protein crystals within opaque amorphous material such as lipidic cubic phase (LCP).…”

Connection of Jones and Mueller Tensors in Second Harmonic Generation and Multi-Photon Fluorescence Measurements

“…While the change in ordering associated with the matrix E is not a fundamental necessity and is a tedious additional step, reordering the indices into the ${\stackrel{false\rightharpoonup }{\chi }}_J^{\ast }\otimes {\stackrel{false\rightharpoonup }{\chi }}_J$ form has two key practical advantages: (i) the location of any particular combination of Jones tensor elements can be easily identified by simple binary counting as described in Appendix IV, and (ii) expressions connecting the ${\stackrel{false\rightharpoonup }{\chi }}_J^{\ast }\otimes {\stackrel{false\rightharpoonup }{\chi }}_J$ ordering to laboratory-frame observables for several different experimental designs are already well-established. 19,46,47 Substitution leads to the following concise expression connecting the Jones and Mueller tensors for rank three nonlinear optical processes. $$\stackrel{\rightharpoonup }{M}=[A\otimes {false(A^{-1}false)}^{\mathrm{T}}\otimes {false(A^{-1}false)}^{\mathrm{T}}]\cdot E\cdot false({\stackrel{false\rightharpoonup }{\chi }}_J^{\ast }\otimes {\stackrel{false\rightharpoonup }{\chi }}_{J}false)$$…”