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

摘要

Abstract: The use of the two-photon excitation (TPE) is believedto be prominent for fluorometric studies with cells. Weevaluated the advantages and limitations of thetwo-photon technique compared to the single photon onewhen it used to detect potent anticancer drugs,camptothecins (CPTs), within single living cancercells. The technique we used was confocalmicrospectrofluorometry amplified with possibility ofthe spectral imaging analysis. We have previouslyreported the use of the florescence emission of CPTs tostudy them qualitatively and quantitatively, namely, tofollow the status of their hydrolyzable lactone moiety.However, the intracellular investigation of CPTs usingmicrospectrofluorometry with single photon UVexcitation (SPE) is hindered by significantinterference of their fluorescence emission withcellular autofluorescence. We attempted to overcomethese problems using the two-photon excitation. Theintracellular single-photon- and two-photon-excitedemission spectra from treated and control cells(HCT-116 line) were recorded using a spectral imagingapproach. The obtained data demonstrate that, apartfrom intrinsically increased three- dimensionalresolution, the two-photon approach was advantageousover the single-photon method with respect to selectivefluorometric detection of intracellular CPTs.Nevertheless, much attention should be paid to avoidany excessive irradiation of the cells with UV and evenNIR light. !23