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In vivo imaging of endogenous enzyme activities using luminescent 1,2-dioxetane compounds.

Tseng JC, Kung AL - J. Biomed. Sci. (2015)

Bottom Line: In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity.In this report, we provide proof-of-concept for CIEEL imaging of in vivo enzymatic activity.In addition, we demonstrate the use of CIEEL energy transfer for visualizing elevated alkaline phosphatase activity associated with tissue inflammation in living animals.

View Article: PubMed Central - PubMed

Affiliation: Lurie Family Imaging Center, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA, 02215, USA. jct223@gmail.com.

ABSTRACT

Background: Here we present a non-invasive imaging method for visualizing endogenous enzyme activities in living animals. This optical imaging method is based on an energy transfer principle termed chemically initiated electron exchange luminescence (CIEEL). The light energy is provided by enzymatic activation of metastable 1,2-dioxetane substrates, whose protective groups are removed by hydrolytic enzymes such as β-galactosidase and alkaline phosphatase. In the presence of a nearby fluorescent recipient, the chemical energy within the activated substrate is then transferred via formation of a charge-transfer complex with the fluorophore, a mechanism closely related to glow stick chemistry.

Results: Efficient CIEEL energy transfer requires close proximity between the trigger enzyme and the fluorescent recipient. Using cells stained with fluorescent dialkylcarbocyanines as the energy recipients, we demonstrated CIEEL imaging of cellular β-galactosidase or alkaline phosphatase activity. In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity.

Conclusions: In this report, we provide proof-of-concept for CIEEL imaging of in vivo enzymatic activity. In addition, we demonstrate the use of CIEEL energy transfer for visualizing elevated alkaline phosphatase activity associated with tissue inflammation in living animals.

No MeSH data available.


Related in: MedlinePlus

CIEEL energy transfer imaging of endogenous phosphatase activity in cells. Human MM1S myeloma cells were stained with DiR prior to CIEEL imaging with CSPD, a chemiluminescent 1,2-dioxetane substrate for alkaline phosphatase detection. Unstained cells were used as negative control. Luminescence emission levels were determined by sequential scanning from 500 to 840 nm. Quantitative representation of the imaging data showed DiR-mediated energy transfer signals in the 720–840 nm NIR region, a finding consistent with the DiR’s fluorescent emission profile
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Fig5: CIEEL energy transfer imaging of endogenous phosphatase activity in cells. Human MM1S myeloma cells were stained with DiR prior to CIEEL imaging with CSPD, a chemiluminescent 1,2-dioxetane substrate for alkaline phosphatase detection. Unstained cells were used as negative control. Luminescence emission levels were determined by sequential scanning from 500 to 840 nm. Quantitative representation of the imaging data showed DiR-mediated energy transfer signals in the 720–840 nm NIR region, a finding consistent with the DiR’s fluorescent emission profile

Mentions: Since phosphatase activities are commonly present in many types of mammalian cells, we tested if such endogenous enzyme activities can be used for substrate activation. Structurally similar to Galacton-plus, CSPD has been widely used as a chemiluminescent substrate for detecting alkaline phosphatase activity in a variety of biological assays (Fig. 1). Hydrolytic phosphatase activity removes the protective phosphate group of CSPD for substrate activation. The substrate then generates a high-energy 1,2-dioxitane intermediate. In order to demonstrate energy capture in cells, we used DiR-stained MM1S human myeloma cells (Fig. 5). Myeloma cells are derived from B lymphoid lineage which is known to express alkaline phosphatase in the plasma membrane [19]. We observed that the endogenous phosphatase triggered CSPD activation and resulted in subsequent energy transfer to DiR. In contrast, without the recipient fluorophore, the unstained control MM1S cells only showed self-decomposition luminescence. These results were consistent with our previous findings where the Galacton-plus was used as the energy source for β-galactosidase imaging (Fig. 3).Fig. 5


In vivo imaging of endogenous enzyme activities using luminescent 1,2-dioxetane compounds.

