Journal articles: 'TIRF'

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Treves, Susan, and Francesco Zorzato. "TIRF." Imaging & Microscopy 11, no. 3 (August 2009): 52–53. http://dx.doi.org/10.1002/imic.200990065.

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Truskey, G. A., J. S. Burmeister, E. Grapa, and W. M. Reichert. "Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances." Journal of Cell Science 103, no. 2 (October 1, 1992): 491–99. http://dx.doi.org/10.1242/jcs.103.2.491.

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A simplified model of TIRF optics was used to quantitate the relative membrane/substratum separation distances from the spatial pattern of TIRF image brightness. Phase-contrast and total internal reflection fluorescence microscopy (TIRFM) images were collected of bovine aortic endothelial cells (BAEC) plated onto glass microscope slides for 15 min, 30 min and 24 h. BAEC adherent for 15 min showed an absence of a focal contact morphology, with the region of closest apposition beneath the cell center. After 30 min, multiple contacts with the surface were established and the morphology became more irregular. BAEC attached for 24 h showed well-defined focal contact regions aligned in characteristically striated patterns. The relative distance between closest and farthest membrane/substratum separations are consistent with reported distance between focal and matrix contacts. Topographical maps of membrane/substratum separation distances over the entire ventral surface of the plated cells were constructed to demonstrate the utility of quantitative TIRF microscopy.

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Fu, Yan, Peter W. Winter, Raul Rojas, Victor Wang, Matthew McAuliffe, and George H. Patterson. "Axial superresolution via multiangle TIRF microscopy with sequential imaging and photobleaching." Proceedings of the National Academy of Sciences 113, no. 16 (April 1, 2016): 4368–73. http://dx.doi.org/10.1073/pnas.1516715113.

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We report superresolution optical sectioning using a multiangle total internal reflection fluorescence (TIRF) microscope. TIRF images were constructed from several layers within a normal TIRF excitation zone by sequentially imaging and photobleaching the fluorescent molecules. The depth of the evanescent wave at different layers was altered by tuning the excitation light incident angle. The angle was tuned from the highest (the smallest TIRF depth) toward the critical angle (the largest TIRF depth) to preferentially photobleach fluorescence from the lower layers and allow straightforward observation of deeper structures without masking by the brighter signals closer to the coverglass. Reconstruction of the TIRF images enabled 3D imaging of biological samples with 20-nm axial resolution. Two-color imaging of epidermal growth factor (EGF) ligand and clathrin revealed the dynamics of EGF-activated clathrin-mediated endocytosis during internalization. Furthermore, Bayesian analysis of images collected during the photobleaching step of each plane enabled lateral superresolution (<100 nm) within each of the sections.

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Foylan, S., W. B. Amos, J. Dempster, L. Kölln, C. G. Hansen, M. Shaw, and G. McConnell. "MesoTIRF: A prism-based Total Internal Reflection Fluorescence illuminator for high resolution, high contrast imaging of large cell populations." Applied Physics Letters 122, no. 11 (March 13, 2023): 113701. http://dx.doi.org/10.1063/5.0133032.

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Total internal reflection fluorescence (TIRF) illumination bypasses the axial diffraction limit of light by using an evanescent field to excite fluorophores close to a sample substrate. However, standard TIRF imaging through the objective requires a high numerical aperture (NA) to generate the evanescent wave. Available lenses have a high magnification with a correspondingly small field of view—ranging from [Formula: see text]50 μm to 1 mm in diameter. Switching to the older prism-TIRF configuration introduced by Axelrod in the 1980s might seem to remove the requirement for high objective NA and allow the use of existing large-field objectives. Unfortunately, these lenses are unsuitable because their throughput of light is too low for TIRF imaging. As such, high sensitivity TIRF imaging over a much larger mesoscopic field has yet to be demonstrated. We have developed a prism-based TIRF illuminator for the Mesolens—a highly corrected objective lens with an unparalleled ratio of NA to magnification. The imaging field of the Mesolens is 204 times larger than that of the TIRF objectives previously described, increasing the optical throughput of the optical system by a factor of 25 compared to an off-the-shelf microscope objective of the same magnification. We demonstrate MesoTIRF imaging of cell specimens and show the multi-wavelength capability of the modality across more than 700 cells in a single image.

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Lin, Jia, and Adam D. Hoppe. "Uniform Total Internal Reflection Fluorescence Illumination Enables Live Cell Fluorescence Resonance Energy Transfer Microscopy." Microscopy and Microanalysis 19, no. 2 (March 11, 2013): 350–59. http://dx.doi.org/10.1017/s1431927612014420.

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AbstractFluorescence resonance energy transfer (FRET) microscopy is a powerful technique to quantify dynamic protein-protein interactions in live cells. Total internal reflection fluorescence (TIRF) microscopy can selectively excite molecules within about 150 nm of the glass-cell interface. Recently, these two approaches were combined to enable high-resolution FRET imaging on the adherent surface of living cells. Here, we show that interference fringing of the coherent laser excitation used in TIRF creates lateral heterogeneities that impair quantitative TIRF-FRET measurements. We overcome this limitation by using a two-dimensional scan head to rotate laser beams for donor and acceptor excitation around the back focal plane of a high numerical aperture objective. By setting different radii for the circles traced out by each laser in the back focal plane, the penetration depth was corrected for different wavelengths. These modifications quell spatial variations in illumination and permit calibration for quantitative TIRF-FRET microscopy. The capability of TIRF-FRET was demonstrated by imaging assembled cyan and yellow fluorescent protein–tagged HIV-Gag molecules in single virions on the surfaces of living cells. These interactions are shown to be distinct from crowding of HIV-Gag in lipid rafts.

