Confocal spectral imaging by microspectrofluorometry using two-photon excitation: application to the study of anticancer drugs within single living cancer cells

Igor Chourpa; Manuela Pereira; Jean-Marc Millot; Hamid Morjani; Michel Manfait

doi:10.1117/12.349215

1 June 1999 Confocal spectral imaging by microspectrofluorometry using two-photon excitation: application to the study of anticancer drugs within single living cancer cells

Igor Chourpa, Manuela Pereira, Jean-Marc Millot, Hamid Morjani, Michel Manfait

Author Affiliations +

Proceedings Volume 3604, Optical Diagnostics of Living Cells II; (1999) https://doi.org/10.1117/12.349215
Event: BiOS '99 International Biomedical Optics Symposium, 1999, San Jose, CA, United States

Abstract

The use of the two-photon excitation (TPE) is believed to be prominent for fluorometric studies with cells. We evaluated the advantages and limitations of the two-photon technique compared to the single photon one when it used to detect potent anticancer drugs, camptothecins (CPTs), within single living cancer cells. The technique we used was confocal microspectrofluorometry amplified with possibility of the spectral imaging analysis. We have previously reported the use of the florescence emission of CPTs to study them qualitatively and quantitatively, namely, to follow the status of their hydrolyzable lactone moiety. However, the intracellular investigation of CPTs using microspectrofluorometry with single photon UV excitation (SPE) is hindered by significant interference of their fluorescence emission with cellular autofluorescence. We attempted to overcome these problems using the two-photon excitation. The intracellular single-photon- and two-photon-excited emission spectra from treated and control cells (HCT-116 line) were recorded using a spectral imaging approach. The obtained data demonstrate that, apart from intrinsically increased three- dimensional resolution, the two-photon approach was advantageous over the single-photon method with respect to selective fluorometric detection of intracellular CPTs. Nevertheless, much attention should be paid to avoid any excessive irradiation of the cells with UV and even NIR light.

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Igor Chourpa, Manuela Pereira, Jean-Marc Millot, Hamid Morjani, and Michel Manfait "Confocal spectral imaging by microspectrofluorometry using two-photon excitation: application to the study of anticancer drugs within single living cancer cells", Proc. SPIE 3604, Optical Diagnostics of Living Cells II, (1 June 1999); https://doi.org/10.1117/12.349215

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