High-numerical-aperture objective lenses and optical system improved objective type total internal reflection fluorescence microscopy

Kawano, Yoshihiro; Abe, Chikara; Kaneda, Teruo; Aono, Yasushi; Abe, Katsuyuki; Tamura, Kouichi; Terakawa, Susumu

doi:10.1117/12.401623

Cited by 11 publications

(8 citation statements)

References 4 publications

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“…The use of TIRF in cellular microscopy increased once an objective‐based TIRF (or prism‐less TIRF; Fig. 4A) system was introduced to the scientific community (Stout & Axelrod, 1989), and increased even more once a commercial solution became available (Kawano et al., 2000). In objective‐based TIRFM, one can easily switch between standard epifluorescence and TIRF by changing the off‐axis position of the beam focus at the objective's back focal plane.…”

Section: The Theory Behind the Techniquementioning

confidence: 99%

Total Internal Reflection Fluorescence (TIRF) Microscopy

Fish

2022

Current Protocols

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Total internal reflection fluorescence (TIRF) microscopy (TIRFM) is an elegant optical technique that provides for the excitation of fluorophores in an extremely thin axial region ("optical section"). The method is based on the principle that when excitation light is completely internally reflected in a transparent solid (e.g., coverglass) at its interface with liquid, an electromagnetic field, called the evanescent wave, is generated in the liquid at the solid-liquid interface and is the same frequency as the excitation light. Since the intensity of the evanescent wave exponentially decays with distance from the surface of the solid, only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited. This overview will review the history, optical theory, and hardware configurations used in TIRFM. In addition, it will provide experimental details and methodological considerations for studying receptors at the plasma membrane in neurons.

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Section: The Theory Behind the Techniquementioning

confidence: 99%

Total Internal Reflection Fluorescence (TIRF) Microscopy

Fish

2022

Current Protocols

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“…The use of TIRF in cellular microscopy increased once an objective-based TIRF (or prism-less TIRF; Fig. 12.18.3A) system was introduced to the scientific community (Stout and Axelrod, 1989), and increased even more once a commercial solution became available (Kawano et al, 2000). In objectivebased TIRFM, one can easily switch between standard epifluorescence and TIRF by changing the off-axis position of the beam focus at the objective's back focal plane.…”

Section: Supplement 50mentioning

confidence: 99%

Total Internal Reflection Fluorescence (TIRF) Microscopy

2009

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Total internal reflection fluorescence (TIRF) microscopy (TIRFM) is an elegant optical technique that provides for the excitation of fluorophores in an extremely thin axial region ("optical section"). The method is based on the principle that when excitation light is totally internally reflected in a transparent solid (e.g., coverglass) at its interface with liquid, an electromagnetic field, called the evanescent wave, is generated in the liquid at the solid-liquid interface and is the same frequency as the excitation light. Since the intensity of the evanescent wave exponentially decays with distance from the surface of the solid, only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited. This unit will briefly review the history, optical theory, and different hardware configurations used in TIRFM. In addition, it will provide experimental details and methodological considerations for studying receptors at the plasma membrane in neurons. Curr.

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“…The higher refractive index of the glass, together with the high numerical aperture of the objective, has a few consequences for TIR microscopy. First, there is a relatively wide range of angles over which TIR can be achieved (Terakawa et al, 1997;Kawano et al, 2000;Table 4.12.1, Fig. 4.12.4B).…”

Section: Objectivesmentioning

confidence: 99%

Total Internal Reflection Fluorescence Microscopy for High‐Resolution Imaging of Cell‐Surface Events

Jaiswal

Simon

2003

CP Cell Biology

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The wavelength of light imposes a physical limit of ∼400 nm on the maximum resolution that can be achieved using light microscopy. This unit will describe the use of total internal reflection fluorescence microscopy (TIR‐FM), or evanescent wave microscopy, an approach that partially overcomes this physical limit and permits one to selectively image just those fluorophores in the optical plane (along the z axis) within 50 nm of the cell surface. TIR‐FM works by means of limiting the depth of penetration of the excitation light within this narrow region. This narrow excitatory plane not only provides a high signal‐to‐noise ratio but also minimizes the photodamage to the cell.

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High-numerical-aperture objective lenses and optical system improved objective type total internal reflection fluorescence microscopy

Cited by 11 publications

References 4 publications

Total Internal Reflection Fluorescence (TIRF) Microscopy

Total Internal Reflection Fluorescence (TIRF) Microscopy

Total Internal Reflection Fluorescence (TIRF) Microscopy

Total Internal Reflection Fluorescence Microscopy for High‐Resolution Imaging of Cell‐Surface Events

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