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Lanni, F., A. S. Waggoner, and D. L. Taylor. "Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy." Journal of Cell Biology 100, no. 4 (April 1, 1985): 1091–102. http://dx.doi.org/10.1083/jcb.100.4.1091.

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We studied the laminar organization of 3T3 fibroblast cells growing on glass slides by use of total internal reflection illumination to excite fluorescence emission (TIRF) from labeled molecules and stained cellular compartments that are very close to the cell-substrate contact region. Mitochondria, distant from the contact regions and stained with the water-soluble cationic dye, dil-C3-(3), fluoresced only as the glass/cytoplasm critical angle was approached. A similar result was obtained when the nuclei were stained with Hoechst dye 33342. From this measured angle a cytoplasmic refractive index in the range 1.358-1.374 was computed. The plasma membrane of 3T3 cells was stained with dil-C18-(3), and the cytoplasmic compartment was stained with fluoresceinyl-dextran (FTC-dextran) or with carboxyfluorescein. We have demonstrated a high degree of correspondence between the low-reflectance zones in the reflection interference image of a live cell and the TIRF images of both the plasma membrane and cytoplasmic compartment. TIRF photometry of selected contact regions of cells provided data from which the absolute separation of cell and substrate was computed. From a population of 3T3 cells microinjected with fluorescein-labeled actin, motile and adherent interphase cells were selected for study. For adherent cells, which displayed fluorescent stress fibers, the TIRF image was composed of intense patches and less intense regions that corresponded, respectively, to the focal contact and close-contact zones of the reflection-interference image. The intense patches corresponded to the endpoints of the stress fibers. Cells of motile morphology, which formed some focal contacts and extensive close-contact zones, gave AF-actin TIRF images of relatively even intensity. Thin lamellar regions of the cytoplasm were found to contain concentrations of actin not significantly different from other close-contact regions of the cell. The major analytical problem of TIRF microscopy is separation of the effects of proximity to substrate, refractive index, and fluorescent probe concentration on the local brightness of the TIRF image. From our results, it appears possible to use TIRF microscopy to measure the proximity of different components of substrate contact regions of cells.

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Morelo Pereira, Douglas Jahir, and Diocelina Torres Castro. "Técnicas e indicadores de rendimiento financiero aplicados al estado de resultados en empresas comerciales y de servicios colombianas." Cuadernos de Contabilidad 22 (August 19, 2021): 1–21. http://dx.doi.org/10.11144/javeriana.cc22.tirf.

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Este artículo está dirigido a identificar el grado de utilización de un conjunto de Técnicas e Indicadores de Rendimiento Financiero (TIRF) que permitan monitorear la sostenibilidad financiera de las empresas comerciales y de servicios. El problema radica en las frecuentes reformas tributarias y la constante lucha contra el contrabando; actividades que en su conjunto conjugan una amenaza a la sostenibilidad financiera de la empresa. En este sentido, la presente investigación busca analizar las características predominantes, diferenciales y correlacionales de las TIRF asociadas al estado de resultados. El estudio se desarrolló bajo el paradigma cuantitativo lo que permitió aplicar un cuestionario de nuevo diseño, la muestra estuvo conformada por 90 expertos con funciones asociadas al análisis de estados financieros vinculados a 55 empresas comerciales y 35 empresas de servicios seleccionadas de un listado de 1.000. Entre las conclusiones se determinó que las empresas de servicios tienden a realizar más proyecciones de tendencia y benchmarking contra un competidor clave que las empresas de comerciales. Situación que permite inferir la versatilidad y el dinamismo de estas TIRF en el análisis de estados financieros.

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Gingell, D., O. S. Heavens, and J. S. Mellor. "General electromagnetic theory of total internal reflection fluorescence: the quantitative basis for mapping cell-substratum topography." Journal of Cell Science 87, no. 5 (June 1, 1987): 677–93. http://dx.doi.org/10.1242/jcs.87.5.677.

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Total internal reflection fluorescence (TIRF) has recently been used to look at the contacts made between cells and a glass surface on which they are spread. Our method utilizes the fluorescence of a water-soluble dye that acts as an extracellular aqueous volume marker. Fluorescence is stimulated by the short-range electric field near the glass surface that exists under conditions of total internal reflection. Since fluorescence is normally generated beneath a spread cell and not beyond it, the fluorescence of the image is related to the size of the cell-glass water gap. The images obtained are remarkable for their detail, contrast and the absence of confusing granularity due to cytoplasmic heterogeneity, which is commonly seen in interference reflection (IRM) images. We here develop a rigorous electromagnetic theory of total internal reflection in layered structures appropriate for cell contacts and apply it to quantitative TIRF. We show that: (1) TIRF, unlike IRM, can report cell-glass gaps in a way that is practically independent of the detailed physical properties of the cell; (2) TIRF is also far more sensitive than IRM for measuring cell-glass water gaps up to approximately equal to 100nm. These striking results explain the image quality seen by TIRF. As the initial step towards verifying our theory we show that measurement of the fluorescence stimulated by total internal reflection at a simple glass-water interface matches theoretical predictions.

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9

Ellefsen, Kyle L., Joseph L. Dynes, and Ian Parker. "Spinning-Spot Shadowless TIRF Microscopy." PLOS ONE 10, no. 8 (August 26, 2015): e0136055. http://dx.doi.org/10.1371/journal.pone.0136055.

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10

Balaa, Karla, Emmanuel Fort, and Nikon Instruments. "Surface Plasmon Enhanced TIRF Imaging." Imaging & Microscopy 11, no. 4 (October 30, 2009): 55–56. http://dx.doi.org/10.1002/imic.200990091.

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11

Fairlamb, Max S., Amy M. Whitaker, Fletcher E. Bain, Maria Spies, and Bret D. Freudenthal. "Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules." Biology 10, no. 7 (June 23, 2021): 571. http://dx.doi.org/10.3390/biology10070571.

