Time Gated Luminescence Imaging of Immunolabeled Human Tissues. 2017

Ting Chen, and Rui Hong, and Darren Magda, and Christopher Bieniarz, and Larry Morrison, and Lawrence W Miller
Department of Chemistry, University of Illinois at Chicago , 845 West Taylor Street, Chicago, Illinois 60607, United States.

Multiplexed immunofluorescence imaging of formalin-fixed, paraffin-embedded tissues is a powerful tool for investigating proteomic profiles and diagnosing disease. However, conventional immunofluorescence with organic dyes is limited in the number of colors that can be simultaneously visualized, is made less sensitive by tissue autofluorescence background, and is usually incompatible with commonly used hematoxylin and eosin staining. Herein, we demonstrate the comparative advantages of using time-gated luminescence microscopy in combination with an emissive Tb(III) complex, Lumi4-Tb, for tissue imaging in terms of sensitivity, multiplexing potential, and compatibility with common immunohistochemistry protocols. We show that time-gated detection of millisecond-scale Tb(III) emission increases signal-to-noise ratio relative to conventional steady-state detection of organic dye fluorescence and permits visualization of low-abundance tissue markers such as Bcl-6 or MSH-6. In addition, temporal separation of long- and short-lifetime (∼nanosecond) signals adds a second dimension for multiplexing and also permits detection of intermolecular Tb(III)-to-dye Förster resonance energy transfer. Furthermore, we demonstrate that the Lumi4-Tb complex is compatible with tyramide signal amplification and, unlike conventional organic dyes, can be reliably used on tissue stained with hematoxylin and eosin. Our results indicate that time-gated luminescence microscopy using Tb(III) labels can provide a sensitive and robust method to perform multiplexed immunofluorescence on archived or clinical tissue specimens.

UI MeSH Term Description Entries
D008853 Microscopy The use of instrumentation and techniques for visualizing material and details that cannot be seen by the unaided eye. It is usually done by enlarging images, transmitted by light or electron beams, with optical or magnetic lenses that magnify the entire image field. With scanning microscopy, images are generated by collecting output from the specimen in a point-by-point fashion, on a magnified scale, as it is scanned by a narrow beam of light or electrons, a laser, a conductive probe, or a topographical probe. Compound Microscopy,Hand-Held Microscopy,Light Microscopy,Optical Microscopy,Simple Microscopy,Hand Held Microscopy,Microscopy, Compound,Microscopy, Hand-Held,Microscopy, Light,Microscopy, Optical,Microscopy, Simple
D005455 Fluorescent Antibody Technique Test for tissue antigen using either a direct method, by conjugation of antibody with fluorescent dye (FLUORESCENT ANTIBODY TECHNIQUE, DIRECT) or an indirect method, by formation of antigen-antibody complex which is then labeled with fluorescein-conjugated anti-immunoglobulin antibody (FLUORESCENT ANTIBODY TECHNIQUE, INDIRECT). The tissue is then examined by fluorescence microscopy. Antinuclear Antibody Test, Fluorescent,Coon's Technique,Fluorescent Antinuclear Antibody Test,Fluorescent Protein Tracing,Immunofluorescence Technique,Coon's Technic,Fluorescent Antibody Technic,Immunofluorescence,Immunofluorescence Technic,Antibody Technic, Fluorescent,Antibody Technics, Fluorescent,Antibody Technique, Fluorescent,Antibody Techniques, Fluorescent,Coon Technic,Coon Technique,Coons Technic,Coons Technique,Fluorescent Antibody Technics,Fluorescent Antibody Techniques,Fluorescent Protein Tracings,Immunofluorescence Technics,Immunofluorescence Techniques,Protein Tracing, Fluorescent,Protein Tracings, Fluorescent,Technic, Coon's,Technic, Fluorescent Antibody,Technic, Immunofluorescence,Technics, Fluorescent Antibody,Technics, Immunofluorescence,Technique, Coon's,Technique, Fluorescent Antibody,Technique, Immunofluorescence,Techniques, Fluorescent Antibody,Techniques, Immunofluorescence,Tracing, Fluorescent Protein,Tracings, Fluorescent Protein
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D013725 Terbium An element of the rare earth family of metals. It has the atomic symbol Tb, atomic number 65, and atomic weight 158.92.
D014066 Palatine Tonsil A round-to-oval mass of lymphoid tissue embedded in the lateral wall of the PHARYNX. There is one on each side of the oropharynx in the fauces between the anterior and posterior pillars of the SOFT PALATE. Tonsil,Tonsil, Palatine,Palatine Tonsils,Tonsils,Tonsils, Palatine
D049408 Luminescent Agents Compound such as LUMINESCENT PROTEINS that cause or emit light (PHYSICAL LUMINESCENCE). Phosphorescent Agents,Agents, Luminescent,Agents, Phosphorescent
D056831 Coordination Complexes Neutral or negatively charged ligands bonded to metal cations or neutral atoms. The number of ligand atoms to which the metal center is directly bonded is the metal cation's coordination number, and this number is always greater than the regular valence or oxidation number of the metal. A coordination complex can be negative, neutral, or positively charged. Metal Complexes,Complexes, Coordination,Complexes, Metal
D031541 Fluorescence Resonance Energy Transfer A type of FLUORESCENCE SPECTROSCOPY using two FLUORESCENT DYES with overlapping emission and absorption spectra, which is used to indicate proximity of labeled molecules. This technique is useful for studying interactions of molecules and PROTEIN FOLDING. Forster Resonance Energy Transfer

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