Using intensity-modulated scanning beams in combination with lock-in detection for recording multiple-labeled fluorescent specimens in confocal laser microscopy

ABSTRACTWe demonstrate how the new IMS (Intensity-modulated Multiple-beam Scanning) technique can be used toimprove simultaneous recording of multiple fluorophores in confocal scanning laser microscopes. Previously usedmethods have often suffered from unacceptably high cross-talk between the recording channels. In the experimentalset-up described two laser beams of different wavelengths, and modulated at different frequencies, illuminate thespecimen simultaneously. These beams predominantly excite one fluorophore each. Light emitted from thespecimen is spectrally separated and detected by two photomultiplier tubes (PMTs). The signals from the PMTs areconnected to lock-in amplifiers, tuned to the modulation frequency of the corresponding laser beam. As a result,improved selectivity in the detection of the fluorophores is obtained, since both the excitation and emissionproperties of the fluorophores are utilized. Previously this has been possible only by scanning the specimen twice,using different excitation wavelengths and detection filters. Compared with scanning the specimen twice, the new