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Single-molecule total internal reflection fluorescence (TIRF) microscopy allows for the real-time visualization of macromolecular dynamics and complex assembly. Prism-based TIRF microscopes (prismTIRF) are relatively simple to operate and can be easily modulated to fit the needs of a wide variety of experimental applications. While building a prismTIRF microscope without expert assistance can pose a significant challenge, the components needed to build a prismTIRF microscope are relatively affordable and, with some guidance, the assembly can be completed by a determined novice. Here, we provide an easy-to-follow guide for the design, assembly, and operation of a three-color prismTIRF microscope which can be utilized for the study of macromolecular complexes, including the multi-component protein–DNA complexes responsible for DNA repair, replication, and transcription. Our hope is that this article can assist laboratories that aspire to implement single-molecule TIRF techniques, and consequently expand the application of this technology.

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Olevsko-Arad, Ilya, Moshe Feldberg, Martin Oheim, and Adi Salomon. "Colour-coded nanoscale calibration and optical quantification of axial fluorophore position." EPJ Web of Conferences 287 (2023): 09034. http://dx.doi.org/10.1051/epjconf/202328709034.

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Total internal reflection fluorescence (TIRF) has come of age, but a reliable and easy-to-use tool for calibrating evanescent-wave penetration depths is missing. We provide a test-sample for TIRF and other axial super-resolution microscopies for emitter axial calibration. Our originality is that nanometer(nm) distances along the microscope’s optical axis are color-encoded in the form of a multi-layered multi-colored transparent sandwich. Emitter layers are excited by the same laser but they emit in different colors. Layers are deposited in a controlled manner onto a glass substrate and protected with a non-fluorescent polymer. Decoding the penetration depth of the exciting evanescent field, by spectrally unmixing of multi-colored samples is presented as well. Our slide can serve as a test sample for quantifying TIRF, but also as an axial ruler for nm-axial distance measurements in single-molecule localization microscopies, supercritical-angle fluorescence, and related super-resolution.

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Keffer, J. L., C. R. Sabanayagam, M. E. Lee, E. F. DeLong, M. W. Hahn, and J. A. Maresca. "Using Total Internal Reflection Fluorescence Microscopy To Visualize Rhodopsin-Containing Cells." Applied and Environmental Microbiology 81, no. 10 (March 13, 2015): 3442–50. http://dx.doi.org/10.1128/aem.00230-15.

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ABSTRACTSunlight is captured and converted to chemical energy in illuminated environments. Although (bacterio)chlorophyll-based photosystems have been characterized in detail, retinal-based photosystems, rhodopsins, have only recently been identified as important mediators of light energy capture and conversion. Recent estimates suggest that up to 70% of cells in some environments harbor rhodopsins. However, because rhodopsin autofluorescence is low—comparable to that of carotenoids and significantly less than that of (bacterio)chlorophylls—these estimates are based on metagenomic sequence data, not direct observation. We report here the use of ultrasensitive total internal reflection fluorescence (TIRF) microscopy to distinguish between unpigmented, carotenoid-producing, and rhodopsin-expressing bacteria.Escherichia colicells were engineered to produce lycopene, β-carotene, or retinal. A gene encoding an uncharacterized rhodopsin, actinorhodopsin, was cloned into retinal-producingE. coli. The production of correctly folded and membrane-incorporated actinorhodopsin was confirmed via development of pink color inE. coliand SDS-PAGE. Cells expressing carotenoids or actinorhodopsin were imaged by TIRF microscopy. The 561-nm excitation laser specifically illuminated rhodopsin-containing cells, allowing them to be differentiated from unpigmented and carotenoid-containing cells. Furthermore, water samples collected from the Delaware River were shown by PCR to have rhodopsin-containing organisms and were examined by TIRF microscopy. Individual microorganisms that fluoresced under illumination from the 561-nm laser were identified. These results verify the sensitivity of the TIRF microscopy method for visualizing and distinguishing between different molecules with low autofluorescence, making it useful for analyzing natural samples.

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Rüttinger, Steffen, Baptiste Lamarre, and Alex E. Knight. "Single Molecule Genotyping by TIRF Microscopy." Journal of Fluorescence 18, no. 5 (June 14, 2008): 1021–26. http://dx.doi.org/10.1007/s10895-008-0386-2.

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Castell, Oliver K., James M. Berridge, and Mark I. Wallace. "TIRF Imaging of Lipid Bilayer Arrays." Biophysical Journal 100, no. 3 (February 2011): 317a. http://dx.doi.org/10.1016/j.bpj.2010.12.1932.

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Tonzani, Stefano. "TIRF: imaging at the cellular edge." Nature Cell Biology 11, S1 (October 2009): S16. http://dx.doi.org/10.1038/ncb1933.

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Greb, Christoph, Ralf Jacob, and Anja Schué. "TIRF-Mikroskopie auf den Kopf gestellt." BIOspektrum 17, no. 3 (May 2011): 314–16. http://dx.doi.org/10.1007/s12268-011-0050-2.

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Denham, Simon, and Deborah Cutchey. "Total Internal Reflection Fluorescence (TIRF) Microscopy." Imaging & Microscopy 11, no. 2 (May 2009): 54–55. http://dx.doi.org/10.1002/imic.200990043.

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Abramova, Natalia, Odeniel Sertil, Sapna Mehta, and Charles V. Lowry. "Reciprocal Regulation of Anaerobic and Aerobic Cell Wall Mannoprotein Gene Expression in Saccharomyces cerevisiae." Journal of Bacteriology 183, no. 9 (May 1, 2001): 2881–87. http://dx.doi.org/10.1128/jb.183.9.2881-2887.2001.

