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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

Comparison of epi-fluorescence (a) and TIRF (b) images of the same HEK-β2AR-GFP cells cultured on glass coverslip. These cells express green fluorescent protein (GFP) tagged β2-adrenergic receptor (β2AR), a prototypical G protein-coupled receptor. Scale bar is 10 µm. In the TIRF image, only GFP-tagged receptors at the cell membrane closest to the cover slip are visible. Furthermore, TIR illumination allows more single molecules to be detected and results in brighter fluorescence than epi-illumination.
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biosensors-05-00223-f003: Comparison of epi-fluorescence (a) and TIRF (b) images of the same HEK-β2AR-GFP cells cultured on glass coverslip. These cells express green fluorescent protein (GFP) tagged β2-adrenergic receptor (β2AR), a prototypical G protein-coupled receptor. Scale bar is 10 µm. In the TIRF image, only GFP-tagged receptors at the cell membrane closest to the cover slip are visible. Furthermore, TIR illumination allows more single molecules to be detected and results in brighter fluorescence than epi-illumination.

Mentions: Since the evanescent field intensity decays exponentially with distance from the interface, TIRFM gives rise to a very narrow excitation depth, suggesting that only a small section at the base of adherent cell is excited. Naturally occurring fluorophores in the cell bulk, such as NADH and flavins, are often out of focus, leading to very high signal-to-background ratios (SBRs). Compared to epi-fluorescence imaging in which both excitation and emission typically involve freely propagating light, TIRFM is unique in that it only produces surface-selective images (Figure 3). The ability to perform such an optical sectioning makes TIRFM the method of choice to visualize single molecules in living cells, in particular fluorescent molecules located at the cell adhesion sites, and in cell membrane and membrane proximal cytoplasmic organelles. TIRFM has been widely used to study dynamic events close to the plasma membrane of living cells, including cell adhesion, endocytosis, exocytosis, ligand binding to cell surface receptor, cytoskeletal remodeling, dynamic membrane microdomains, and forward trafficking (i.e., targeting of intracellular proteins to cell membrane) (reviewed in [4,5,46,47,48]). This section reviews recent progress of TIRF techniques for cell analysis.


Total internal reflection fluorescence quantification of receptor pharmacology.

Fang Y - Biosensors (Basel) (2015)

Comparison of epi-fluorescence (a) and TIRF (b) images of the same HEK-β2AR-GFP cells cultured on glass coverslip. These cells express green fluorescent protein (GFP) tagged β2-adrenergic receptor (β2AR), a prototypical G protein-coupled receptor. Scale bar is 10 µm. In the TIRF image, only GFP-tagged receptors at the cell membrane closest to the cover slip are visible. Furthermore, TIR illumination allows more single molecules to be detected and results in brighter fluorescence than epi-illumination.
© Copyright Policy
Related In: Results  -  Collection

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

biosensors-05-00223-f003: Comparison of epi-fluorescence (a) and TIRF (b) images of the same HEK-β2AR-GFP cells cultured on glass coverslip. These cells express green fluorescent protein (GFP) tagged β2-adrenergic receptor (β2AR), a prototypical G protein-coupled receptor. Scale bar is 10 µm. In the TIRF image, only GFP-tagged receptors at the cell membrane closest to the cover slip are visible. Furthermore, TIR illumination allows more single molecules to be detected and results in brighter fluorescence than epi-illumination.
Mentions: Since the evanescent field intensity decays exponentially with distance from the interface, TIRFM gives rise to a very narrow excitation depth, suggesting that only a small section at the base of adherent cell is excited. Naturally occurring fluorophores in the cell bulk, such as NADH and flavins, are often out of focus, leading to very high signal-to-background ratios (SBRs). Compared to epi-fluorescence imaging in which both excitation and emission typically involve freely propagating light, TIRFM is unique in that it only produces surface-selective images (Figure 3). The ability to perform such an optical sectioning makes TIRFM the method of choice to visualize single molecules in living cells, in particular fluorescent molecules located at the cell adhesion sites, and in cell membrane and membrane proximal cytoplasmic organelles. TIRFM has been widely used to study dynamic events close to the plasma membrane of living cells, including cell adhesion, endocytosis, exocytosis, ligand binding to cell surface receptor, cytoskeletal remodeling, dynamic membrane microdomains, and forward trafficking (i.e., targeting of intracellular proteins to cell membrane) (reviewed in [4,5,46,47,48]). This section reviews recent progress of TIRF techniques for cell analysis.

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