Waveguide evanescent field fluorescence microscopy: Thin film fluorescence intensities and its application in cell biology

Hassanzadeh, Abdollah; Nitsche, Michael; Mittler, Silvia; Armstrong, Souzan; Dixon, S. Jeffrey; Langbein, U.

doi:10.1063/1.2937840

Cited by 47 publications

(40 citation statements)

References 14 publications

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“…This limits the field-of-view down to around 100 x 100 μm 2 . Another way of setting up an evanescent field for TIRF microscopy is by using optical waveguides [12,14,15,17,18]. In waveguide chip-based microscopy, the illumination and collection light paths are efficiently decoupled, opening several opportunities for bio-imaging.…”

Section: Si 3 N 4 Waveguide Platform For Tirf Microscopymentioning

confidence: 99%

Silicon nitride waveguide platform for fluorescence microscopy of living cells

Tinguely¹,

Helle²,

Ahluwalia³

2017

Opt. Express

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Waveguide chip-based microscopy reduces the complexity of total internal reflection fluorescence (TIRF) microscopy, and adds features like large field of view illumination, decoupling of illumination and collection path and easy multimodal imaging. However, for the technique to become widespread there is a need of low-loss and affordable waveguides made of high-refractive index material. Here, we develop and report a low-loss silicon nitride (Si 3 N 4 ) waveguide platform for multi-color TIRF microscopy. Single mode conditions at visible wavelengths (488-660 nm) were achieved using shallow rib geometry. To generate uniform excitation over appropriate dimensions waveguide bends were used to filter-out higher modes followed by adiabatic tapering. Si 3 N 4 material is finally shown to be biocompatible for growing and imaging living cells.

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Section: Si 3 N 4 Waveguide Platform For Tirf Microscopymentioning

confidence: 99%

Silicon nitride waveguide platform for fluorescence microscopy of living cells

Tinguely¹,

Helle²,

Ahluwalia³

2017

Opt. Express

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“…TIRF is typically achieved by coupling excitation light to the surface through either a prism or the microscope objective [5]. More recently, excitation light has been coupled into a waveguide and microscopic measurements have been performed on the waveguide surface [6]. In our experiments, light is coupled directly into a 2.5 x 7 x 0.2 cm slide fabricated from cyclo-olefin polymer, known commercially as Zeonor¨, to allow for characterization of surface coatings by simply counting the adsorbed particles on the substrate.…”

Section: Introductionmentioning

confidence: 99%

TIRF microscopy as a screening method for non-specific binding on surfaces

Charlton

Gubala

Gandhiraman

et al. 2011

Journal of Colloid and Interface Science

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We report a method for studying nanoparticle-biosensor surface interactions based on Total Internal Reflection Fluorescence (TIRF) Microscopy. We demonstrate that this simple technique allows for high throughput screening of non-specific adsorption (NSA) of nanoparticles on surfaces of different chemical composition. Binding events between fluorescent nanoparticles and functionalized Zeonor¨ surfaces are observed in real-time, giving a measure of the attractive or repulsive properties of the surface and the kinetics of the interaction. Three types of coatings have been studied: one containing a polymerized aminosilane network with terminal ÐNH 2 groups, a second film with a high density of Ð COOH surface groups and the third with sterically restraining branched poly(ethylene)glycol (PEG) functionality. TIRF microscopy revealed that the NSA of nanoparticles with negative surface charge on such modified coatings decreased in the following order ÐNH 2 > -branched PEG > -COOH. The surface specificity of the technique also allows discrimination of the degree of NSA of the same surface at different pH. KeywordsTotal Internal Reflection, Fluorescence, Microscopy, Non-Specific Binding, SurfaceParticle Interaction IntroductionCurrent research in biomedical diagnostics is directed at inexpensive, highly sensitive, miniaturized devices, in which usually disposable substrates or chips are used. Such devices are required to measure analytes in microliter volume samples, typically at picomolar level. In fluorescence-based assays, the signal can be greatly enhanced with the use of dye doped nanoparticles as fluorescent labels [1][2][3][4]. However, in a successful diagnostics device, signal-to-noise ratio is paramount to its performance and effectively controls the sensitivity. Controlling the noise and background contribution, dictated by non-specific binding of the non-analyte constituents of the sample remains very challenging. The chemical composition of the substrate surface and its interactions with the active surface area of the dye-doped nanoparticles, therefore, play a key role in the performance of diagnostic platform. In this work, we focus on surface functionalization of the substrate ZeonorÒ, a cyclo-olefin polymer (COP), which is characterized by ease of injection molding for biochip fabrication and relatively low autofluorescence, which is an important factor for fluorescence-based applications including TIRF microscopy.Total Internal Reflection Fluorescence (TIRF) microscopy [5] is presented here as a very useful and fast method to compare non-specific adsorption of detection molecules on different surfaces. This method allowed for rapid screening of surfaces to find their ability to prevent non-specific binding. Total internal reflection occurs an any interface between materials of different refractive index inside the material of higher refractive index provided the angle of incidence of the light is above the critical angle defined as:where n 1 is the refractive index of the high refractive index mat...

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“…Indeed, the demand for evanescent wave micronano-sensors has significantly increased in the last few years, with the strong development of nanotechnology in fields such as medical and biological sciences. In that sense, the evanescent field of a propagating mode can be used as an illumination source for the excitation of attached elements on the surface of the sensor [2,3]. Besides, the same principle is also applied in total internal reflection microscopy (TIRF) or reverse symmetry waveguide sensing [4].…”

Section: Introductionmentioning

confidence: 97%