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ABSTRACT The DAN/TIR genes encode nine cell wall mannoproteins in Saccharomyces cerevisiae which are expressed during anaerobiosis (DAN1,DAN2, DAN3, DAN4,TIR1, TIR2, TIR3,TIR4, and TIP1). Most are expressed within an hour of an anaerobic shift, but DAN2 andDAN3 are expressed after about 3 h. At the same time, CWP1 and CWP2, the genes encoding the major mannoproteins, are down-regulated, suggesting that there is a programmed remodeling of the cell wall in which Cwp1 and Cwp2 are replaced by nine anaerobic counterparts. TIP1,TIR1, TIR2, and TIR4 are also induced during cold shock. Correspondingly, CWP1 is down-regulated during cold shock. As reported elsewhere, Mox4 is a heme-inhibited activator, and Mot3 is a heme-induced repressor of theDAN/TIR genes (but not of TIP1). We show that CWP2 (but not CWP1) is controlled by the same factors, but in reverse fashion—primarily by Mot3 (which can function as either an activator or repressor) but also by Mox4, accounting for the reciprocal regulation of the two groups of genes. Disruptions of TIR1, TIR3, orTIR4 prevent anaerobic growth, indicating that each protein is essential for anaerobic adaptation. The Dan/Tir and Cwp proteins are homologous, with the greatest similarities shown within three subgroups: the Dan proteins, the Tip and Tir proteins, and, more distantly, the Cwp proteins. The clustering of homology corresponds to differences in expression: the Tip and Tir proteins are expressed during hypoxia and cold shock, the Dan proteins are more stringently repressed by oxygen and insensitive to cold shock, and the Cwp proteins are oppositely regulated by oxygen and temperature.

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Knight, Alex E. "Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)." Pure and Applied Chemistry 86, no. 8 (August 20, 2014): 1303–20. http://dx.doi.org/10.1515/pac-2012-0605.

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AbstractTotal internal reflection fluorescence (TIRF) is a popular illumination technique in microscopy, with many applications in cell and molecular biology and biophysics. The chief advantage of the technique is the high contrast that can be achieved by restricting fluorescent excitation to a thin layer. We summarise the optical theory needed to understand the technique and various aspects required for a practical implementation of it, including the merits of different TIRF geometries. Finally, we discuss a variety of applications including super-resolution microscopy and high-throughput DNA sequencing technologies.

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Jaykumar, Ankita Bachhawat, Paulo S. Caceres, Ibrahim Sablaban, Bakhos A. Tannous, and Pablo A. Ortiz. "Real-time monitoring of NKCC2 endocytosis by total internal reflection fluorescence (TIRF) microscopy." American Journal of Physiology-Renal Physiology 310, no. 2 (January 15, 2016): F183—F191. http://dx.doi.org/10.1152/ajprenal.00104.2015.

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The apical Na-K-2Cl cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The amount of NKCC2 at the apical membrane of TAL cells is determined by exocytic delivery, recycling, and endocytosis. Surface biotinylation allows measurement of NKCC2 endocytosis, but it has low time resolution and does not allow imaging of the dynamic process of endocytosis. We hypothesized that total internal reflection fluorescence (TIRF) microscopy imaging of labeled NKCC2 would allow monitoring of NKCC2 endocytosis in polarized Madin-Darby canine kidney (MDCK) and TAL cells. Thus we generated a NKCC2 construct containing a biotin acceptor domain (BAD) sequence between the transmembrane domains 5 and 6. Once expressed in polarized MDCK or TAL cells, surface NKCC2 was specifically biotinylated by exogenous biotin ligase (BirA). We also demonstrate that expression of a secretory form of BirA in TAL cells induces metabolic biotinylation of NKCC2. Labeling biotinylated surface NKCC2 with fluorescent streptavidin showed that most apical NKCC2 was located within small discrete domains or clusters referred to as “puncta” on the TIRF field. NKCC2 puncta were observed to disappear from the TIRF field, indicating an endocytic event which led to a decrease in the number of surface puncta at a rate of 1.18 ± 0.16%/min in MDCK cells, and a rate 1.09 ± 0.08%/min in TAL cells ( n = 5). Treating cells with a cholesterol-chelating agent (methyl-β-cyclodextrin) completely blocked NKCC2 endocytosis. We conclude that TIRF microscopy of labeled NKCC2 allows the dynamic imaging of individual endocytic events at the apical membrane of TAL cells.

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Park, Young IL, Seung Ha Lee, and Jeong-Hwan Kim. "Study of Applicability of Triangular Impulse Response Function for Ultimate Strength of LNG Cargo Containment Systems under Sloshing Impact Loads." Applied Sciences 13, no. 5 (February 23, 2023): 2883. http://dx.doi.org/10.3390/app13052883.

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The LNG cargo containment system used in membrane-type LNG cargo tanks must have sufficient dynamic strength to withstand the impact of sloshing loads. However, performing direct dynamic nonlinear transient finite element assessments against design sloshing impact loads with different design specifications can be complicated and time-consuming. To address this, it is effective to use linear superposition methods, such as the triangular impulse response function (TIRF) method, to conduct dynamic transient FE assessments of LNG cargo containment systems. However, as LNG cargo containment systems have a high level of nonlinearities in terms of geometry, material, and boundary effects, it is necessary to evaluate the applicability of the TIRF method in advance. This study investigates the dynamic responses of an LNG cargo containment system using the TIRF method and compares the ultimate value of the structural responses and impulses with that obtained using direct dynamic nonlinear transient assessments. Based on a comparison of a series of FE analyses, the study proposes a design for the partial safety factors for calculating the ultimate bending and shear capacities of an LNG cargo containment system, taking into consideration the dynamic impact of sloshing loads using the TIRF method. Finally, the ultimate shear and bending capacities are calculated using the proposed method and compared with those obtained through direct dynamic nonlinear transient assessments. The results show that the proposed method provides conservative estimates against direct nonlinear finite element simulations, with a difference of around 10% for the mean minus two standard deviations. This approach can be practically applied for early basic design purposes in the shipbuilding industry.

