skip to main content

Title: Imaging of Protein Crystals with Two-Photon Microscopy

Second-order nonlinear optical imaging of chiral crystals (SONICC), which portrays second-harmonic generation (SHG) by noncentrosymmetric crystals, is emerging as a powerful imaging technique for protein crystals in media opaque to visible light because of its high signal-to-noise ratio. Here we report the incorporation of both SONICC and two-photon excited fluorescence (TPEF) into one imaging system that allows visualization of crystals as small as 10 {mu}m in their longest dimension. Using this system, we then documented an inverse correlation between the level of symmetry in examined crystals and the intensity of their SHG. Moreover, because of blue-green TPEF exhibited by most tested protein crystals, we also could identify and image SHG-silent protein crystals. Our experimental data suggest that the TPEF in protein crystals is mainly caused by the oxidation of tryptophan residues. Additionally, we found that unspecific fluorescent dyes are able to bind to lysozyme crystals and enhance their detection by TPEF. We finally confirmed that the observed fluorescence was generated by a two-photon rather than a three-photon process. The capability for imaging small protein crystals in turbid or opaque media with nondamaging infrared light in a single system makes the combination of SHG and intrinsic visible TPEF a powerful toolmore » for nondestructive protein crystal identification and characterization during crystallization trials.« less
Authors:
; ; ; ;  [1] ;  [2]
  1. (Case Western)
  2. (
Publication Date:
OSTI Identifier:
1038277
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry (Eaton); Journal Volume: 51; Journal Issue: 8
Research Org:
Advanced Photon Source (APS), Argonne National Laboratory (ANL), Argonne, IL (US)
Sponsoring Org:
NIH
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; CRYSTALLIZATION; DETECTION; DYES; EXCITATION; FLUORESCENCE; FLUORESCENCE SPECTROSCOPY; HARMONIC GENERATION; IMAGES; INFRARED RADIATION; LYSOZYME; MICROSCOPY; MULTI-PHOTON PROCESSES; OPTICAL PROPERTIES; OXIDATION; PROTEINS; RESIDUES; SIGNAL-TO-NOISE RATIO; SYMMETRY; TRYPTOPHAN; VISIBLE SPECTRA