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Two-photon excited UV fluorescence for protein crystal detection
Jeremy T Madden1, Emma L DeWalt, Garth J Simpson
1Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA.
Acta Crystallographica. Section D, Biological Crystallography
|September 21, 2011
Summary
Two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy offers sensitive protein-crystal detection. This advanced technique complements chiral crystal imaging, providing superior signal and reduced scattering compared to conventional methods.
Area of Science:
- Biophysics
- Crystallography
- Microscopy
Background:
- Conventional ultraviolet fluorescence (UVF) relies on intrinsic aromatic residue fluorescence for contrast.
- Protein crystals typically exhibit higher fluorescence than mother liquor due to concentrated proteins.
Purpose of the Study:
- To explore two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy for sensitive protein-crystal detection.
- To evaluate TPE-UVF as a complementary technique to second-order nonlinear optical imaging of chiral crystals (SONICC).
Main Methods:
- Utilized TPE-UVF microscopy for imaging protein crystals.
- Compared TPE-UVF performance against conventional UVF and SONICC.
Main Results:
- TPE-UVF demonstrates insensitivity to optical scattering, enabling imaging in turbid matrices.
- TPE-UVF offers improved signal-to-noise ratio through background reduction.
- Visible light excitation in TPE-UVF avoids damaging out-of-plane UV excitation.
Conclusions:
- TPE-UVF is a sensitive and advantageous method for protein-crystal detection.
- TPE-UVF integrates well with existing SONICC instrumentation.
- TPE-UVF overcomes limitations of conventional UVF for crystal imaging.
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