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Young, Laurence J., Gabriele S. Kaminski Schierle, and Clemens F. Kaminski. "Imaging Aβ(1–42) fibril elongation reveals strongly polarised growth and growth incompetent states." Phys. Chem. Chem. Phys. 19, no. 41 (2017): 27987–96. http://dx.doi.org/10.1039/c7cp03412a.

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Laasfeld, Tõnis, Robin Ehrminger, Maris-Johanna Tahk, Santa Veiksina, Karl Rene Kõlvart, Mart Min, Sergei Kopanchuk, and Ago Rinken. "Budded baculoviruses as a receptor display system to quantify ligand binding with TIRF microscopy." Nanoscale 13, no. 4 (2021): 2436–47. http://dx.doi.org/10.1039/d0nr06737g.

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Won, Jong Hak, and David I. Yule. "Measurement of Ca2+ signaling dynamics in exocrine cells with total internal reflection microscopy." American Journal of Physiology-Gastrointestinal and Liver Physiology 291, no. 1 (July 2006): G146—G155. http://dx.doi.org/10.1152/ajpgi.00003.2006.

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In nonexcitable cells, such as exocrine cells from the pancreas and salivary glands, agonist-stimulated Ca2+ signals consist of both Ca2+ release and Ca2+ influx. We have investigated the contribution of these processes to membrane-localized Ca2+ signals in pancreatic and parotid acinar cells using total internal reflection fluorescence (TIRF) microscopy (TIRFM). This technique allows imaging with unsurpassed resolution in a limited zone at the interface of the plasma membrane and the coverslip. In TIRFM mode, physiological agonist stimulation resulted in Ca2+ oscillations in both pancreas and parotid with qualitatively similar characteristics to those reported using conventional wide-field microscopy (WFM). Because local Ca2+ release in the TIRF zone would be expected to saturate the Ca2+ indicator (Fluo-4), these data suggest that Ca2+ release is occurring some distance from the area subjected to the measurement. When acini were stimulated with supermaximal concentrations of agonists, an initial peak, largely due to Ca2+ release, followed by a substantial, maintained plateau phase indicative of Ca2+ entry, was observed. The contribution of Ca2+ influx and Ca2+ release in isolation to these near-plasma membrane Ca2+ signals was investigated by using a Ca2+ readmission protocol. In the absence of extracellular Ca2+, the profile and magnitude of the initial Ca2+ release following stimulation with maximal concentrations of agonist or after SERCA pump inhibition were similar to those obtained with WFM in both pancreas and parotid acini. In contrast, when Ca2+ influx was isolated by subsequent Ca2+ readmission, the Ca2+ signals evoked were more robust than those measured with WFM. Furthermore, in parotid acinar cells, Ca2+ readdition often resulted in the apparent saturation of Fluo-4 but not of the low-affinity dye Fluo-4-FF. Interestingly, Ca2+ influx as measured by this protocol in parotid acinar cells was substantially greater than that initiated in pancreatic acinar cells. Indeed, robust Ca2+ influx was observed in parotid acinar cells even at low physiological concentrations of agonist. These data indicate that TIRFM is a useful tool to monitor agonist-stimulated near-membrane Ca2+ signals mediated by Ca2+ influx in exocrine acinar cells. In addition, TIRFM reveals that the extent of Ca2+ influx in parotid acinar cells is greater than pancreatic acinar cells when compared using identical methodologies.

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Drawbond, Rachel, and Kathrin Spendier. "TIRF Microscope Image Sequences of Fluorescent IgE-FcεRI Receptor Complexes inside a FcεRI-Centric Synapse in RBL-2H3 Cells." Data 4, no. 3 (July 28, 2019): 111. http://dx.doi.org/10.3390/data4030111.

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Total internal reflection fluorescence (TIRF) microscope image sequences are commonly used to study receptors in live cells. The dataset presented herein facilitates the study of the IgE-FcεRI receptor signaling complex (IgE-RC) in rat basophilic leukemia (RBL-2H3) cells coming into contact with a supported lipid bilayer with 25 mol% N-dinitrophenyl-aminocaproyl phosphatidylethanolamine, modeling an immunological synapse. TIRF microscopy was used to image IgE-RCs within this FcεRI-centric synapse by loading RBL-2H3 cells with fluorescent anti-dinitrophenyl (anti-DNP) immunoglobulin E (IgE) in suspension for 24 h. Fluorescent anti-DNP IgE (IgE488) concentrations of this suspension increased from 10% to 100% and corresponding non-fluorescent anti-DNP IgE concentrations decreased from 90% to 0%. After the removal of unbound anti-DNP IgE, multiple image sequences were taken for each of these ten conditions. Prior to imaging, anti-DNP IgE-primed RBL-2H3 cells were either kept for a few minutes, for about 30 min, or for about one hour in Hanks buffer. The dataset contains 482 RBL-2H3 model synapse image stacks, dark images to correct for background intensity, and TIRF illumination profile images to correct for non-uniform TIRF illumination. After background subtraction, non-uniform illumination correction, and conversion of pixel units from analog-to-digital units to photo electrons, the average pixel intensity was calculated. The average pixel intensity within FcεRI-centric synapses for all three Hanks buffer conditions increased linearly at a rate of 0.42 ± 0.02 photo electrons per pixel per % IgE488 in suspension. RBL-2H3 cell degranulation was tested by detecting β-hexosaminidase activity. Prolonged RBL-2H3 cell exposure to Hanks buffer inhibited exocytosis in RBL-2H3 cells.

