Abstract
Deep-ultraviolet excitation fluorescence microscopy has enabled molecular imaging having an optical sectioning capability with a wide-field configuration and its usefulness for slide-free pathology has been shown in recent years. Here, we report usefulness of terbium ions as RNA-specific labeling probes for slide-free pathology with deep-ultraviolet excitation fluorescence. On excitation in the wavelength range of 250–300 nm, terbium ions emitted fluorescence after entering cells. Bright fluorescence was observed at nucleoli and cytoplasm while fluorescence became weak after RNA decomposition by ribonuclease prior to staining. It was also found that the fluorescence intensity at nucleoplasm increased with temperature during staining and that this temperature-dependent behavior resembled temperature-dependent hypochromicity of DNA due to melting. These findings indicated that terbium ions stained single-stranded nucleic acid more efficiently than double-stranded nucleic acid. We further combined terbium ions and DNA-specific dyes for dual-color imaging. In the obtained image, nucleolus, nucleoplasm, and cytoplasm were distinguished. We demonstrated the usefulness of dual-color imaging for rapid diagnosis of surgical specimen by showing optical sectioning of unsliced tissues. The present findings can enhance deep-ultraviolet excitation fluorescence microscopy and consequently expand the potential of fluorescence microscopy in life sciences.
Highlights
Fluorescence microscopes are invaluable tools in the life sciences
No study has been dedicated to establishing a labeling probe for DUV excitation fluorescence microscopy, but just a portion of the fluorescence dyes generally used for visible light excitation (e.g., eosin, Rhodamine, Hoechst, and propidium iodide (PI)) has been diverted[11,12,13,14,15]
We explored the potential usefulness of the multicolor imaging for slide-free pathology as one advantage of DUV excitation fluorescence microscopy is the capability of optical sectioning of the sample surface with a wide-field microscope configuration[9,10,11,12]
Summary
Fluorescence microscopes are invaluable tools in the life sciences. They have been widely used by life scientists for analyzing distributions of molecules and cells of interest, as well as cellular and subcellular structures. Advanced techniques, such as super resolution imaging[1,2,3], three-dimensional imaging[4,5], and high-speed imaging[6,7], have been increasing the importance of fluorescence microscopy in life sciences In such fluorescence microscopes, visible light (400–700 nm in wavelength, λ) and longer-wavelength ultraviolet (UV) A light (λ = 360–400 nm) are used in general for exciting labeling probes. By staining a cell with a mixture of Tb3+ and commercial DNA-specific staining dyes, a multicolor fluorescence image where nucleoplasm, nucleoli, and cytoplasm are distinguishable is acquired at DUV excitation As this multiplex staining does not require any time-consuming and complex procedure, DUV excitation multicolor imaging is useful as an alternative to conventional staining in histopathology for rapid structural imaging. We expect that the present study enhancing DUV excitation fluorescence microscopy can expand the potential of fluorescence microscopy by enabling biomedical applications that cannot be implemented with visible and NIR excitation
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