Illuminating protein interactions in tissue using confocal and two-photon excitation fluorescent resonance energy transfer microscopy

Mills, James D.; Stone, James L.; Rubin, David G.; Melon, David E.; Okonkwo, David O.; Periasamy, Ammasi; Helm, Gregory A.

doi:10.1117/1.1584443

Cited by 50 publications

(37 citation statements)

References 31 publications

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“…As mentioned in the literature, the intensity based or steady-state protein-protein interaction imaging introduces errors in estimating the distance between the donor and acceptor molecules. Moreover, the spectral bleed-through, or cross talk, is a problem to recognize whether one is observing true sensitized emission, the bleed-through signals, or a combination of both Gordon et al, 1998;Mills et al, 2003;Wallrabe et al, 2003). The strength of the signal also depends on the excitation intensity and the fluorophore concentration.…”

Section: Resultsmentioning

confidence: 98%

Characterization of two‐photon excitation fluorescence lifetime imaging microscopy for protein localization

Chen

Periasamy

2003

Microscopy Res & Technique

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Two-photon excitation fluorescence resonance energy transfer (2P-FRET) imaging microscopy can provide details of specific protein molecule interactions inside living cells. Fluorophore molecules used for 2P-FRET imaging have characteristic absorption and emission spectra that introduce spectral cross-talk (bleed-through) in the FRET signal that should be removed in the 2P-FRET images, to establish that FRET has actually occurred and to have a basis for distance estimations. These contaminations in the FRET signal can be corrected using a mathematical algorithm to extract the true FRET signal. Another approach is 2P-FRET fluorescence lifetime imaging (FLIM). This methodology allows studying the dynamic behavior of protein-protein interactions in living cells and tissues. 2P-FRET-FLIM was used to study the dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). Results show that the reduction in donor lifetime in the presence of acceptor reveals the dimerization of the protein molecules and also determines more precisely the distance between the donor and acceptor. We describe the development and characterization of the 2P-FRET-FLIM imaging system with the Bio-Rad Radiance2100 confocal/multiphoton microscopy system.

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Section: Resultsmentioning

confidence: 98%

Characterization of two‐photon excitation fluorescence lifetime imaging microscopy for protein localization

Chen

Periasamy

2003

Microscopy Res & Technique

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156

128

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“…We developed a method by which one can reliably detect protein-protein interactions within fixed teeth sections through conventional immunohistochemical approaches (Mills et al 2003). FRET detects the proximity of fluorescently labeled molecules over distances <10 nm and is known to be a reliable technique for in situ proteintarget interactions (Wouters et al 1998;Zal et al 2002).…”

Section: Discussionmentioning

confidence: 99%

“…The doubly labeled P8 tissue sections (Appendix Scheme 2; Mills et al 2003) were examined under a 63× 1.4-NA oil immersion objective and 7× zoom. FRET was measured with the acceptor photobleaching method in Leica Microsystems LASAF FRET AB Wizard (Bastiaens and Jovin 1996;Wouters et al 1998;Day et al 2001;Kenworthy 2001;Zal et al 2002).…”

