Nondestructive quantum dot-based intracellular serotonin imaging in intact cells

https://doi.org/10.1016/j.ab.2009.01.041Get rights and content

Abstract

In recent years, quantum dots (Qdot), with their unique physical, chemical, and optical properties, have been used extensively as probes to visualize several cell membrane receptors and extracellular biomolecules. However, Qdot-based intracellular imaging has always been associated with vital lacunas. High affinity between quantum dots may induce serious aggregation in the cytoplasm; as a result, quantum dot aggregates are usually misinterpreted as quantum dot-probed intracellular molecules. Moreover, due to the more viscous nature of the cytoplasm versus the extracellular aqueous media, aggregation issues become more severe during intracellular studies. In this work, we suggest direct nondestructive serotonin imaging in an intact cell using the quantum dot-based immunoassay with a rapid tunable multicolor imaging system based on the acousto-optic tunable filter. Any false-positive intracellular serotonin molecules that appeared due to the aggregation of quantum dots could be completely discriminated from the real intracellular serotonin granules through multicolor cellular imaging. The developed method is quick and has wide applicability in targeting various intracellular proteins, coenzymes, and micronutrients.

Section snippets

Cells and serotonin uptake

Human choriocarcinoma cells (JAR) were cultured in RPMI 1640 medium (30041, JRS, Inc.). Cultures were supplemented with 10% v/v fetal bovine serum (FBS; 16000-044, Gibco), 60 μg/ml penicillin, and 100 μg/ml streptomycin (P0781, Sigma) and maintained in 25 cm2 cell culture flasks (Nunclon) at 37 °C in a 5% CO2 atmosphere. After 48 h in culture, cells were used for uptake experiments. The culture medium was removed and cells were washed briefly in 1× phosphate-buffered saline (PBS) and preincubated

Results and discussion

Native autofluorescence of JAR cells can be seen in Fig. 2a. Cells were excited at 488 nm (in the absence of Qdot-antibody conjugates and serotonin) and autofluorescence was detected at 523 nm. Numerous intracellular fluorescent molecules that emit light at specified wavelengths have been reported as biomarkers to distinguish normal cells from neoplastic cells. These biomarkers include a large number of proteins, coenzymes, and micronutrients. Among the native intracellular fluorescent molecules,

Conclusion

This study provides a sophisticated approach that directly detects intracellular components such as serotonin granules in intact cells. The serotonin neurotransmitter is directly visualized via a nondestructive multicolor immunofluorescence assay. A set of cellular images as a function of wavelength is obtained without much cellular destruction. This multicolor imaging technique clearly does away with aggregation issues that emerge from the use of quantum dots as probes for intracellular

Acknowledgments

This work was supported by a Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean Ministry of Science and Technology (MOST) under contract M10640040002-08N4004-00211 and M10644080003-08N4408-00310.

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