The Physical Basis of Total Internal Reflection Fluorescence (TIRF) Microscopy and Its Cellular Applications

Poulter, Natalie S.; Pitkeathly, William T. E.; Smith, Philip J.; Rappoport, Joshua Z.

doi:10.1007/978-1-4939-2080-8_1

Cited by 50 publications

(36 citation statements)

References 53 publications

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“…Having observed these effects exerted by SMIs on cell adhesion and based on previous knowledge that cell surface TG2 promotes integrin clustering (20), we next evaluated how targeting TG2-FN interaction impacts integrins' organization at the interface with FN. TIRF microscopy, which allows high-resolution scanning at the cell-glass substrate interface through an evanescent light wave effect (21,22) demonstrated that b1 integrins distribute evenly after OVCAR5 cells' attachment to FN-coated substrate (Fig. 4D, top; Supplementary Fig.…”

Section: Smis Prevent the Formation Of Stable Cell Contacts With The Fnmentioning

confidence: 99%

Small Molecules Target the Interaction between Tissue Transglutaminase and Fibronectin

Sima

Yakubov

Zhang

et al. 2019

Molecular Cancer Therapeutics

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Tissue transglutaminase (TG2) is a multifunctional protein with enzymatic, GTP-ase, and scaffold properties. TG2 interacts with fibronectin (FN) through its N-terminus domain, stabilizing integrin complexes, which regulate cell adhesion to the matrix. Through this mechanism, TG2 participates in key steps involved in metastasis in ovarian and other cancers. High-throughput screening identified several small molecule inhibitors (SMI) for the TG2/FN complex. Rational medicinal chemistry optimization of the hit compound (TG53) led to second-generation analogues (MT1-6). ELISA demonstrated that these analogues blocked TG2/FN interaction, and bio-layer interferometry (BLI) showed that the SMIs bound to TG2. The compounds also potently inhibited cancer cell adhesion to FN and decreased outside-in signaling mediated through the focal adhesion kinase. Blockade of TG2/FN interaction by the small molecules caused membrane ruffling, delaying the formation of stable focal contacts and mature adhesions points and disrupted organization of the actin cytoskeleton. In an in vivo model measuring intraperitoneal dissemination, MT4 and MT6 inhibited the adhesion of ovarian cancer cells to the peritoneum. Pretreatment with MT4 also sensitized ovarian cancer cells to paclitaxel. The data support continued optimization of the new class of SMIs that block the TG2/FN complex at the interface between cancer cells and the tumor niche.

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Section: Smis Prevent the Formation Of Stable Cell Contacts With The Fnmentioning

confidence: 99%

Small Molecules Target the Interaction between Tissue Transglutaminase and Fibronectin

Sima

Yakubov

Zhang

et al. 2019

Molecular Cancer Therapeutics

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“…Based on the intrinsic features of evanescent illumination, the TIRF image depicts the ventral side of the plasma membrane without interference coming from intracellular fluorescence signals (Fig. S1C) (Poulter et al, 2015). The brightness of the monomeric and dimeric mEGFP tags was calibrated using two control constructs encoding glycosylphosphatidylinositol (GPI)anchored mEGFP and mEGFP-mEGFP, that served as reliable standards in previous studies on cell surface receptors (Hellriegel et al, 2011).…”

Section: Ncam and Fgf2 Induce The Formation Of Fgfr1 Complexes With Dmentioning

confidence: 99%

“…FGFR1-mEGFP population on the ventral cell membrane was imaged by TIRF microscopy, taking advantage of the short penetration of evanescent illumination (Poulter et al, 2015) HeLa cells were serum-starved overnight and imaging started immediately after adding the ligands. Cells with exceedingly high fluorescence (i.e.…”

Section: Tirf Microscopymentioning

confidence: 99%

Number and brightness analysis in live cells reveals that NCAM and FGF2 elicit different assembly and dynamics of FGFR1

Zamai

Trullo

Giordano

et al. 2018

Journal of Cell Science

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Both fibroblast growth factor-2 (FGF2) and neural cell adhesion molecule (NCAM) trigger FGF receptor 1 (FGFR1) signaling; however, they induce remarkably distinct receptor trafficking and cellular responses. The molecular basis of such a dichotomy and the role of distinct types of ligand-receptor interaction remain elusive. Number of molecules and brightness (N&B) analysis revealed that FGF2 and NCAM promote different FGFR1 assembly and dynamics at the plasma membrane. NCAM stimulation elicits long-lasting cycles of short-lived FGFR1 monomers and multimers, a behavior that might reflect a rapid FGFR1 internalization and recycling. FGF2, instead, induces stable dimerization at the dose that stimulates cell proliferation. Reducing the occupancy of FGFR1 in response to low FGF2 doses causes a switch towards cyclically exposed and unstable receptor dimers, consistently with previously reported biphasic response to FGF2 and with the divergent signaling elicited by different ligand concentrations. Similar instability was observed upon altering the endocytic pathway. Thus, FGF2 and NCAM induce differential FGFR1 clustering at the cell surface, which might account for the distinct intracellular fate of the receptor and, hence, for the different signaling cascades and cellular responses.

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“…Depending on refractive indices, the AOI, the wavelength and the polarization of the light source, this evanescent field reaches into the probe with a typical penetration depth of 40200 nm, and a single exponential decay. Since its introduction in fluorescence microscopy by Daniel Axelrod , TIRF microscopy has demonstrated favorable properties in numerous applications, applying living cells or reconstituted proteins (for review see Reference ). TIRF illumination through objectives with high numerical aperture, commonly referred to as objective‐type TIRF microscopy, has become a widespread standard to image signaling events during stimulation of cells with neurotransmitters, hormones, or other modulators .…”

Section: Introductionmentioning

confidence: 99%

Theoretical background of light‐emitting diode total internal reflection fluorescence microscopy and photobleaching lifetime analysis of membrane‐associated proteins—Part II

Schaefer

2020

Journal of Biophotonics

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The selective microscopic imaging of the plasma membrane and adjacent structures by total internal reflection fluorescence (TIRF) microscopy is a versatile and frequently used technique in cell biology. A reduction of imaging artifacts in objective-type TIRF microscopy can be achieved by circular or multi-spot laser illumination or by using noncoherent light sources that are projected into the back focal plane as a light annulus. Light-emitting diode (LED)-based TIRF excitation is a recent advancement of the latter strategy. While some basic principles of LED-TIRF remain the same as in laser-based methods, the calculation of penetration depth, the flatness of illumination and the amount of available illumination power differ. This study provides the theoretical framework for the construction and adjustment of LED-TIRF. Using state-of-the art high power LED emitters, LED-TIRF achieves excitation efficiencies that are comparable to laser-based systems and homogenously illuminate the entire field of view, thus, allowing variation of the penetration depth or quantitative photobleaching-assisted imaging protocols. Using autofluorescent transmembrane, soluble and membrane-attached fusion proteins, we provide examples for a photobleaching-based assessment of the exchange kinetics of proteins within living human endothelial cells.

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The Physical Basis of Total Internal Reflection Fluorescence (TIRF) Microscopy and Its Cellular Applications

Cited by 50 publications

References 53 publications

Small Molecules Target the Interaction between Tissue Transglutaminase and Fibronectin

Small Molecules Target the Interaction between Tissue Transglutaminase and Fibronectin

Number and brightness analysis in live cells reveals that NCAM and FGF2 elicit different assembly and dynamics of FGFR1

Theoretical background of light‐emitting diode total internal reflection fluorescence microscopy and photobleaching lifetime analysis of membrane‐associated proteins—Part II

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