Section: Acceptor Photobleaching Fretmentioning

confidence: 99%

Amelogenin-Ameloblastin Spatial Interaction around Maturing Enamel Rods

et al. 2016

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Amelogenin and ameloblastin are 2 extracellular matrix proteins that are essential for the proper development of enamel. We recently reported that amelogenin and ameloblastin colocalized during the secretory stage of enamel formation when nucleation of enamel crystallites occurs. Direct interactions between the 2 proteins have been also demonstrated in our in vitro studies. Here, we explore interactions between their fragments during enamel maturation. We applied in vivo immunofluorescence imaging, quantitative colocalization analysis, and a new FRET (fluorescence resonance energy transfer) technique to demonstrate ameloblastin and amelogenin interaction in the maturing mouse enamel. Using immunochemical analysis of protein samples extracted from 8-d-old (P8) first molars from mice as a model for maturation-stage enamel, we identified the ~17-kDa ameloblastin (Ambn-N) and the TRAP (tyrosine-rich amelogenin peptide) fragments. We used Ambn-N18 and Ambn-M300 antibodies raised against the N-terminal and C-terminal segments of ameloblastin, as well as Amel-FL and Amel-C19 antibodies against full-length recombinant mouse amelogenin (rM179) and C-terminal amelogenin, respectively. In transverse sections, co-localization images of N-terminal fragments of amelogenin and ameloblastin around the prism boundary revealed the "fish net" pattern of the enamel matrix. Using in vivo FRET microscopy, we further demonstrated spatial interactions between amelogenin and ameloblastin N-terminal fragments. In the maturing mouse enamel, the association of these residual protein fragments created a discontinuity between enamel rods, which we suggest is important for support and maintenance of enamel rods and eventual contribution to unique enamel mechanical properties. We present data that support cooperative functions of enamel matrix proteins in mediating the structural hierarchy of enamel and that contribute to our efforts to design and develop enamel biomimetic material.

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“…TIR-FRET was used to visualize protein -protein interactions on cell membranes and insulin secreting cells (Lam et al 2010;Sohn et al 2010). Two-photon FRET was applied to visualize protein -protein colocalization (Mills et al 2003) and free versus clustered receptor-ligand complexes in the membrane (Wallrabe et al 2003). …”

Section: Applications Of Fret In Lipid Biologymentioning

confidence: 99%

Fluorescence Techniques to Study Lipid Dynamics

Sezgin¹,

Schwille²

2011

Cold Spring Harbor Perspectives in Biology

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Biological research has always tremendously benefited from the development of key methodology. In fact, it was the advent of microscopy that shaped our understanding of cells as the fundamental units of life. Microscopic techniques are still central to the elucidation of biological units and processes, but equally important are methods that allow access to the dimension of time, to investigate the dynamics of molecular functions and interactions. Here, fluorescence spectroscopy with its sensitivity to access the single-molecule level, and its large temporal resolution, has been opening up fully new perspectives for cell biology. Here we summarize the key fluorescent techniques used to study cellular dynamics, with the focus on lipid and membrane systems.T o elucidate cellular processes in their native dynamic environment has been one of the main issues in cell biology over the past decades. The lack of appropriate techniques has long been the main limiting step for the research on dynamic systems, because it was impossible to acquire real time information with the well-known biochemical techniques. The key challenge in dynamically observing biological systems is to combine the ability to resolve moderate to very low concentrations of molecules-because they are simply limited in living cells-on relevant timescales. Relevant timescales in cell biology can be minutes and hours, on a systemic level of cell metabolism, down to the microsecond and even nanosecond regime in which molecular and intramolecular rearrangements take place. With respect to lipidic systems, relevant dynamics range from the local movements of lipids by diffusion to the mechanical transformations of whole membranes, spanning several orders of magnitude in time to be covered. Like for other cellular processes, the investigation of lipids and membranes also in general benefited greatly from the introduction of fluorescence microscopy and spectroscopy to biology. After the 1960s, great technological inventions based on the phenomenon of fluorescence were made, such as confocal microscopy, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), total internal reflection fluorescence (TIRF), and two-photon microscopy, that not only revolutionized imaging but also yielded access to dynamics on previously inaccessible timescales. Another very big step was certainly taken after the introduction of fluorescent proteins, which again accelerated the use of these techniques in living cells and organisms. Nowadays, the technical advancements of fluorescence-based methods allow us to explore systems as small as single molecules with temporal resolution down to the nanoseconds regime. Lately, even the resolution limit of optical microscopy, for a long time being one of the fundamental barriers in elucidating cellular processes, has been overcome by smart applications of the phenomenon of fluorescence.This article aims at giving a short overview on mainly fluorescence-based metho...

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Illuminating protein interactions in tissue using confocal and two-photon excitation fluorescent resonance energy transfer microscopy

Cited by 50 publications

References 31 publications

Characterization of two‐photon excitation fluorescence lifetime imaging microscopy for protein localization

Characterization of two‐photon excitation fluorescence lifetime imaging microscopy for protein localization

Amelogenin-Ameloblastin Spatial Interaction around Maturing Enamel Rods

Fluorescence Techniques to Study Lipid Dynamics

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