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Boguslavsky, Shlomit, Tim Chiu, Kevin P. Foley, Cesar Osorio-Fuentealba, Costin N. Antonescu, K. Ulrich Bayer, Philip J. Bilan, and Amira Klip. "Myo1c binding to submembrane actin mediates insulin-induced tethering of GLUT4 vesicles." Molecular Biology of the Cell 23, no. 20 (October 15, 2012): 4065–78. http://dx.doi.org/10.1091/mbc.e12-04-0263.

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GLUT4-containing vesicles cycle between the plasma membrane and intracellular compartments. Insulin promotes GLUT4 exocytosis by regulating GLUT4 vesicle arrival at the cell periphery and its subsequent tethering, docking, and fusion with the plasma membrane. The molecular machinery involved in GLUT4 vesicle tethering is unknown. We show here that Myo1c, an actin-based motor protein that associates with membranes and actin filaments, is required for insulin-induced vesicle tethering in muscle cells. Myo1c was found to associate with both mobile and tethered GLUT4 vesicles and to be required for vesicle capture in the total internal reflection fluorescence (TIRF) zone beneath the plasma membrane. Myo1c knockdown or overexpression of an actin binding–deficient Myo1c mutant abolished insulin-induced vesicle immobilization, increased GLUT4 vesicle velocity in the TIRF zone, and prevented their externalization. Conversely, Myo1c overexpression immobilized GLUT4 vesicles in the TIRF zone and promoted insulin-induced GLUT4 exposure to the extracellular milieu. Myo1c also contributed to insulin-dependent actin filament remodeling. Thus we propose that interaction of vesicular Myo1c with cortical actin filaments is required for insulin-mediated tethering of GLUT4 vesicles and for efficient GLUT4 surface delivery in muscle cells.

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Machado, Bendesky, Brown, Spendier, and Hagen. "Imaging Membrane Curvature inside a FcεRI-Centric Synapse in RBL-2H3 Cells Using TIRF Microscopy with Polarized Excitation." Journal of Imaging 5, no. 7 (July 4, 2019): 63. http://dx.doi.org/10.3390/jimaging5070063.

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Total internal reflection fluorescence microscopy with polarized excitation (P-TIRF) can be used to image nanoscale curvature phenomena in live cells. We used P-TIRF to visualize rat basophilic leukemia cells (RBL-2H3 cells) primed with fluorescent anti-dinitrophenyl (anti-DNP) immunoglobulin E (IgE) coming into contact with a supported lipid bilayer containing mobile, monovalent DNP, modeling an immunological synapse. The spatial relationship of the IgE-bound high affinity IgE receptor (FcεRI) to the ratio image of P-polarized excitation and S-polarized excitation was analyzed. These studies help correlate the dynamics of cell surface molecules with the mechanical properties of the plasma membrane during synapse formation.

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Nishizaka, T., T. Okuno, J. Okawa, T. Ogura, M. Tsumuraya, H. Noji, and K. Oiwa. "Exploration and application of innovative TIRF microscopy." Seibutsu Butsuri 43, supplement (2003): S229. http://dx.doi.org/10.2142/biophys.43.s229_3.

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Shen, Hao, Eric Huang, Tapaswini Das, Hongxing Xu, Mark Ellisman, and Zhaowei Liu. "TIRF microscopy with ultra-short penetration depth." Optics Express 22, no. 9 (April 25, 2014): 10728. http://dx.doi.org/10.1364/oe.22.010728.

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Tedeschi, Lorena, Claudio Domenici, Arti Ahluwalia, Francesco Baldini, and Andrea Mencaglia. "Antibody immobilisation on fibre optic TIRF sensors." Biosensors and Bioelectronics 19, no. 2 (November 2003): 85–93. http://dx.doi.org/10.1016/s0956-5663(03)00173-8.

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Popescu, Alina L., Kathryn K. Gersh, Dan Safer, and John W. Weisel. "Single Molecule TIRF Study of Fibrinogen Polymerization." Biophysical Journal 100, no. 3 (February 2011): 153a. http://dx.doi.org/10.1016/j.bpj.2010.12.1049.

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Hoying, James B., and Stuart K. Williams. "Endothelial cell monolayers viewed by TIRF microscopy." In Vitro Cellular & Developmental Biology - Animal 30, no. 1 (January 1994): 1–3. http://dx.doi.org/10.1007/bf02631406.

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Zong, Weijian, Xiaoshuai Huang, Chi Zhang, Tianyi Yuan, Ling-ling Zhu, Ming Fan, and Liangyi Chen. "Shadowless-illuminated variable-angle TIRF (siva-TIRF) microscopy for the observation of spatial-temporal dynamics in live cells." Biomedical Optics Express 5, no. 5 (April 15, 2014): 1530. http://dx.doi.org/10.1364/boe.5.001530.

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Fu, Meifang, Luru Dai, Qiao Jiang, Yunqing Tang, Xiaoming Zhang, Baoquan Ding, and Junbai Li. "Observation of intracellular interactions between DNA origami and lysosomes by the fluorescence localization method." Chemical Communications 52, no. 59 (2016): 9240–42. http://dx.doi.org/10.1039/c6cc00484a.

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Rangnekar, Vinay M., John T. Foley, and Philip B. Oldham. "Investigation of Chromatographic Surface Viscosity Using Total Internal Reflection Fluorescence." Applied Spectroscopy 46, no. 5 (May 1992): 827–31. http://dx.doi.org/10.1366/0003702924124600.

