In this research, we demonstrate the enhanced autofluorescence and high-sensitivity bioimaging of intracellular organelles using DUV-SPR. The Kretschmann configuration is used for excitation of DUV-SPR. We used an aluminum thickness of 24 nm. The alumina surface was estimated to be 6 nm by comparison between the experimental and calculated results. Reflectance after culturing of cells was measured. DUV-SPR is excited at an incident angle of 52° after the biological samples are cultured.
MC3T3-E1 cells as Label-free cells are directly cultured on an aluminum and glass surfaces, and they were cultured on the both substrates in an incubator. Autofluorescence spectra excited of the label-free MC3T3-E1 cells was measured by 266-nm exictation. The autofluorescence intensity for the aluminum is higher than that for the glass. In the autofluorescence spectra, MC3T3-E1 cells exhibited two fluorescence peaks, which were located around 330 and 500 nm. These 330 and 500 nm emissions indicate aromatic amino acid and mitochondria, respectively. Both of the ehnahcement factors were 8 times.
We also observed autofluorescence of aromatic amino acid and mitochondrial NADH in the label-free MC3T3-E1 cells cultured on the aluminum and glass surfaces. In the autofluorescence image with DUV-SPR, organelles can be clearly observed in the MC3T3-E1 cells. On the other hand, the autofluorescence intensity is very weak in the image without DUV-SPR.
Accordingly, DUV-SPR can facilitate the observation of proteins, DNA in nucleus, and other structures that cannot be excited by visible light. DUV-SPR is shown to be a powerful technique for acquiring high-sensitivity label-free observation of biological samples.
Yoshimasa Kawata, Masakazu Kikawada, Atsushi Ono, and Wataru Inami, "Sensitive imaging of organelles in label-free cells by surface plasmon resonance in deep-ultraviolet region
(Conference Presentation)," Proc. SPIE 9926, UV and Higher Energy Photonics: From Materials to Applications, 99260O (Presented at SPIE Nanoscience + Engineering: August 30, 2016; Published: 11 November 2016); https://doi.org/10.1117/12.2236857.5161498121001.
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