Denaturing of single electrospun fibrinogen fibers studied by deep ultraviolet fluorescence microscopy.
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.
Kim
Jeongyong
J
Department of Physics, University of Incheon, Incheon 406-772, South Korea. jeongyong@incheon.ac.kr.
Song
Hugeun
H
Park
Inho
I
Carlisle
Christine R
CR
Bonin
Keith
K
Guthold
Martin
M
D000596:Amino Acids / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008860:Microscopy, Ultraviolet / Q000295:instrumentation* / Q000379:methods*; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods
D000596:Amino Acids; D005340:Fibrinogen