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Total internal reflection fluorescence quantification of receptor pharmacology.

Fang Y - Biosensors (Basel) (2015)

Bottom Line: Total internal reflection fluorescence (TIRF) microscopy has been widely used as a single molecule imaging technique to study various fundamental aspects of cell biology, owing to its ability to selectively excite a very thin fluorescent volume immediately above the substrate on which the cells are grown.Inspired by the recent demonstration of label-free evanescent wave biosensors for cell phenotypic profiling and drug screening with high throughput, we had hypothesized and demonstrated that TIRF imaging is also amenable to receptor pharmacology profiling.This paper reviews key considerations and recent applications of TIRF imaging for pharmacology profiling.

View Article: PubMed Central - PubMed

Affiliation: Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, NY 14831, USA. fangy2@corning.com.

ABSTRACT
Total internal reflection fluorescence (TIRF) microscopy has been widely used as a single molecule imaging technique to study various fundamental aspects of cell biology, owing to its ability to selectively excite a very thin fluorescent volume immediately above the substrate on which the cells are grown. However, TIRF microscopy has found little use in high content screening due to its complexity in instrumental setup and experimental procedures. Inspired by the recent demonstration of label-free evanescent wave biosensors for cell phenotypic profiling and drug screening with high throughput, we had hypothesized and demonstrated that TIRF imaging is also amenable to receptor pharmacology profiling. This paper reviews key considerations and recent applications of TIRF imaging for pharmacology profiling.

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Related in: MedlinePlus

Three types of evanescent wave-excited fluorescence microscopy. (a) Through-the-objective TIRFM, wherein a high numerical aperture (NA) objective lens is used to simultaneously generate the evanescent field at the cell-glass interface and view the cell. A laser light is directed and focused on the back focal plane, which then creates a refracted parallel beam approaching the interface in the small gap between objective and glass coverslip. TIR is achieved when the angle is greater than the critical angle (θ > θc); (b) Prism-based surface plasmon-excited TIRFM, wherein an incident light is directed onto a gold film via a prism, creating an electromagnetic field penetrating into the cell under resonance condition. The reflected beam is detected via a photodetector or imager for SPR measurement, while the excited fluorescence is collected using a separate objective; (c) Resonant waveguide grating-based TIRFM, wherein a grating is used to couple light into the waveguide, creating an evanescent field resulting from total internal reflection of the light beam. The excited fluorescence is collected using a CCD camera.
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biosensors-05-00223-f001: Three types of evanescent wave-excited fluorescence microscopy. (a) Through-the-objective TIRFM, wherein a high numerical aperture (NA) objective lens is used to simultaneously generate the evanescent field at the cell-glass interface and view the cell. A laser light is directed and focused on the back focal plane, which then creates a refracted parallel beam approaching the interface in the small gap between objective and glass coverslip. TIR is achieved when the angle is greater than the critical angle (θ > θc); (b) Prism-based surface plasmon-excited TIRFM, wherein an incident light is directed onto a gold film via a prism, creating an electromagnetic field penetrating into the cell under resonance condition. The reflected beam is detected via a photodetector or imager for SPR measurement, while the excited fluorescence is collected using a separate objective; (c) Resonant waveguide grating-based TIRFM, wherein a grating is used to couple light into the waveguide, creating an evanescent field resulting from total internal reflection of the light beam. The excited fluorescence is collected using a CCD camera.

Mentions: TIRFM uses an evanescent electromagnetic field to selectively excite and visualize fluorescent molecules in the close vicinity of a substrate. TIRFM generally uses three distinct configurations: glass coverslip/sample, glass/gold film/sample (SPR), and glass/grating waveguide film/sample (RWG) (Figure 1). For cell biology applications the sample is adherent cells in aqueous solution.


Total internal reflection fluorescence quantification of receptor pharmacology.

Fang Y - Biosensors (Basel) (2015)

Three types of evanescent wave-excited fluorescence microscopy. (a) Through-the-objective TIRFM, wherein a high numerical aperture (NA) objective lens is used to simultaneously generate the evanescent field at the cell-glass interface and view the cell. A laser light is directed and focused on the back focal plane, which then creates a refracted parallel beam approaching the interface in the small gap between objective and glass coverslip. TIR is achieved when the angle is greater than the critical angle (θ > θc); (b) Prism-based surface plasmon-excited TIRFM, wherein an incident light is directed onto a gold film via a prism, creating an electromagnetic field penetrating into the cell under resonance condition. The reflected beam is detected via a photodetector or imager for SPR measurement, while the excited fluorescence is collected using a separate objective; (c) Resonant waveguide grating-based TIRFM, wherein a grating is used to couple light into the waveguide, creating an evanescent field resulting from total internal reflection of the light beam. The excited fluorescence is collected using a CCD camera.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493547&req=5

biosensors-05-00223-f001: Three types of evanescent wave-excited fluorescence microscopy. (a) Through-the-objective TIRFM, wherein a high numerical aperture (NA) objective lens is used to simultaneously generate the evanescent field at the cell-glass interface and view the cell. A laser light is directed and focused on the back focal plane, which then creates a refracted parallel beam approaching the interface in the small gap between objective and glass coverslip. TIR is achieved when the angle is greater than the critical angle (θ > θc); (b) Prism-based surface plasmon-excited TIRFM, wherein an incident light is directed onto a gold film via a prism, creating an electromagnetic field penetrating into the cell under resonance condition. The reflected beam is detected via a photodetector or imager for SPR measurement, while the excited fluorescence is collected using a separate objective; (c) Resonant waveguide grating-based TIRFM, wherein a grating is used to couple light into the waveguide, creating an evanescent field resulting from total internal reflection of the light beam. The excited fluorescence is collected using a CCD camera.
Mentions: TIRFM uses an evanescent electromagnetic field to selectively excite and visualize fluorescent molecules in the close vicinity of a substrate. TIRFM generally uses three distinct configurations: glass coverslip/sample, glass/gold film/sample (SPR), and glass/grating waveguide film/sample (RWG) (Figure 1). For cell biology applications the sample is adherent cells in aqueous solution.

Bottom Line: Total internal reflection fluorescence (TIRF) microscopy has been widely used as a single molecule imaging technique to study various fundamental aspects of cell biology, owing to its ability to selectively excite a very thin fluorescent volume immediately above the substrate on which the cells are grown.Inspired by the recent demonstration of label-free evanescent wave biosensors for cell phenotypic profiling and drug screening with high throughput, we had hypothesized and demonstrated that TIRF imaging is also amenable to receptor pharmacology profiling.This paper reviews key considerations and recent applications of TIRF imaging for pharmacology profiling.

View Article: PubMed Central - PubMed

Affiliation: Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, NY 14831, USA. fangy2@corning.com.

ABSTRACT
Total internal reflection fluorescence (TIRF) microscopy has been widely used as a single molecule imaging technique to study various fundamental aspects of cell biology, owing to its ability to selectively excite a very thin fluorescent volume immediately above the substrate on which the cells are grown. However, TIRF microscopy has found little use in high content screening due to its complexity in instrumental setup and experimental procedures. Inspired by the recent demonstration of label-free evanescent wave biosensors for cell phenotypic profiling and drug screening with high throughput, we had hypothesized and demonstrated that TIRF imaging is also amenable to receptor pharmacology profiling. This paper reviews key considerations and recent applications of TIRF imaging for pharmacology profiling.

Show MeSH
Related in: MedlinePlus