Tseng JC, Kung AL - J. Biomed. Sci. (2015)

CIEEL energy transfer imaging of endogenous phosphatase activity in cells. Human MM1S myeloma cells were stained with DiR prior to CIEEL imaging with CSPD, a chemiluminescent 1,2-dioxetane substrate for alkaline phosphatase detection. Unstained cells were used as negative control. Luminescence emission levels were determined by sequential scanning from 500 to 840 nm. Quantitative representation of the imaging data showed DiR-mediated energy transfer signals in the 720–840 nm NIR region, a finding consistent with the DiR’s fluorescent emission profile
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4477311&req=5

Fig5: CIEEL energy transfer imaging of endogenous phosphatase activity in cells. Human MM1S myeloma cells were stained with DiR prior to CIEEL imaging with CSPD, a chemiluminescent 1,2-dioxetane substrate for alkaline phosphatase detection. Unstained cells were used as negative control. Luminescence emission levels were determined by sequential scanning from 500 to 840 nm. Quantitative representation of the imaging data showed DiR-mediated energy transfer signals in the 720–840 nm NIR region, a finding consistent with the DiR’s fluorescent emission profile
Mentions: Since phosphatase activities are commonly present in many types of mammalian cells, we tested if such endogenous enzyme activities can be used for substrate activation. Structurally similar to Galacton-plus, CSPD has been widely used as a chemiluminescent substrate for detecting alkaline phosphatase activity in a variety of biological assays (Fig. 1). Hydrolytic phosphatase activity removes the protective phosphate group of CSPD for substrate activation. The substrate then generates a high-energy 1,2-dioxitane intermediate. In order to demonstrate energy capture in cells, we used DiR-stained MM1S human myeloma cells (Fig. 5). Myeloma cells are derived from B lymphoid lineage which is known to express alkaline phosphatase in the plasma membrane [19]. We observed that the endogenous phosphatase triggered CSPD activation and resulted in subsequent energy transfer to DiR. In contrast, without the recipient fluorophore, the unstained control MM1S cells only showed self-decomposition luminescence. These results were consistent with our previous findings where the Galacton-plus was used as the energy source for β-galactosidase imaging (Fig. 3).Fig. 5

Bottom Line: In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity.In this report, we provide proof-of-concept for CIEEL imaging of in vivo enzymatic activity.In addition, we demonstrate the use of CIEEL energy transfer for visualizing elevated alkaline phosphatase activity associated with tissue inflammation in living animals.

View Article: PubMed Central - PubMed

Affiliation: Lurie Family Imaging Center, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA, 02215, USA. jct223@gmail.com.

ABSTRACT

Background: Here we present a non-invasive imaging method for visualizing endogenous enzyme activities in living animals. This optical imaging method is based on an energy transfer principle termed chemically initiated electron exchange luminescence (CIEEL). The light energy is provided by enzymatic activation of metastable 1,2-dioxetane substrates, whose protective groups are removed by hydrolytic enzymes such as β-galactosidase and alkaline phosphatase. In the presence of a nearby fluorescent recipient, the chemical energy within the activated substrate is then transferred via formation of a charge-transfer complex with the fluorophore, a mechanism closely related to glow stick chemistry.

Results: Efficient CIEEL energy transfer requires close proximity between the trigger enzyme and the fluorescent recipient. Using cells stained with fluorescent dialkylcarbocyanines as the energy recipients, we demonstrated CIEEL imaging of cellular β-galactosidase or alkaline phosphatase activity. In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity.

Conclusions: In this report, we provide proof-of-concept for CIEEL imaging of in vivo enzymatic activity. In addition, we demonstrate the use of CIEEL energy transfer for visualizing elevated alkaline phosphatase activity associated with tissue inflammation in living animals.

No MeSH data available.


Related in: MedlinePlus