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Total internal reflection fluorescence (TIRF) spectroscopy was employed to determine the viscosity of planar chromatographic surfaces. Various n-alkyldimethylchlorosilanes were used to modify the surface of a UV-quartz plate, so as to obtain a covalently attached monomeric C1, C3, C8, or C18 surface. TIRF anisotropy data of 1, 6-diphenylhexatriene (DPH) were collected on the unmodified and modified surfaces in the presence of overlaying solvents covering a range of viscosities. The results indicate that DPH adsorbs onto C1 and C3, but clearly exhibits partitioning with the C18 phase. The C8 surface data indicate intermediate behavior between adsorption and partitioning. On the basis of these preliminary data, the estimated microviscosity of a planar C18 surface is approximately 18 cP.

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Richter, Verena, Peter Lanzerstorfer, Julian Weghuber, and Herbert Schneckenburger. "Super-Resolution Live Cell Microscopy of Membrane-Proximal Fluorophores." International Journal of Molecular Sciences 21, no. 19 (September 26, 2020): 7099. http://dx.doi.org/10.3390/ijms21197099.

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Here, we present a simple and robust experimental setup for the super-resolution live cell microscopy of membrane-proximal fluorophores, which is comparably easy to perform and to implement. The method is based on Structured Illumination Microscopy (SIM) with a switchable spatial light modulator (SLM) and exchangeable objective lenses for epi-illumination and total internal reflection fluorescence (TIRF) microscopy. While, in the case of SIM (upon epi-illumination), cell layers of about 1–2 µm in close proximity to the plasma membrane can be selected by software, layers in the 100 nm range are assessed experimentally by TIRF-SIM. To show the applicability of this approach, both methods are used to measure the translocation of the glucose transporter 4 (GLUT4) from intracellular vesicles to the plasma membrane upon stimulation by insulin or insulin-mimetic compounds, with a lateral resolution of around 100 nm and an axial resolution of around 200 nm. While SIM is an appropriate method to visualize the intracellular localization of GLUT4 fused with a green fluorescent protein, TIRF-SIM permits the quantitative evaluation of its fluorescence in the plasma membrane. These imaging methods are discussed in the context of fluorescence lifetime kinetics, providing additional data for the molecular microenvironment.

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Mantil, Elisabeth, Trinda Crippin, Anatoli Ianoul, and Tyler J. Avis. "Experimental Parameters Leading to Optimal Bilayers for Total Internal Reflection Fluorescence Microscopy Visualization." Microscopy and Microanalysis 23, no. 1 (February 2017): 97–112. http://dx.doi.org/10.1017/s1431927617000083.

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AbstractSupported lipid bilayer systems were evaluated following various experimental procedures in an effort to determine their appropriateness for visualization using total internal reflection fluorescence (TIRF) microscopy. The incorporation and distribution of Texas Red® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE) was studied when incorporated into bilayers of variable lipid composition using different forms of mechanical shearing. Results showed that 0.8 mol% TR-DHPE provides the most optimum TIRF images. At this concentration, a sufficient level of photostability can be achieved without an undesirable increase in TR-DHPE aggregates caused by excess probe molecules. Solutions composed of a 3:1 molar ratio of DOPC:DPPC with 0.8 mol% TR-DHPE produce bilayers that consistently display clear, distinct, rounded domains, whereas other lipid compositions did not. This optimum phase separation appears to be influenced by an increase in mechanical shearing during the vesicle formation process, when the lipid solutions were exposed to sonication and extrusion processes. The combination of a sonication and extrusion process also helped with eliminating the presence of TR-DHPE aggregates within the model membranes. It was also shown that bilayers formed on conditioned glass, placed on a slide, produced more highly detailed bilayers in which distinct lipid phase separation could be optimally visualized using TIRF microscopy.

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Liras, M., S. Simoncelli, A. Rivas-Aravena, O. García, J. C. Scaiano, E. I. Alarcon, and A. Aspée. "Nitroxide amide-BODIPY probe behavior in fibroblasts analyzed by advanced fluorescence microscopy." Organic & Biomolecular Chemistry 14, no. 17 (2016): 4023–26. http://dx.doi.org/10.1039/c6ob00533k.

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Soni, Gautam V., and Amit Meller. "Progress toward Ultrafast DNA Sequencing Using Solid-State Nanopores." Clinical Chemistry 53, no. 11 (November 1, 2007): 1996–2001. http://dx.doi.org/10.1373/clinchem.2007.091231.

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Abstract Background: Measurements of the ionic current flowing through nanometer-scale pores (nanopores) have been used to analyze single DNA and RNA molecules, with the ultimate goal of achieving ultrafast DNA sequencing. However, attempts at purely electronic measurements have not achieved the signal contrast required for single nucleotide differentiation. In this report we propose a novel method of optical detection of DNA sequence translocating through a nanopore. Methods: Each base of the target DNA sequence is 1st mapped onto a 2-unit code, 2 10-bp nucleotide sequence, by biochemical conversion into Designed DNA Polymers. These 2-unit codes are then hybridized to complementary, fluorescently labeled, and self-quenching molecular beacons. As the molecular beacons are sequentially unzipped during translocation through a <2-nm-wide nanopore, their fluorescent tags are unquenched and are detected by a custom-built dual-color total internal reflection fluorescence (TIRF) microscope. The 2-color optical signal is then correlated to the target DNA sequence. Results: A dual-color TIRFM microscope with single-molecule resolution was constructed, and controlled fabrication of 1-dimensional and 2-dimensional arrays of solid-state nanopores was performed. A nanofluidic cell assembly was constructed for TIRF-based optical detection of voltage-driven DNA translocation through a nanopore. Conclusions: We present a novel nanopore-based DNA sequencing technique that uses an optical readout of DNA translocating unzipping through a nanopore. Our technique offers better single nucleotide differentiation in sequence readout, as well as the possibility of large-scale parallelism using nanopore arrays.

