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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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The label-free and TIRF profiles of receptor activation in HEK293-β2AR-GFP cells. (a–c) Real-time DMR signals induced by 10 µM epinephrine and 10 µM isoproterenol (a); 32nM EGF (b); and 10 µM TBB (c); (d–f) Real-time TIRF signals induced by 10 µM epinephrine and 10 µM isoproterenol (d); 32nM EGF (e); and 10 µM TBB (f); (g–i) TIRF images before (g), and 2 min (h) and 10 min (i) after the stimulation with 10 µM epinephrine. The data in (a–c) shows mean ± s.d. of eight replicates. Scale bar in g–i is 10 µm. This figure is reproduced with permission from Ref. [25].
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biosensors-05-00223-f005: The label-free and TIRF profiles of receptor activation in HEK293-β2AR-GFP cells. (a–c) Real-time DMR signals induced by 10 µM epinephrine and 10 µM isoproterenol (a); 32nM EGF (b); and 10 µM TBB (c); (d–f) Real-time TIRF signals induced by 10 µM epinephrine and 10 µM isoproterenol (d); 32nM EGF (e); and 10 µM TBB (f); (g–i) TIRF images before (g), and 2 min (h) and 10 min (i) after the stimulation with 10 µM epinephrine. The data in (a–c) shows mean ± s.d. of eight replicates. Scale bar in g–i is 10 µm. This figure is reproduced with permission from Ref. [25].

Mentions: Given that optical biosensors and TIRF all utilize surface-bound evanescent wave for cell assays, it is reasonable to hypothesize that TIRF can also be used for drug pharmacology profiling in a manner complementary to label-free biosensors. Using an engineered HEK293 cell line that stably expresses β2-AR with green fluorescent protein (GFP) at its C-terminal (β2AR-GFP) as the model, we compared the label-free DMR signals arising from the activation of several receptors with their respective TIRF signals [25]. The fluorescence intensity of β2AR-GFP was measured using a conventional TIRFM (Nikon Instruments Inc., Melville, NY, USA) and used as a cell surface fluorescence reporter for receptor activation. The DMR profiles of agonists were obtained using Epic® BT system, a whole microplate-based RWG biosensor imager (Corning Incorporated, Corning, NY, USA) [32]. Results showed that the treatment with several ligands all triggered robust DMR and TIRF signals, but with distinct kinetics and amplitudes (Figure 5a–f). The two β2AR agonists epinephrine and isoproterenol triggered a transit positive DMR (Figure 5a), while epidermal growth factor (EGF), an agonist for the EGF receptor, also triggered a transit positive DMR but with much greater amplitude (Figure 5b). In contrast, the multi-kinase inhibitor TBB (4,5,6,7-tetrabromobenzotriazole) led to a sustained negative DMR (Figure 5c). Consistent with the fact that both label-free DMR and TIRF use surface bound evanescent wave for detection, we found that the signature of every ligand-induced TIRF kinetic profile obtained closely resembles its corresponding DMR signal (comparing Figure 4d with 4a for the two β2AR agonists, comparing Figure 4e with Figure 4b for EGF, and comparing Figure 4f with Figure 4c for TBB). Interestingly, the DMR and TIRF signatures of each agonist also display distinct fine features. This is because the RWG biosensor is mostly sensitive to dynamic redistribution of cellular components within the penetration depth (~150 nm) [75], while TIRF is a direct function of the distances of fluorescent receptors at the cell surface but within 100nm near the glass surface [1]. TIRF only detects the fluorescent receptors located at the cell surface membrane near the substrate surface (Figure 1g–i). Collectively, these results suggest that the TIRF measurements can provide useful information for assessing receptor pharmacology.


Total internal reflection fluorescence quantification of receptor pharmacology.

Fang Y - Biosensors (Basel) (2015)

The label-free and TIRF profiles of receptor activation in HEK293-β2AR-GFP cells. (a–c) Real-time DMR signals induced by 10 µM epinephrine and 10 µM isoproterenol (a); 32nM EGF (b); and 10 µM TBB (c); (d–f) Real-time TIRF signals induced by 10 µM epinephrine and 10 µM isoproterenol (d); 32nM EGF (e); and 10 µM TBB (f); (g–i) TIRF images before (g), and 2 min (h) and 10 min (i) after the stimulation with 10 µM epinephrine. The data in (a–c) shows mean ± s.d. of eight replicates. Scale bar in g–i is 10 µm. This figure is reproduced with permission from Ref. [25].
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Related In: Results  -  Collection

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biosensors-05-00223-f005: The label-free and TIRF profiles of receptor activation in HEK293-β2AR-GFP cells. (a–c) Real-time DMR signals induced by 10 µM epinephrine and 10 µM isoproterenol (a); 32nM EGF (b); and 10 µM TBB (c); (d–f) Real-time TIRF signals induced by 10 µM epinephrine and 10 µM isoproterenol (d); 32nM EGF (e); and 10 µM TBB (f); (g–i) TIRF images before (g), and 2 min (h) and 10 min (i) after the stimulation with 10 µM epinephrine. The data in (a–c) shows mean ± s.d. of eight replicates. Scale bar in g–i is 10 µm. This figure is reproduced with permission from Ref. [25].
Mentions: Given that optical biosensors and TIRF all utilize surface-bound evanescent wave for cell assays, it is reasonable to hypothesize that TIRF can also be used for drug pharmacology profiling in a manner complementary to label-free biosensors. Using an engineered HEK293 cell line that stably expresses β2-AR with green fluorescent protein (GFP) at its C-terminal (β2AR-GFP) as the model, we compared the label-free DMR signals arising from the activation of several receptors with their respective TIRF signals [25]. The fluorescence intensity of β2AR-GFP was measured using a conventional TIRFM (Nikon Instruments Inc., Melville, NY, USA) and used as a cell surface fluorescence reporter for receptor activation. The DMR profiles of agonists were obtained using Epic® BT system, a whole microplate-based RWG biosensor imager (Corning Incorporated, Corning, NY, USA) [32]. Results showed that the treatment with several ligands all triggered robust DMR and TIRF signals, but with distinct kinetics and amplitudes (Figure 5a–f). The two β2AR agonists epinephrine and isoproterenol triggered a transit positive DMR (Figure 5a), while epidermal growth factor (EGF), an agonist for the EGF receptor, also triggered a transit positive DMR but with much greater amplitude (Figure 5b). In contrast, the multi-kinase inhibitor TBB (4,5,6,7-tetrabromobenzotriazole) led to a sustained negative DMR (Figure 5c). Consistent with the fact that both label-free DMR and TIRF use surface bound evanescent wave for detection, we found that the signature of every ligand-induced TIRF kinetic profile obtained closely resembles its corresponding DMR signal (comparing Figure 4d with 4a for the two β2AR agonists, comparing Figure 4e with Figure 4b for EGF, and comparing Figure 4f with Figure 4c for TBB). Interestingly, the DMR and TIRF signatures of each agonist also display distinct fine features. This is because the RWG biosensor is mostly sensitive to dynamic redistribution of cellular components within the penetration depth (~150 nm) [75], while TIRF is a direct function of the distances of fluorescent receptors at the cell surface but within 100nm near the glass surface [1]. TIRF only detects the fluorescent receptors located at the cell surface membrane near the substrate surface (Figure 1g–i). Collectively, these results suggest that the TIRF measurements can provide useful information for assessing receptor pharmacology.

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