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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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Microplate-compatible TIRF imaging system. (a) Instrument setup. A 488 nm laser light is directed to illuminate the glass substrate after it has been guided through a right angle prism. Under TIR condition the evanescent wave excited fluorescence is collected using an objective lens, passed through a filter and focused via a tube lens onto a CCD camera; (b) The back image of a glass bottom microplate to show the geometry of alumina stripes; (c) The excitation light propagates within the glass substrate after it illuminates the well D4; (d) Whole plate TIRF observed within multi-wells by placing a filter above the CCD camera; (e) Epi-fluorescence image of HEK-β2AR-GFP cells; (f) TIRF image of the cells. Scale bar in (e,f) is 40 µm. This figure was reproduced with permission from [25].
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biosensors-05-00223-f002: Microplate-compatible TIRF imaging system. (a) Instrument setup. A 488 nm laser light is directed to illuminate the glass substrate after it has been guided through a right angle prism. Under TIR condition the evanescent wave excited fluorescence is collected using an objective lens, passed through a filter and focused via a tube lens onto a CCD camera; (b) The back image of a glass bottom microplate to show the geometry of alumina stripes; (c) The excitation light propagates within the glass substrate after it illuminates the well D4; (d) Whole plate TIRF observed within multi-wells by placing a filter above the CCD camera; (e) Epi-fluorescence image of HEK-β2AR-GFP cells; (f) TIRF image of the cells. Scale bar in (e,f) is 40 µm. This figure was reproduced with permission from [25].

Mentions: Most TIRFM systems use laser beams as incident light. However, TIRFM varies greatly in instrument setup, which generally falls into: through-the-objective, through-the-prism, RWG-based configurations (Figure 1), and around-the-objective (Figure 2a). Currently, most TIRF microscopes are objective-based.


Total internal reflection fluorescence quantification of receptor pharmacology.

Fang Y - Biosensors (Basel) (2015)

Microplate-compatible TIRF imaging system. (a) Instrument setup. A 488 nm laser light is directed to illuminate the glass substrate after it has been guided through a right angle prism. Under TIR condition the evanescent wave excited fluorescence is collected using an objective lens, passed through a filter and focused via a tube lens onto a CCD camera; (b) The back image of a glass bottom microplate to show the geometry of alumina stripes; (c) The excitation light propagates within the glass substrate after it illuminates the well D4; (d) Whole plate TIRF observed within multi-wells by placing a filter above the CCD camera; (e) Epi-fluorescence image of HEK-β2AR-GFP cells; (f) TIRF image of the cells. Scale bar in (e,f) is 40 µm. This figure was reproduced with permission from [25].
© Copyright Policy
Related In: Results  -  Collection

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

biosensors-05-00223-f002: Microplate-compatible TIRF imaging system. (a) Instrument setup. A 488 nm laser light is directed to illuminate the glass substrate after it has been guided through a right angle prism. Under TIR condition the evanescent wave excited fluorescence is collected using an objective lens, passed through a filter and focused via a tube lens onto a CCD camera; (b) The back image of a glass bottom microplate to show the geometry of alumina stripes; (c) The excitation light propagates within the glass substrate after it illuminates the well D4; (d) Whole plate TIRF observed within multi-wells by placing a filter above the CCD camera; (e) Epi-fluorescence image of HEK-β2AR-GFP cells; (f) TIRF image of the cells. Scale bar in (e,f) is 40 µm. This figure was reproduced with permission from [25].
Mentions: Most TIRFM systems use laser beams as incident light. However, TIRFM varies greatly in instrument setup, which generally falls into: through-the-objective, through-the-prism, RWG-based configurations (Figure 1), and around-the-objective (Figure 2a). Currently, most TIRF microscopes are objective-based.

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