Fluorogenic Protein‐Based Strategies for Detection, Actuation, and Sensing

Gautier, Arnaud; Tebo, Alison G.

doi:10.1002/bies.201800118

Cited by 16 publications

(19 citation statements)

References 95 publications

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“…5 In contrast, fluorogen activating proteins (FAPs) interact non-covalently with their cognate fluorogens to generate a fluorescent complex. 6 Contrary to many self-labeling tags, the fluorogenic nature of these systems means that a fluorophore is initially in a non-fluorescent state, but become fluorescent upon binding. Separate washing steps to remove unbound dye are not required, and therefore dynamic processes can be followed more easily.…”

Section: Introductionmentioning

confidence: 99%

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Tebo

Moeyaert

Thauvin

et al. 2020

Preprint

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Fluorescence microscopy is an indispensable tool in biological research, allowing subsecond and sub-micrometer mapping of molecules or processes inside living cells.Moreover, using spectrally separated fluorophores, one can observe multiple targets simultaneously, leading to a deeper understanding of the dynamic molecular interplays that regulate cell function and fate. Chemogenetic systems, which combine a protein tag and a synthetic fluorophore, provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. However, the fluorophore promiscuity of chemogenetic systems renders two-color applications challenging. Here, we present the engineering of a set of spectrally orthogonal fluorogen activating tags based on the Fluorescence Activating and absorption Shifting Tag (FAST), that are compatible with two-color, live cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one-and two-photon absorption profiles. A two-color cell cycle sensor based on greenFAST and redFAST is capable of detecting very short, early cell cycles in zebrafish development which had previously been difficult to image. Furthermore, this pair of orthogonal tags can be developed into split complementation systems that are capable of detecting multiple protein-protein interactions by live cell fluorescence microscopy. 3

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Section: Introductionmentioning

confidence: 99%

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Tebo

Moeyaert

Thauvin

et al. 2020

Preprint

Self Cite

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“…It is the process which visualizes fluorescent dyes or proteins as labels to study the biological molecules and phenomena [23]. The visualization of these tissues are usually achieved by labelling the proteins via a nanobody, antibody or bio-specific ligand [24]. Fluorescent labelling [25] binds fluorescent dyes covalently to biomolecules, such as proteins, so that they can be differentiated from non-fluorescent (non-bound) biomolecules.…”

Section: Introductionmentioning

confidence: 99%

A Fluorescence-Based Wireless Capsule Endoscopy System for Detecting Colorectal Cancer

Alam

Vedaei

Wahid

2020

Cancers

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Wireless capsule endoscopy (WCE) has been widely used in gastrointestinal (GI) diagnosis that allows the physicians to examine the interior wall of the human GI tract through a pain-free procedure. However, there are still several limitations of the technology, which limits its functionality, ultimately limiting its wide acceptance. Its counterpart, the wired endoscopic system is a painful procedure that demotivates patients from going through the procedure, and adversely affects early diagnosis. Furthermore, the current generation of capsules is unable to automate the detection of abnormality. As a result, physicians are required to spend longer hours to examine each image from the endoscopic capsule for abnormalities, which makes this technology tiresome and error-prone. Early detection of cancer is important to improve the survival rate in patients with colorectal cancer. Hence, a fluorescence-imaging-based endoscopic capsule that automates the detection process of colorectal cancer was designed and developed in our lab. The proof of concept of this endoscopic capsule was tested on porcine intestine and liquid phantom. The proposed WCE system offers great possibilities for future applicability in selective and specific detection of other fluorescently labelled cancers.

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“…With microscopy, cells in the biomaterials are visible by either a genetically transfected fluorescent protein or a chemical probe . Although these labeling techniques are available, not all cell types possess a transfected fluorescent protein and chemical probes are not entirely protein specific . For scientists interested in tracking a specific cell type, protein bound to its membrane, or understanding a cell's secretome in a biomaterial, these experiments rely on immunolabeling with antibodies.…”

Section: Introductionmentioning

confidence: 99%

“…[4,5] Although these labeling techniques are available, not all cell types possess a transfected fluorescent protein and chemical probes are not entirely protein specific. [4,6] For scientists interested in tracking a specific cell type, protein bound to its membrane, or understanding a cell's secretome in a biomaterial, these experiments rely on immunolabeling with antibodies. At present, visualization of immunolabeled proteins by microscopy is to some degree possible in translucent biomaterials (hyaluronic acid, [7] collagen, [5] and Matrigel gels [8] ) although light refraction and/or antibody diffusion are limitations.…”

Section: Introductionmentioning

confidence: 99%

3D Culture Histology Cryosectioned Well Insert Technology Preserves the Structural Relationship between Cells and Biomaterials for Time‐Lapse Analysis of 3D Cultures

Charbonneau

Al‐Samadi

Salo

et al. 2019

Biotechnology Journal

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When performing histology of softer biomaterials, aspiration disrupts the cellular and molecular location information. This study aims to develop a cryosectionable well insert able to preserve the biomaterial and cell's original 3D conformation from the well to histology analysis. The well insert is composed of a paraffin‐coated gelatine pill. Within the coated capsule, the human epithelial cell line (NS‐SV‐AC) is cultured in Matrigel, GrowDex, Myogel, Myogel + GrowDex, or cell culture media for 14 days. At 0 and 14 days, the samples are frozen in liquid nitrogen and cryotome is used to create sections. The slides are stained by Sirius Red and immunohistochemistry using antibodies human collagens I–V and human Ki‐67. Sirius Red shows pink shades of biomaterials and the best cellular vertical distribution throughout the sagittal section of the well is achieved with Matrigel, GrowDex, and Myogel + GrowDex; in Myogel and media, the cells sink. For collagen protein expression, only Matrigel induces a notable difference while in the other materials, collagen staining is weak or difficult to distinguish from endogenous collagens. Ki‐67 expression is maintained over time. The 3D‐cryo well insert provides a new time‐lapse histology perspective of analysis for liquid or gel cultures that maintains cells and macromolecules in their unaltered in‐well configuration.

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Fluorogenic Protein‐Based Strategies for Detection, Actuation, and Sensing

Cited by 16 publications

References 95 publications

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

A Fluorescence-Based Wireless Capsule Endoscopy System for Detecting Colorectal Cancer

3D Culture Histology Cryosectioned Well Insert Technology Preserves the Structural Relationship between Cells and Biomaterials for Time‐Lapse Analysis of 3D Cultures

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