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Title: Imaging of Protein Crystals with Two-Photon Microscopy

Abstract

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:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1038277
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry (Eaton); Journal Volume: 51; Journal Issue: 8
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

Citation Formats

Padayatti, Pius, Palczewska, Grazyna, Sun, Wenyu, Palczewski, Krzysztof, Salom, David, and Polgenix). Imaging of Protein Crystals with Two-Photon Microscopy. United States: N. p., 2012. Web. doi:10.1021/bi201682q.
Padayatti, Pius, Palczewska, Grazyna, Sun, Wenyu, Palczewski, Krzysztof, Salom, David, & Polgenix). Imaging of Protein Crystals with Two-Photon Microscopy. United States. doi:10.1021/bi201682q.
Padayatti, Pius, Palczewska, Grazyna, Sun, Wenyu, Palczewski, Krzysztof, Salom, David, and Polgenix). Wed . "Imaging of Protein Crystals with Two-Photon Microscopy". United States. doi:10.1021/bi201682q.
@article{osti_1038277,
title = {Imaging of Protein Crystals with Two-Photon Microscopy},
author = {Padayatti, Pius and Palczewska, Grazyna and Sun, Wenyu and Palczewski, Krzysztof and Salom, David and Polgenix)},
abstractNote = {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 tool for nondestructive protein crystal identification and characterization during crystallization trials.},
doi = {10.1021/bi201682q},
journal = {Biochemistry (Eaton)},
number = 8,
volume = 51,
place = {United States},
year = {Wed May 02 00:00:00 EDT 2012},
month = {Wed May 02 00:00:00 EDT 2012}
}
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