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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 enzyme activities in cells. a Chemical structures and optical properties of three membrane-targeting fluorescent dialkylcarboncyanines, DiI, DiD, and DiR. The blue shaded region indicates the self-decomposition light emission of Galacton-plus. b Human HCT116/LacZ colon cancer cells were single-stained with DiI, DiD, DIR or double-stained with both DiD and DiR prior to CIEEL imaging with Glalacton-plus. The cells were stably transfected with a LacZ construct for bacterial β-galactosidase expression and Galacton-plus activation. The total luminescence emission was imaged without an emission filter (open), and then scanned from 500 to 680 nm. Fluorescence imaging was also performed to determine the input levels of dyes. The DiD + DiR double-stained cells contained half fluorescence intensity for each dye in comparison with the corresponding single-stained cells. c Quantitative representation of the luminescence emission profiles as in b
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Fig3: CIEEL energy transfer imaging of enzyme activities in cells. a Chemical structures and optical properties of three membrane-targeting fluorescent dialkylcarboncyanines, DiI, DiD, and DiR. The blue shaded region indicates the self-decomposition light emission of Galacton-plus. b Human HCT116/LacZ colon cancer cells were single-stained with DiI, DiD, DIR or double-stained with both DiD and DiR prior to CIEEL imaging with Glalacton-plus. The cells were stably transfected with a LacZ construct for bacterial β-galactosidase expression and Galacton-plus activation. The total luminescence emission was imaged without an emission filter (open), and then scanned from 500 to 680 nm. Fluorescence imaging was also performed to determine the input levels of dyes. The DiD + DiR double-stained cells contained half fluorescence intensity for each dye in comparison with the corresponding single-stained cells. c Quantitative representation of the luminescence emission profiles as in b

Mentions: To further test if the resonance mechanism plays a role in the intermolecular CIEEL energy transfer, we took advantage of three closely related long-chain dialkylcarbocyanines, DiI [DiIC18(3)], DiD [DiIC18(5)] and DiR [DiIC18(7)], as the fluorescent recipients (Fig. 3a) [17]. These lipophilic fluorophores have similar structures and only show strong fluorescence when bound to plasma membrane [17]. Depending on the number of ethelenyl (−HC = CH−) groups in the linker between the two carbocyanines, their emission fluorescent spectra range from visible yellow-green to the NIR region (Fig. 3a). DiI has the shortest 3-carbon linker and a yellow-green spectrum (λex/λem = 549/565 nm). DiD has a 5-carbon linker and a red fluorescent spectrum (λex/λem = 644/663 nm). DiR has the longest 7-carbon linker and a NIR spectrum (λex/λem = 748/780 nm). Of note, among all three dialkylcarboncyanines, only DiI has an excitation spectrum overlapping with the blue emission spectrum of Galacton-plus. DiD and DiR have no appreciable spectral overlap for resonance energy transfer. Thus, if the resonance-based mechanism is the major contributor to the observed energy transfer light production, we would expect DiI to demonstrate the highest energy transfer efficiency and DiR should be the least efficient.Fig. 3


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 enzyme activities in cells. a Chemical structures and optical properties of three membrane-targeting fluorescent dialkylcarboncyanines, DiI, DiD, and DiR. The blue shaded region indicates the self-decomposition light emission of Galacton-plus. b Human HCT116/LacZ colon cancer cells were single-stained with DiI, DiD, DIR or double-stained with both DiD and DiR prior to CIEEL imaging with Glalacton-plus. The cells were stably transfected with a LacZ construct for bacterial β-galactosidase expression and Galacton-plus activation. The total luminescence emission was imaged without an emission filter (open), and then scanned from 500 to 680 nm. Fluorescence imaging was also performed to determine the input levels of dyes. The DiD + DiR double-stained cells contained half fluorescence intensity for each dye in comparison with the corresponding single-stained cells. c Quantitative representation of the luminescence emission profiles as in b
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4477311&req=5

Fig3: CIEEL energy transfer imaging of enzyme activities in cells. a Chemical structures and optical properties of three membrane-targeting fluorescent dialkylcarboncyanines, DiI, DiD, and DiR. The blue shaded region indicates the self-decomposition light emission of Galacton-plus. b Human HCT116/LacZ colon cancer cells were single-stained with DiI, DiD, DIR or double-stained with both DiD and DiR prior to CIEEL imaging with Glalacton-plus. The cells were stably transfected with a LacZ construct for bacterial β-galactosidase expression and Galacton-plus activation. The total luminescence emission was imaged without an emission filter (open), and then scanned from 500 to 680 nm. Fluorescence imaging was also performed to determine the input levels of dyes. The DiD + DiR double-stained cells contained half fluorescence intensity for each dye in comparison with the corresponding single-stained cells. c Quantitative representation of the luminescence emission profiles as in b
Mentions: To further test if the resonance mechanism plays a role in the intermolecular CIEEL energy transfer, we took advantage of three closely related long-chain dialkylcarbocyanines, DiI [DiIC18(3)], DiD [DiIC18(5)] and DiR [DiIC18(7)], as the fluorescent recipients (Fig. 3a) [17]. These lipophilic fluorophores have similar structures and only show strong fluorescence when bound to plasma membrane [17]. Depending on the number of ethelenyl (−HC = CH−) groups in the linker between the two carbocyanines, their emission fluorescent spectra range from visible yellow-green to the NIR region (Fig. 3a). DiI has the shortest 3-carbon linker and a yellow-green spectrum (λex/λem = 549/565 nm). DiD has a 5-carbon linker and a red fluorescent spectrum (λex/λem = 644/663 nm). DiR has the longest 7-carbon linker and a NIR spectrum (λex/λem = 748/780 nm). Of note, among all three dialkylcarboncyanines, only DiI has an excitation spectrum overlapping with the blue emission spectrum of Galacton-plus. DiD and DiR have no appreciable spectral overlap for resonance energy transfer. Thus, if the resonance-based mechanism is the major contributor to the observed energy transfer light production, we would expect DiI to demonstrate the highest energy transfer efficiency and DiR should be the least efficient.Fig. 3

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