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Published on: June 2, 2019
Denaturing of single electrospun fibrinogen fibers studied by deep ultraviolet fluorescence microscopy
Jeongyong Kim1, Hugeun Song, Inho Park
1Department of Physics, University of Incheon, Incheon 406-772, South Korea. jeongyong@incheon.ac.kr.
Abstract:
Deep ultraviolet (DUV) microscopy is a fluorescence microscopy technique to image unlabeled proteins via the native fluorescence of some of their amino acids. We constructed a DUV fluorescence microscope, capable of 280 nm wavelength excitation by modifying an inverted optical microscope. Moreover, we integrated a nanomanipulator-controlled micropipette into this instrument for precise delivery of picoliter amounts of fluid to selected regions of the sample. In proof-of-principle experiments, we used this instrument to study, in situ, the effect of a denaturing agent on the autofluorescence intensity of single, unlabeled, electrospun fibrinogen nanofibers. Autofluorescence emission from the nanofibers was excited at 280 nm and detected at ∼350 nm. A denaturant solution was discretely applied to small, select sections of the nanofibers and a clear local reduction in autofluorescence intensity was observed. This reduction is attributed to the dissolution of the fibers and the unfolding of proteins in the fibers.
Insights
Deep ultraviolet (DUV) microscopy images unlabeled proteins using native fluorescence. This study demonstrated DUV microscopy with a nanomanipulator to observe protein denaturation in nanofibers.
Area of Science:
- Biophysics
- Materials Science
- Analytical Chemistry
Background:
- Deep ultraviolet (DUV) microscopy utilizes intrinsic amino acid fluorescence for imaging unlabeled proteins.
- Native fluorescence imaging avoids labeling artifacts and simplifies sample preparation.
- Studying protein behavior in nanostructures requires high-resolution techniques with precise manipulation capabilities.
Purpose of the Study:
- To develop and demonstrate a DUV fluorescence microscope integrated with a nanomanipulator for localized sample analysis.
- To investigate the in situ effect of a denaturing agent on the autofluorescence of single fibrinogen nanofibers.
- To correlate changes in autofluorescence intensity with protein structural changes.
Main Methods:
- Construction of a DUV fluorescence microscope with 280 nm excitation capability by modifying an inverted optical microscope.
- Integration of a nanomanipulator-controlled micropipette for precise picoliter fluid delivery.
- Excitation of autofluorescence at 280 nm and detection around 350 nm from electrospun fibrinogen nanofibers.
Main Results:
- A localized reduction in autofluorescence intensity was observed upon application of a denaturant to specific sections of fibrinogen nanofibers.
- The observed autofluorescence decrease is attributed to the dissolution of nanofibers and unfolding of proteins.
- The DUV microscopy system successfully imaged and analyzed the localized effect of the denaturant on protein structure.
Conclusions:
- The developed DUV fluorescence microscope with integrated nanomanipulation is effective for studying localized protein behavior in nanostructures.
- This technique allows for in situ observation of protein denaturation and fiber dissolution at the nanoscale.
- DUV microscopy offers a valuable tool for analyzing unlabeled protein dynamics and structural changes in materials.