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Reichert, W. M., and G. A. Truskey. "Total internal reflection fluorescence (TIRF) microscopy. I. Modelling cell contact region fluorescence." Journal of Cell Science 96, no. 2 (June 1, 1990): 219–30. http://dx.doi.org/10.1242/jcs.96.2.219.

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Total Internal Reflection Fluorescence (TIRF) is a powerful technique for visualizing focal and close contacts between the cell and the surface. Practical application of TIRF has been hampered by the lack of straightforward methods to calculate separation distances. The characteristic matrix theory of thin dielectric films was used to develop simple exponential approximations for the fluorescence excited in the cell-substratum contact region during a TIRF experiment. Two types of fluorescence were examined: fluorescently labeled cell membranes, and a fluorescent water-soluble dye. By neglecting the refractive index of the cell membrane, the fluorescence excited in the cell membrane was modelled by a single exponential function while the fluorescence in the membrane/substratum water gap followed a weighted sum of two exponentials. The error associated with neglecting the cell membrane for an incident angle of 70 degrees never exceeded 2.5%, regardless of the cell-substratum separation distance. Comparisons of approximated fluorescence intensities to more exact solutions of the fluorescence integrals for the three-phase model indicated that the approximations are accurate to about 1% for membrane/substratum gap thicknesses of less than 50 nm if the cytoplasmic and water gap refractive indices are known. The intrinsic error of this model in the determination of membrane/substratum separations was 10% as long as the uncertainties in the water gap and cytoplasmic refractive indices were less than 1%.

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Peng, Weichen, Luo Chen, Xue Ouyang, and Wei Xiong. "A Time-Identified R-Tree: A Workload-Controllable Dynamic Spatio-Temporal Index Scheme for Streaming Processing." ISPRS International Journal of Geo-Information 13, no. 2 (February 4, 2024): 49. http://dx.doi.org/10.3390/ijgi13020049.

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Many kinds of spatio-temporal data in our daily lives, such as the trajectory data of moving objects, stream natively. Streaming systems exhibit significant advantages in processing streaming data due to their distributed architecture, high throughput, and real-time performance. The use of streaming processing techniques for spatio-temporal data applications is a promising research direction. However, due to the strong dynamic nature of data in streaming processing systems, traditional spatio-temporal indexing techniques based on relatively static data cannot be used directly in stream-processing environments. It is necessary to study and design new spatio-temporal indexing strategies. Hence, we propose a workload-controllable dynamic spatio-temporal index based on the R-tree. In order to restrict memory usage, we formulate an INSERT and batch-REMOVE (I&BR) method and append a collection mechanism to the traditional R-tree. To improve the updating performance, we propose a time-identified R-tree (TIR). Moreover, we propose a distributed system prototype called a time-identified R-tree farm (TIRF). Experiments show that the TIR could work in a scenario with a controllable usage of memory and a stable response time. The throughput of the TIRF could reach 1 million points per second. The performance of a range search in the TIRF is many times better than in PostgreSQL, which is a widely used database system for spatio-temporal applications.

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Matsui, K. "Optical aberration correction for TIRF single molecule imaging." Seibutsu Butsuri 43, supplement (2003): S17. http://dx.doi.org/10.2142/biophys.43.s17_1.

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Hingorani, Manju M. "TIRF(ing) reveals Msh2-Msh6 surfing on DNA." Nature Structural & Molecular Biology 14, no. 12 (December 2007): 1124–25. http://dx.doi.org/10.1038/nsmb1207-1124.

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Holden, Seamus J., Stephan Uphoff, David Yadin, Johannes Hohlbein, Ludovic Le Reste, Oliver J. Britton, and Achillefs N. Kapanidis. "TIRF-Based FRET with One Base-Pair Resolution." Biophysical Journal 98, no. 3 (January 2010): 413a. http://dx.doi.org/10.1016/j.bpj.2009.12.2230.

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Lee, Jinwoo, and Sungchul Hong Hohng. "Alex Single Molecule Three Color in TIRF Microscopy." Biophysical Journal 98, no. 3 (January 2010): 747a. http://dx.doi.org/10.1016/j.bpj.2009.12.4096.

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Hildebrandt, Lasse L., Søren Preus, and Victoria Birkedal. "Quantitative single molecule FRET efficiencies using TIRF microscopy." Faraday Discussions 184 (2015): 131–42. http://dx.doi.org/10.1039/c5fd00100e.

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Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2–10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as other correction factors can compensate for inaccurate values of these parameters. We give here guidelines, that can be used through tools within the iSMS software (http://www.isms.au.dk), for determining quantitative FRET and assess uncertainties linked with the procedure. Our results provide insights into the experimental parameters governing quantitative FRET determination, which is essential for obtaining accurate structural information from a wide range of biomolecules.

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Boisvert, Jacques, Joel Ryan, Abbas Padeganeh, Valerie De Rop, Paul S. Maddox, and Jonas F. Dorn. "Quantitative Imaging of Protein Complexes using TIRF Microscopy." Biophysical Journal 104, no. 2 (January 2013): 524a. http://dx.doi.org/10.1016/j.bpj.2012.11.2899.

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49

Fiolka, Reto. "Clearer view for TIRF and oblique illumination microscopy." Optics Express 24, no. 26 (December 13, 2016): 29556. http://dx.doi.org/10.1364/oe.24.029556.

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Filippov, Leonid K. "Kinetic-Diffusive-Convective Adsorption in TIRF Flow Cells." Journal of Colloid and Interface Science 174, no. 1 (September 1995): 32–39. http://dx.doi.org/10.1006/jcis.1995.1360.

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Journal articles on the topic 'TIRF'

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